USF Libraries
USF Digital Collections

The novel use of recombinant granulocyte-macrophage colony-stimulating factor (gm-csf) to reverse cerebral amyloidosis a...

MISSING IMAGE

Material Information

Title:
The novel use of recombinant granulocyte-macrophage colony-stimulating factor (gm-csf) to reverse cerebral amyloidosis and cognitive impairment in alzheimer's disease mouse models : insights from the investigation of rheumatoid arthritis as a negative risk factor for alzheimer's disease
Physical Description:
Book
Language:
English
Creator:
Boyd, Timothy
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
G-CSF
M-CSF
Neuroinflammation
Amyloid beta
Radial Arm Water Maze
Cognitive Interference task
Transgenic mice
Intrahippocampal
Subcutanteous
Dissertations, Academic -- Molecular Medicine -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: For many years, it has been known that Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). It has been commonly assumed that RA patients' usage of non-steroidal anti-inflammatory drugs (NSAIDs) have helped prevent the onset and progression of AD pathogenesis. Furthermore, experiments in animal models of Alzheimer's disease have looked to inhibit inflammation, and have demonstrated some efficacy against AD-like pathology in these models. Thus many NSAID clinical trials have been performed over the years, but all have proven unsuccessful in AD patients. This suggested that intrinsic factors within RA pathogenesis itself may underlie RA's protective effect. My dissertation research goal was to investigate this inverse relationship between RA and AD, in order to more precisely pinpoint critical events in AD pathogenesis toward developing therapeutic strategies against AD. It seemed improbable that any secreted factors, produced in RA pathogenesis, could maintain high enough concentrations in the circulatory system to cross the blood brain barrier and inhibit AD pathogenesis, without affecting all other organ systems. It did seem possible that the leukocyte populations induced in RA, could traverse the circulatory system, extravasate into the brain parenchyma, and impede or reverse AD pathogenesis. We thus investigated the colony-stimulating factors, which are up‑regulated in RA and which induce most of RA's leukocytosis, on the pathology and behavior of transgenic AD mice. We found that G‑CSF and more significantly, GM-CSF, reduced amyloidosis throughout the treated brain hemisphere one week following bolus intrahippocampal administration into AD mice. We then found that 20 days of subcutaneous injections of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) significantly reduced brain amyloidosis and completely reversed cognitive impairment in aged cognitively-impaired mice, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, the recombinant human GM‑CSF analogue, in elderly leukopenic patients, suggested that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD patients. It was also implied that age-related depressed hematopoiesis may be etiological for AD pathogenesis.
Thesis:
Dissertation (PHD)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Timothy Boyd.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains X pages.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
usfldc doi - E14-SFE0004662
usfldc handle - e14.4662
System ID:
SFS0027977:00001


This item is only available as the following downloads:


Full Text

PAGE 1

The Novel Use of Recombinant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) to Reverse Cerebral Amyloidosis and Cognitive Impairment in Alzheimers Disease Mouse Models: Insights from the Investigation of Rheumatoid Arthritis as a Negative Risk Factor for Alzheimers Disease by Timothy David Boyd A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Hunt ington Potter, Ph.D. Inge Wefes, Ph.D. Ray Widen, Ph.D. Ronald Keller, Ph.D. Andreas Seyfang, Ph.D. Juan Sanchez-Ramos, Ph.D., M.D. Date of Approval: July 2, 2010 Keywords: G-CSF, M-CSF, neuroinflammation, A Radial Arm Water Maze, Cognitive Interference task, transgenic mice, intrahippocampal, subcutaneous Copyright 2010, Timothy David Boyd

PAGE 2

DEDICATION This dissertation is dedicated to my father, who passed on July 4th, 2008, almost two years to the date of my defense. I would also like to dedicate this dissertation to Luz Bau tista, who welcomed me into her home as family when I first moved to Florida and started USF, and who also passed away during my time in this degree program. Without either the support of my dad or Lu z, I would never have achieved a doctorate degree at USF, and I will always treasure their memories and the influences that they had on my life.

PAGE 3

ACKNOWLEDGMENTS I would like to first thank my entire dissert ation committee for thei r help in guiding me through my studies at the University of South Florida and for helping me be successful in my dissertation defense. I es pecially want to thank my mentor, Dr. Huntington Potter, who gave me the opportunity, s upport, and encouragement to pursue my research project. I also am very appreciative to Dr. Inge Wefe s for all of her help and guidance in both the Ph.D. and the Masters in Biotechnology. Additionally, Dr. Jaya Padmanabhan has been a dear friend and collaborator in the lab for wh ich I am extremely grateful. I would like to thank all of my other colleagues and friends in the lab, such as Dr. Peter Neame, Antoneta Granic, Michelle Norden, Tina Fi orelli, Lisa Hornbeck, Dr. Sergiy Borysov, and Monique Levy, who have helped make th is degree very rewarding. There are many professors, such as Dr. Ronald K. Keller, Dr. Samuel Saporta, Dr. Herman Friedman, and Dr. Burt Anderson that have also been frie nds and mentors throughout my degree. I want to thank Dr. Ray Widen and Dr. Shannon Moroney for also mentoring me and giving me the chance to work in the lab at Tampa Ge neral Hospital after my undergraduate years, which helped guide me on the path of the docto rate degree. I am especially appreciative to Ann Brooks, who allowed me to volunteer at the Melting Pot Restaurant for St. Jude Childrens Research Hospital. Unfortunately, th ere are too many friends to list, but I am very appreciative to have had them all in my life during the time of my degree. Lastly, but not least of all, I would like to give my colleague and close friend, Dr. Steve Bennett, as well as his wife Laura, many thanks for all of their help and influence throughout the last five years while achieving this degree. Ag ain, thanks to all and to any that I have inadvertantly failed to mention.

PAGE 4

i TABLE OF CONTENTS LIST OF FIGURES iv ABSTRACT vi CHAPTER ONE INTRODUCTION 1 The Inverse Relationship Between Rheu matoid Arthritis and Alzheimers Disease 1 Epidemilogical Studies 1 NSAID Usage 2 Leprosy and Dementia 5 Rheumatoid Arthritis 7 Pathogenesis of Rheumatoid Arthritis 7 Alzheimers Disease 9 History of Alzheimers Disease 9 Statistics of Alzheimers Disease 10 Pathological Hallmarks of Alzheimers Disease 11 Overview 11 Amyloidosis 11 Amyloid-Associated Proteins 14 Tauopathy 15 Cerebrovascular Dysfunction 17 Downs Sydrome, Aneuploidy, Cell Cycle, and Alzheimers Disease 19 Conclusions and Research Theory 28 References 31 CHAPTER TWO RESEARCH RATIONALE 47 Introduction 47 Secreted Factors in RA and AD 46 Leukocytes in RA and AD 50 Complement System and its Involvement in AD 51 Overview 51 Classical Pathway 51 Lectin Pathway 53 Alternative Pathway 54 C3-Convertase 55 C3 and AD 56 C3 Receptors 58 Formation of the Membrane Attack Complex 59 Regulation of the MAC 59 Vitronectin 59 Protectin 60 Clusterin 61

PAGE 5

ii Proteolytic Fragments of Complement 64 Conclusion 66 Leukocyte Recruitment Into the Brain 67 Bone Marrow-Derived Cells (BMDCs) in the CNS 67 BMDCs and AD 69 Research Plan 72 References 77 CHAPTER THREE DEVELOPMENT OF BILATERAL BRAIN INFUSION METHODOLOGY 89 Introduction 89 Available Commercial Catheters 90 Bilateral Brain Infusion Catheter Construction 91 Transgenic Mice 92 Materials 92 Intracranial Infusions 93 Histology and Immunohistochemistry 94 Results 95 Animal Recovery 95 Bilateral Brain Infusions 97 Interaction of ACT and Amyloid 98 Conclusions 98 References 106 CHAPTER FOUR INTRAHIPPOCAMPA L ADMINISTRATION OF CSFS 108 Introduction 108 Methodology of Transgenic Mouse Studies Involving Intrahippocampal Administration of CSFs 109 Transgenic Mice 109 Intrahippocampal Injections of CSFs 109 Immunohistochemistry and Image Analysis of Intrahippocampal-injected Mice 110 Microscopy and Method of Imag e J Analysis of Amyloid Deposition 110 Results 112 Bolus Intrahippocampal Injections of CSFs 112 Conclusion 113 References 126 CHAPTER FIVE SUBCU TANEOUS ADMINISTRATION OF GM-CSF 128 Introduction 128 Methodology of Behavioral Transg enic Mouse Study Involving Daily Subcutaneous Treatment with GM-CSF 128 Transgenic Mice 128 Radial Arm Water Maze (RAWM) and Cognitive Interference Tasks 129

PAGE 6

iii Immunohistochemistry and Image Analysis of Subcutaneous GM-CSF-treated Mice 132 Results 133 Daily Subcutaneous Injections of GM-CSF 133 Conclusions 135 References 146 CHAPTER SIX DISCUSSION 147 Introduction 147 Chronology of Initial Experiments 148 Bilateral Infusion Catheters 149 M-CSF and AD 152 G-CSF and AD 154 GM-CSF and AD 155 Angiogenic Functions of CSFs in the AD Brain 156 Introduction 156 Angiogenic Mediators 158 Macrophages 158 Myeloid-Derived Suppressor Cells 159 Neutrophils 160 Dendritic Cells 160 Conclusion 161 Neuroprotective Functions of CSFs in the AD Brain 163 GM-CSF 163 G-CSF 164 M-CSF 164 Neurogenic Functions of CSFs in the AD Brain 165 Research Theory Summary 166 Clinical Implications 167 Conclusion 168 References 168 APPENDIX A: A NOVEL TECHNIQUE FOR SIMULTANEOUS BILATERAL INFUSIONS IN A MOUSE MODEL OF NEURODEGENERATIVE DISEASE 176 APPENDIX B: GM-CSF UP-REGULAT ED IN RHEUMATOID ARTHRITIS REVERSES COGNITIVE IMPAIRMENT AND AMYLOIDOSIS IN ALZHEIMERS MICE 184 APPENDIX C: COPYRI GHT STATEMENTS 244 APPENDIX D: IMAGEJ ANALYSIS PROTOCOL 247 APPENDIX E: MAGNETIC I MMUNOHISTOCHEMICAL STAINING DEVICE AND METHODS OF USE 261 APPENDIX F: METHODS OF TREA TING COGNITIVE IMPAIRMENT 285 ABOUT THE AUTHOR End Page

PAGE 7

iv LIST OF FIGURES Figure 1. Extensive Cerebrovascular Damage in AD ...............................................30 Figure 2. Overview of the Complement System .......................................................75 Figure 3. Overview of the Hematopoietic System ....................................................76 Figure 4. Commercial Alzet catheter......................................................................91 Figure 5. Significant Variation of Amyloi d Plaque Load Between Mice Infused with M-CSF .......................................................................100 Figure 6. Significant Variation of Amyloi d Plaque Load Between mice ................101 Figure 7. Scatter-plot of Plaque Load Between Mice .............................................102 Figure 8. Catheter Assembly ...................................................................................103 Figure 9. Catheters Usage in vivo ............................................................................ 103 Figure 10. Double Immunohistochemistry of A and ACT ......................................104 Figure 11. Recombinant Peptides Remain Localized to Infused Hemispheres .........105 Figure 12. Replication of Transgenic Animal Models ..............................................106 Figure 13. Schematic of the Intrahippocampal Injection Sites ..................................114 Figure 14. Magnetic Immunohist ochemical Device .................................................115 Figure 15. Method of Identifying Individual Photomicrographs for Analysis ..........116 Figure 16. Method of ImageJ Analysis of Amyloid Plaques ....................................117 Figure 17. Intrahippocampal Injection of M-CSF ....................................................119 Figure 18. Intrahippocampal Injection of G-CSF ......................................................120 Figure 19. Intrahippocampal Injection of GM-CSF ..................................................122 Figure 20. Overall Percent Reductions in A Burden by GM-CSF .................................... 124

PAGE 8

v Figure 21. Individual Parameter Reductions in A Burden by GM-CSF ........................... 125 Figure 22. Radial Arm Water Maze Task .................................................................137 Figure 23. Behavioral Analysis Follow ing Daily Subcutaneous GM-CSF Injections .........................................................................................138 Figure 24. Amyloid Deposition in Subcutan eous GM-CSF-injected Mice ..............140 Figure 25. Microglial Immunostaining in S ubcutaneous GM-CSF-injected Mice ................................................................................................142 Figure 26. Synaptophysin Immunostaini ng in Subcutaneous GM-CSFinjected Mice ...................................................................................144

PAGE 9

vi The Novel Use of Recombinant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) to Reverse Cerebral Am yloidosis and Cognitive Impairment in Alzheimers Disease Mouse Models: Insights from the Investigation of Rheumatoid Arthritis as a Negative Risk Factor for Alzheimers Disease Timothy David Boyd ABSTRACT For many years, it has been known that Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzhei mers disease (AD). It has been commonly assumed that RA patients usage of non-st eroidal anti-inflammatory drugs (NSAIDs) have helped prevent the onset and progr ession of AD pathogenesis. Furthermore, experiments in animal models of Alzh eimers disease have looked to inhibit inflammation, and have demonstrated some efficacy against AD-like pathology in these models. Thus many NSAID clinical trials ha ve been performed ove r the years, but all have proven unsuccessful in AD patients. This suggests that intrinsic factors within RA pathogenesis itself may underlie RAs protective effect. My dissertation research goal was to inve stigate this inverse relationship between RA and AD, in order to more precisely pinpoint critical events in AD pathogenesis toward developing therapeutic strategies ag ainst AD. It seemed improbable that any secreted factors, produced in RA pa thogenesis, could maintain high enough concentrations in the circulatory system to cross the blood brain barrier and inhibit AD pathogenesis, without affecting all other organ systems. It did seem possible that the

PAGE 10

vii leukocyte populations induced in RA, could tr averse the circulatory system, extravasate into the brain parenchyma, and impede or re verse AD pathogenesis. We thus investigated the colony-stimulating factors, which are upregulated in RA and which induce most of RAs leukocytosis, on the pathology and behavior of transgenic AD mice. We found that G-CSF and more significantly, GM-CSF, re duced amyloidosis throughout the treated brain hemisphere one week following bolus intrahippocampal administration into AD mice. We then found that 20 days of subcut aneous injections of GM-CSF (the most amyloid-reducing CSF in the bolus experime nt) significantly reduced brain amyloidosis and completely reversed cognitive impairme nt in aged cognitively-impaired AD mice, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, the recombinant human GM-CSF analogue, in elderly leukopenic patients, suggested that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD patients. It was also implied that age-related depressed hematopoiesis may contribute to AD pathogenesis.

PAGE 11

1 CHAPTER 1 INTRODUCTION The Inverse Relationship Between Rheuma toid Arthritis and Alzheimers Disease Epidemiological Studies Several case-control a nd population-based studies suggest that patients with Rheumatoid arthritis (RA) are protected from developing Alzheimers disease (AD). The case-control st udy of Jenkinson et al., a survey of 96 AD and 92 non-AD cases, looking for the presence or absence of RA, found that only 2% of AD patients had RA as compared to 13% of control cases (Jenkinson, Bliss, Brain, & Scott, 1989). In a population-based study of McGeer et al., of 973 RA patients, who were 65 years and older and had extensive c linical history records, only 4 (0.41%) were demented (P. L. McGeer, McGeer, Rogers & Sibley, 1990). Similarly, the authors examined the discharge records of 7,490 RA pa tients, 65 years or older, and found that only 0.39% had concomitant AD. However, us ing data gathered in the Rochester AD Incidence cohort collected between 1950 to 1975, Beard et al. reported that only 23 (4.4%) of the 521 RA cases subsequently developed AD (Beard, Kokman, & Kurland, 1991). Another population-based study, examini ng the causes of death within the whole Finnish population covering th e year of 1989, found that only 2 (0.12%) RA patients died with concomitant AD (Myllykangas-Luosujarvi & Isomaki, 1994). The study also revealed that 227 of the Finnish people th at died in 1989, or onl y 0.54% of the whole population cohort, had been diagnosed with AD. To put these numbers into perspective today, it has recently been reported in the Un ited States, that there was a 46.1% increase in deaths attributable to Alzheimers di sease from year 2000 to 2006, with an overall average of 22.6% mortality rate (Alzheimer's_Association, 2010).

PAGE 12

2 Although it was concluded that RA is a ne gative risk factor for AD, the small population of patients with concomitant RA a nd AD suggests that genetic predispositions for AD may override RAs protective effects. There have not been any epidemiological studies that have examined this inverse re lationship between RA and AD in regards to AD-positive genetic risk factors, such as Apolipoprotein E type 4 (ApoE4) positivity, amyloid precursor protein (APP) or presen ilin-1 (PS1) mutations found in Familial Alzheimers disease (FAD), or mutations in genes that encode immune system components, such as complement receptor1(CR1) or Clusterin. However, two studies have examined the prevalence of ApoE4 in RA patients with systemic reactive amyloidosis, and both concluded that ApoE4 may be a risk fa ctor for developing reactive amyloidosis (Hasegawa et al., 1996; Maury et al., 2001). Whether ApoE4 or other genetic polymorphisms influence cerebral amyloidosis in RA patients will need to be specifically addressed in future epidemiological studi es, although the very small population of concomitant RA and AD patients may make these analyses di fficult to conduct. NSAID Usage Concurrent with the studies that examined RA as a negative risk factor for AD, were similar epidemiological studies that investigated nonsteroidal antiinflammatory drugs (NSAIDs), which RA patients chronically used to ameliorate RA symptoms, as protective against AD. A meta -analysis study reviewed 17 epidemiological studies in which RA or NSAIDs were considered as risk factors for AD (P. L. McGeer, Schulzer, & McGeer, 1996). In the meta-analysi s, there were severa l case-control and 3 population-based studies that concluded that NSAID usage may be protective against AD onset. The 3 population-based studies examined the NSAID and AD relationship by different approaches. In an Italian study, 195 AD patients were surveyed as to whether

PAGE 13

3their NSAID usage was greater or less than in people of similar age (Lucca, Tettamanti, Forloni, & Spagnoli, 1994). The authors found that NSAID usage was much less than expected in AD patients as compared to NSAID usage in an elderly control group. Another population-based study in Rotterdam examined 365 NSAID users, which had only 1.4% afflicted with AD, and concluded th at NSAIDs possibly pr evented the risk of developing AD (Andersen et al., 1995). In a third population-based study of 210 patients in the Johns Hopkins Alzheimer Disease Resear ch Center, a review of medical records of 32 AD patients who used NSAIDs compar ed to 177 AD patients who did not use NSAIDs (Rich et al., 1995). The clinical, c ognitive, and psychiatric measures of each group were compared to examine which group progressed faster with AD within a one year longitudinal study. The authors concl uded that the AD patients who took NSAIDs had a slower progression of AD pathogene sis. Altogether in the meta-analysis examination of McGeer et al. (1996), it was likewise concluded that NSAID usage was protective against developing AD, although th e data did not dedu ce which specific NSAID, or combination of NS AIDs, would be most protectiv e against AD. It was also concluded that anti-inflammatory drugs would reduce neuroinflammation, which was commonly assumed to drive the pathogenesis of AD, and that clinical trials were needed to investigate NSAIDs in order to halt the onset or progression of AD. Since the meta-analysis of McGeer (1996), many pre-clinical a nd clinical research studies have focused on investigating the reduction of inflammation as a therapeutic approach to ameliorate AD pa thogenesis. Animal studies have shown some promise in lowering inflammation and reducing AD-like symp toms (Heneka et al., 2005; Jantzen et al., 2002; Kukar et al., 2005; Lim et al., 2000; Q. Yan et al., 2003). However, large

PAGE 14

4NSAID clinical trials have been disappointing, and some have failed to show efficacy or have even been discontinued due to a dverse events (Aisen et al., 2003; Aisen, Schmeidler, & Pasinetti, 2002; P. L. McGeer, Rogers, & McGeer, 2006; Reines et al., 2004; Rogers et al., 1993; Scharf, Mander, Ugoni, Vajda, & Christophidis, 1999). Moreover, the recent Alzheimers Disease An ti-inflammatory Prevention Trial (ADAPT) study, that investigated celecoxib and naproxen, was halted due to adverse cardio/cerebrovascular events by celecoxib and because test subjects who already had mild cognitive impairment at the beginning of the trial tended to have accelerated impairment and progression to AD by naproxe n (Meinert, McCaffrey, & Breitner, 2009; Strobel, 2009). However, a two-year follow-up study of the ADAPT participants, who were cognitively normal at the beginning of the trial, and who took naproxen versus placebo, were found to be significantly protected against incidence of AD, as well as having a more favorable biomarker profile (Tau/A 1-42, proteins explained below). It was suggested by Dr. Brietner at the 2009 Intern ational Conference of Alzheimers Disease (ICAD) that Primary prevention, not diseas e modification, is the ultimate cure, and timing is everything (Strobel, 2009). This suggestion echoes the findings of another large epidemiology study of veterans over th e age of 55 years who were on long-term NSAID usage, particularly ibuprofen, and who were also protected against AD onset (Vlad, Miller, Kowall, & Felson, 2008). NSAIDs act mainly to inhibit the ac tion of cyclooxygenases (Cox), which are enzymes that catalyze the formation of eicosanoids, such as thromboxanes or prostaglandins, from an arachadonic aci d precursor. There ar e two isoforms of cyclooxygenases,Cox-1 and Cox-2, which diffe r in their expression and biological

PAGE 15

5functions (Kaufmann, Andreasson, Isaks on, & Worley, 1997; O'Banion, Sadowski, Winn, & Young, 1991). Cox-1 and Cox-2 are cons titutively expressed at low levels in most tissues, and induced e xpression of Cox-2 occurs dur ing inflammation. Traditional NSAIDs, such as aspirin or ibuprofen, ar e non-selective and inhibit both Cox-1 and Cox-2. In the brain and esp ecially around AD-vulnerable pyr amidal neurons, Cox-2 is constitutively expressed at higher levels th an Cox-1, and has enhanced expression in AD brain (Seibert et al., 1994; Yasojima, Schwab, McGeer, & McGeer, 1999; Yermakova, Rollins, Callahan, Rogers, & O'Banion, 1999). However, it has been found that Cox-2 is not detectable in microglia a nd astrocytes in AD, but it is instead expressed in neurons. Exposure of human microglial cultures to in flammatory mediators over-expressed in AD brain, such as interleukin (IL)-1, IL-6 tumor necrosis factor alpha (TNF ), and amyloid beta 1-42 (A 1-42), did not induce Cox-2 activity, a nd it was only after exposure to bacterial lipopolysaccharide (LPS) that the microglia express Cox-2 (Hoozemans et al., 2001). On the other hand, Cox-1 is up-regul ated in AD-activated microglia and may provide a more selective target to reduce AD-associated neuroinflammation (Hoozemans et al., 2002). Therefore, it can be predicte d that the Cox-2-selective NSAIDs, such as celecoxib, would act mainly to inhibit periphe ral pro-inflammatory effects, instead of acting directly against AD-related neur oinflammation. As of today, there are no clinically-approved selective Cox-1 inhib itors, and traditional NSAIDs are the only available options for inhibiting Cox-1. Leprosy and Dementia It has been well known among physicians in Japan that elderly leprosy patients who inhabit wellmonitored leprosy hospitals do not tend to develop cognitive impairment. Bacterial infection by Mycobacterium leprae causes a

PAGE 16

6chronic granulomatous disease of the skin and chronic inflammati on of the peripheral nervous system (Dastur & Razzak, 1971; Jacobs, Shetty, & Antia, 1987; Job, 1971; Save, Shetty, Shetty, & Antia, 2004; Swift, 1974). A study by McGeer et al. in 1992 surveyed living leprosy patients in Japan, aged 65 and older, for the usage of anti-leprosy drugs and for dementia. In the 1,410 leprosy patients that had been treated with anti-leprosy drugs, there was a 2.9% prevalence of demen tia compared to 6.25% of the 1,761 leprosy patients that had not been treated for at le ast 5 years. Since dapsone and other antileprosy drugs have anti-inflammatory activ ity, it was proposed that these drugs might also be protective against AD, similar to th e proposed efficacy of NSAIDs against AD (P. L. Mcgeer, Harada N., Kimura H., McGeer E. G., Schulzer M., 1992; P. L. McGeer et al., 1996). However, another epidemiological st udy examining inpatients in a Japanese leprosarium with an average age of 70 a nd who were free from active leprosy did not show protection against AD as compared to previous Japanese general population-based data (Goto et al., 1995). The la test study that examined whether anti-leprosy drugs were efficacious against AD showed that anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin, or ofloxacin) did not have any protective effect on the neurotoxicity of amyloid beta (A ) peptides, the main component of the cerebral amyloidosis in AD (Endoh, Kunishita, & Tabira, 1999). The author s also reviewed the medical records of 196 lepromatous patients, and found that there was no significant difference in prevalence of dementia of those patients th at were treated with anti-leprosy drugs compared to untreated lepromatous patients. Taking into account their previous study that did show significantly lower cerebral amyl oidosis in elderly lepromatous patients compared to controls (Chui, Tabira, Izumi, Koya, & Ogata, 1994), the authors concluded

PAGE 17

7that intrinsic mechanisms of Mycobacterium leprae infection itself in the central nervous system (CNS) may underlie the protection ag ainst AD pathogenesis in active lepromatous patients. Indeed it is a striking similarity with RA, that another chronic peripheral inflammatory disease, such as Mycobacterium leprae infection, also c onfers a reported decreased risk of AD onset, independen t of anti-inflammatory pharmaceutical intervention. Rheumatoid Arthritis Pathogenesis of Rheumatoid Arthritis. Rheumatoid arthritis is the most common systemic inflammatory disorder and the most serious and disabling type of arthritis. RA is an autoimmune disease in which the joint synovium becomes inflamed and a highly vascularized inflammatory pa nnus forms that irreparably damages the cartilage and bone, causing joint disfigurement and excruciating pain. This systemic autoimmunity is characterized by autoantibod ies against a wide ra nge of autoantigens. The first autoantibody, described in 1940 and named rheumatoid factor (RF) (Waaler, 1940), was later found to have specificity fo r the Fc region of IgG immunoglobulins. Since then, other RA autoantigens, such as cartilage components, stress proteins, enzymes, nuclear proteins, and citrullinated proteins have been discovered (Reviewed in (Duskin & Eisenberg)). The autoimmunity of RA involves not only the B lymphocytic arm of the adaptive immune system, but also a recently-described class of T helper (Th) cell lymphocytes. This new pro-inflammatory CD4+ effector cell lineage, separate from Th1, Th2, and Treg differentiations (Harring ton et al., 2005; H. Park et al., 2005; Wynn, 2005) is termed Th17, due to their releas e of cytokines, IL-17A/F. These Th17 lymphocytes also secrete many other proinf lammatory cytokines and chemokines that

PAGE 18

work in the recruitment and act ivation of innate leukocytes, as well as the pr oliferation of more autoimmune T and B lymphocytes. RA is not the only autoim mune disease with a Th17 phenotype, as Th17 lymphocytes have also been found to be greatly implicated in the pathogenesis of Multiple Sclerosis, Crohns disease, Inflammatory Bowel Disease and Systemic Lupus Erythematosus (Furuzawa-Carballeda, Vargas-Rojas, & Cabral, 2007; Nalbandian, Crispin, & Tsokos, 2009; Sarkar, Tesmer, Hindnavis, Endres, & Fox, 2007; Thakker et al., 2007). Of the innate leukocytes involved in RA, differentiated myeloid-lineage monocytic cells are major contributors to the pathogenesis of RA. The hematopoietic growth factor, M-CSF (Macrophage Colony-St imulating Factor), produced by synovial endothelial cells and macrophages, is highl y expressed and drives macrophage and osteoclast production and differentiation. The TNF family member, RANKL (receptor activator of NFB ligand), secreted by bone marrow stromal cells and T lymphocytes, binds to their RANK receptors on these macr ophage/osteoclast progenitors and activates an intracellular transduction cascade involving TRAFs, JNK, and NFB in order to induce functional osteoclasts. Secreted oste oprotegerin (OPG) is i nduced in RA synovial fluid to work as a negative regulator of th is process (Karsenty, 1999). Besides increased bone resorption due to osteoclasts in RA, the innate leukocytes work in combination with the above-mentioned autoimmune lymphocytes in the production of inflammatory-related cytokines,chemokines, hematopoietic factors, en zymes, and other solubl e factors, such as TNF IL-1, IL-6, IL-8, IL-15, IL-17A/F, IL -18, CXCL16, ADAM10, M-CSF, G-CSF, GM-CSF, MCP-1 (monocyte chemotactic protein 1), and MIF (macrophage migration inhibitory factor). There is also elevated expression of angiogenic factors, VEGF-A 8

PAGE 19

9(vascular endothelial growth factor A) a nd IGF-1 (insulin-like growth factor 1), by synovial macrophages, fibroblasts, and vascul ar smooth muscle cells, as well as, the increased expression of their recepto rs, VEGFR-1,2 on endothelial cells and macrophages, and secreted IGF Binding Prot ein (IGFBP)-3 (Matsumoto & Tsurumoto, 2002; Murakami et al., 2006). Thus the combin ed innate and adaptive autoimmunity in the inflammatory pannus of RA cause a chro nic feed-forward mechanism to increase leukocyte populations, further cytokine and chemokine induction, increased osteoclastogenesis, resultant synovia l joint deformation, and extensive neovascualarization as the pannus grows. Alzheimers Disease History of Alzheimers Disease. Alzheimers disease was first described at a scientific meeting in November 1906 by Germ an physician, Alois Alzheimer. He was the physician of a 51 year old patient, Frau Auguste D., who was brought to him by her family for memory problems, unfounded su spicions, and increasing difficulty in the ability to process new events. During Dr. Alzheimers care, these symptoms rapidly progressed until the death of Mrs. Auguste D. in the spring of 1906. Since Dr. Alzheimer had never encountered these symptoms in a patient, he requeste d the permission to perform an autopsy on Mrs. Auguste D. Examin ation of her brain revealed atrophy of the cerebral cortex, fatty deposits in the cerebral microvascul ature, abnormal extracellular and intracellular deposits, and evidence of active neurodegeneration. Dr. Alzheimer published his work in 1907, and the disease wa s named after him in 1910 (Graeber et al., 1997; Graeber, Kosel, Grasbon-Frodl, Moller, & Mehraein, 1998).

PAGE 20

10Statistics of Alzheimers Disease Alzheimers disease is the most common type of dementia today, accounting for 60 80% of all cases of dementia, followed secondly by vascular dementia, although many patient s may have hallmark pathologies of both (Alzheimer's_Association, 2010). Approximately 1 out of 8 people over the age of 65, and 1 out of 2 over the age of 85 will be di agnosed with AD. As of 2010, there were over 5.3 million Alzheimers patients in the Un ited States, costing $172 Billion in annual costs. AD is the 7th leading cause of death, and although its prevalence is increasing, the mortality from cardiovascular disease, cancer, and other di seases are decreasing. It is estimated that by 2030, there will be 7.7 milli on Alzheimers patients over the age of 65, and between 11 and 16 million by 2050. The diseas e is characterized initially by shortterm forgetfulness with the inability of the individual to learn any new things, along with fluctuations in the individua ls mood and attention span. As the disease progresses, the AD patient gradually wit hdraws from daily life, can become easily agitated, and starts to forget the names and identities of family and loved ones. Slowly and inexorably, the ability of carry out familiar tasks, to reason and exercise judgment, and to carry on conversation declines to the state of bedrest, followed by certain fatality. The lifespan of Alzheimers patients averages about 10 year s from diagnosis, and the overall physical condition and age of onset may influence how fast the disease progresses. The cause of death is usually by AD directly, such as by dehydration and malnutrition from the inability of patients to swallow, or by other diseases, such as pneumonia, stroke, etc., as the bodys general health slowly deteriorates (Alzheimer's_Association, 2010).

PAGE 21

11Pathological Hallmarks of Alzheimers Disease. Overview Alzheimers disease is a fatal, age-related, and progressive neurodegenerative disorder that presents clinic ally in patients as a progressive decline in cognitive and executive functions. Its dementia is associated with extracellular deposits of amyloid, intracellular aggregates of ne urofibrillary tangles (NFTs), and cerebral microvascular occlusions and other ischem ic defects. The hippocampus, amygdala, and cerebral cortex are the major areas of the br ain in which these hallmark pathologies occur (Hopper & Vogel, 1976; Woodard, 1966). Alth ough Dr. Alzheimer described these pathologies in his 1907 publication, it took ov er 7 decades to develop the laboratory techniques and tools in which to identify the principle component s of the then-called senile plaques and neurofibri llary tangles (Glenner & Wong, 1984). It is now known that oxidative stress, neuroinflammation, excito toxicity, apoptosis, and aneuploidy may all contribute to the pathogenesis of AD. Amyloidosis Amyloid beta (A ) peptides are the primar y component of amyloid deposits in the Alzheimers disease brain. A is proteolytically derived from the amyloid precursor protein (APP), a transmembrane pr otein expressed in almost all tissues (Glenner & Wong, 1984). Three APP isof orms (APP695, APP751, and APP 770) are found in mammals, with APP695 being the is oform mainly expressed on the surface of neurons (Tanaka et al., 1988; Tanaka et al ., 1989). Whether APP contains a chondroitin sulfate glycosaminoglycan site or a Kunitz pr otease inhibitor domain differentiates the three isoforms from each othe r (Sisodia & Price, 1995). APP is a synaptic receptor whose functi on has not been elucidated completely, but its processing is thought to influence dendritic spine development and maintenance

PAGE 22

12(Dewji & Singer, 1996; Ho & Sudhof, 2004; K. J. Lee et al., 2010; Lorenzo et al., 2000; P. Wang et al., 2005). The processing of APP occurs by three enzymes, , and secretases, and depending on whether or -secretase cleaves the APP will determine whether the resultant peptides are non-amyloidogenic or am yloidogenic, respectively. For the amyloidogenic pathway, cleavage by -secretase is the rate-limiting step for A generation (Hussain et al., 1999; Sinha et al ., 1999; Vassar et al., 1999; R. Yan et al., 1999). There are two isoforms of -secretase, BACE1 and BACE2, and although both isoforms compete for the same substrate and cleave at the same -site of APP, BACE2 activity is much less abundant and thought to be masked in the brain (Ahmed et al., 2010; Basi et al., 2003; Farzan, Sc hnitzler, Vasilieva, Leung, & Choe, 2000; Hussain et al., 2000; Sun et al., 2005; R. Yan, Munzner, S huck, & Bienkowski, 2001). Cleavage of APP by BACE1 results in the extracellular release of soluble APP beta (sAPP ), a large N-terminal ectodomain of APP with unknown function, leaving a transmembrane C-terminal fragment of 99 amino acids (C99), which is then intramembranely-cleaved by -secretase to produce extracellular A peptides of either 39, 40, or 42 amino acid residues in length. The C-terminal APP intrace llular domain (AICD) is thought to play a feed-back regulatory role in the transcrip tion of genes implicated in AD pathogenesis (Slomnicki & Lesniak, 2008). For the non-amyloidogenic pathway, cleavage by -secretase at the APP -site, which lies within the A sequence, generates a transmembrane C-terminal fragment of 83 amino acids in length (C83), with the extracellular release of the large N-terminal soluble APP alpha (sAPP ). It is interesting to note that the gene for BACE2 is located in the Downs syndrome critical region of Chromosome 21, and although purif ied BACE2 cleaves at the -site of APP, it can also

PAGE 23

13process APP downstream of the -site (R. Yan et al., 2001). Overexpression of BACE2 in a human embryonic kidney (HEK) cell line stably-expressing the Swedish mutant of APP, resulted in reduced A production, but an increase in soluble APP species (sAPP, which includes sAPP sAPP and BACE2-generated sAPP) (Sun et al., 2005). Although the authors did not determ ine relative levels of each type of sAPP, they did find that knockdown of BACE2 results in an in crease of APP C83, indicative of the nonamyloidogenic pathway. Although the authors co ncluded that BACE2 is not involved in the formation of amyloid plaques, Downs patients chronically overexpressing BACE2, as well as BACE1, would have both sp ecies of BACE competing against -secretase, which may have yet undescribed physiological effects, since the biological functions of sAPP and over-expressed BACE2-generated sAPP have not been completely elucidated. The -secretase is a multiprotein intram embrane-cleaving aspartyl protease complex, which consisits of Presenilin (PS, wh ich can be either of two isofoms, PS1 or PS2), Pen2, Aph1 (anterior pharynx-defectiv e 1 protein), and nicastrin (NCT). PS contains the catalytic site (D e Strooper et al., 1998; Y. M. Li et al., 2000; Wolfe et al., 1999), Pen2 and Aph1 have functions that have not been fully elucidated, and NCT is thought to contain the substrat e binding site (Shah et al., 2005), as well as stabilizing -secretase, since the trimeric PS/Pen2/A ph1 complex is highly unstable (Zhao, Liu, Ilagan, & Kopan, 2010). Mutations in PS1 and PS2, as well as the APP gene, have been found to lead to increased production of pathogenic A and cause early onset familial AD (FAD) (Chartier-Harlin et al., 1991; Fidani et al., 1992; Goldgaber, Lerman, McBride, Saffiotti, & Gajdusek, 1987; St George-Hys lop et al., 1990; Tanzi, George-Hyslop, &

PAGE 24

14Gusella, 1991). There are over 160 known mutations that predispose individuals to FAD, with PS1 mutations accounting fo r the majority of FAD. Amyloid-associated Proteins Over 30 other proteins have also been found to be associated with extracellular amyloid plaques, including complement proteins, complement regulatory proteins, growth factors, proteases, protease inhibitors, coagulation factors, proteoglycans, acute pha se reactants, receptors, adhesion molecules, and other soluble factors (P. L. McGeer, Kl egeris, Walker, Yasuhara, & McGeer, 1994). The majority of these proteins are directly associated with the immune system, but the characterization and determination of each proteins role in amyloid deposition and subsequent AD pathogenic pathwa ys have not yet been fully elucidated. It is known that the plaque-associated proteins, apolipoprot ein E (ApoE) and Alpha-1-antichymotrypsin (ACT) work to catalyze the formation of A filaments in vitro (Ma, Brewer, & Potter, 1996; Wisniewski & Frangione, 1992) and formation of amyloid beta-pleated sheets in vivo (Abraham, Selkoe, & Potter, 1988; Bales et al., 1999; Ma, Yee, Brewer, Das, & Potter, 1994; Nilsson et al ., 2001; Potter, Wefes, & Nilsson, 2001). ApoE acts systemically in the transport of lipid proteins, but in the brain, it is released by astrocytes and microglia during neuroinflammation. ApoE allele 4 (ApoE4) confers a very high risk for developing AD, ApoE allele 2 (ApoE2) is associated with low risk and high age of onset, and ApoE allele 3 (ApoE3) confers intermediate risk and age of onset (Strittmatter et al., 1993). ACT is an acute-phase serine protease inhib itor (serpin) that is released systemically by hepatocytes and m onocytes during inflammatory responses, and by neutrophils to work as a suicide inhibitor of Cathepsi n G during vascular remodeling (Kalsheker, 1996). However, in the brain, ACT is induced and released by astrocytes in

PAGE 25

15response to microglial release of IL-1 trauma, infection, or amyloidosis (Abraham, 2001; Das & Potter, 1995). Not only does AC T work to catalyze amyloid plaque formation, it also indu ces intracellular neuronal Tau hyperphosphorylation (Padmanabhan, Levy, Dickson, & Potter, 2006). Tauopathy The hyperphosprorylation of Tau l eads to paired helical filaments (PHFs), which themselves aggregate intracel lularly to form neurofibrillary tangles (NFTs), another major hallmark pathology of AD(Grundke-Iqbal, Iqbal, Quinlan et al., 1986; Grundke-Iqbal, Iqbal, Tung et al., 1986). Tau is a cytosolic protein that is involved in the assembly and stabilization of micr otubules (MT), and the hyperphosphorylation of Tau, impedes Tau from binding MTs and performing this role, since it undergoes misfolding, dissociation from MTs, and a ggregation (Chung, 2009; V. M. Lee, Balin, Otvos, & Trojanowski, 1991). Tau is primarily expressed in neurons and mutations in the Tau gene, encoded by 16 exons on Chromosome 17, leads to tauopathies, such as Progressive Supranuclear Pals y (PSP), Corticobasal Degene ration, Picks disease, and parkinsonism linked to chromosome 17 (FTD P-17) (Baker et al ., 1997). Although, these tauopathies also present with progressive dementia, Tau mutations have not been identified in AD pathogenesis, and the vari ous taupathies presen t in different brain regions and specific types of neurons (Ari ma, Murayama, Oyanagi, Akashi, & Inose, 1992; Delisle et al., 1999; Galloway, 1988; Hauw et al., 1990; Kertesz et al., 2000; Ksiezak-Reding et al., 1994; Mori, Nishimura, Namba, & Oda, 1994; Nasreddine et al., 1999; Poorkaj et al., 1998; Schneider & Mandelkow, 2008; Spillantin i et al., 2000). There are 85 reported phosphorylation sites that have been identified on Tau, and 45 of these have been identified to cont ribute to AD pathogenesis. Ta u is primarily phosphorylated

PAGE 26

16by glycogen synthase kinase-3beta (GSK-3 ), cyclin-dependent kinase 5 (cdk5), calcium/calmodulin-dependent protein kinase 2 (CaMKII) and secondarily by extracellular signal-related kinase (ERK), casein kinase s 1 (CK1) (Singh, Grundke-Iqbal, & Iqbal, 1995; Singh, Grundke-I qbal, McDonald, & Iqbal, 1994), and cAMP-dependent protein Kinase A (PKA) (G. Li, Yin, & Kuret, 2004; F. Liu et al., 2006; Pei et al., 2002; Trojanowski, Mawal-Dewan, Schmidt, Martin, & Lee, 1993 ). Regulation and dephosphorylation occurs by known phosphatases, PP2A, PP1, and PP2B. A peptidyl-prolyl cis-isomerase, Pin-1, is involve d in the assembly, folding, and transport of Tau. For example in AD, Pin-1 binds Tau when Tau is phosphorylated at its Threonine residue 231 (Thr231), in order to facili tate dephosphorylation of Thr231 by PP2A (Butterfield et al., 2006). The mechanisms into the hype rphosphorylation of Tau, and its subsequent aggregation into NF Ts, are currently a very active area of research, as levels of NFTs correlate closely with AD prognosis. Cerebrovasculature Dysfunction. Another major hallmark pathology of AD is the extensive cerebrovascular damage that occurs as AD progresses. Cerebrovascular damage was noted as early as 1911, by Dr. Alois Alzh eimer from his second senile dementia case study of a 56 year-old demented man, Johann Feigel, who was admitted to the Royal Psychiatric Clinic in Munich on November 12, 1907 with possi ble vascular dementia and who died on October 3, 1910 with pneumonia-li ke symptoms, and was described by Dr. Alzheimer to have degeneration of the smalle r cerebral blood vessels (Graeber et al., 1997). An immunohistochemical study that l ooked at deceased AD patients and agematched control subjects found that over 90% of those with AD had an absence of endothelial staining for capi llaries, even though it app eared that their basement

PAGE 27

17membranes remained, suggesting that the ca pillaries had collapsed and degenerated, concomitant with A deposition (Kalaria & Hedera, 1995). Other studies have shown in the cerebrovasculature of AD patients that the basement membranes thicken, that the pericytes degenerate, that endothelial cell morphology is altered, and that luminal buckling occurs (Buee et al., 1994; Claudio, 1996; Mancardi, Perdelli, Rivano, Leonardi, & Bugiani, 1980; Perlmutter, Myers, & Barron, 1994; Prasher et al., 1998; Scheibel, 1987; Scheibel, Duong, & Tomiyasu, 1987; Zarow, Barron, Chui, & Perlmutter, 1997). These microvascular abnormalities and degeneration of capillary vessels result in reduced cerebral blood flow and deficient delivery of oxygen and glucose to the brain (de la Torre, 2004, 2009, 2010; de la Torre & Stefano, 2000) As discussed later, this damage to the cerebral vasculature may also im pair the clearance mechanisms of A from the brain (Chapters 2 and 6). Although these capillary abnormalities can be examined post-mortem in AD patients, non-invasive techniques are not available to longitudinally study these microvascular effects in vivo However, experimental animal models, such as chronic cerebral hypoperfusion in aged rats over one year, induced by permanent surgical occlusion of both common carotid arteries (two-vessel occlusion), results in almost identical microvascular pat hology as in AD patients, such as basement membrane thickening, deposition of collagen, dist ortion of endothelial cell morphology, degeneration of pericytes, and vessel lume n buckling, all within the CA1 hippocampal region and which also correlates with a progr essive decline in spatial memory function (De Jong et al., 1999; Farkas, De Jong et al ., 2000; Farkas, De Vos, Jansen Steur, & Luiten, 2000). The immense cerebrovascular damage found in AD pathogenesis can also

PAGE 28

18be demonstrated by vascular corrosion cas ting (VCC) of Tg AD mice. A recent study which used the VCC technique, employed a new polyurethane lowviscosity resin and other special resins to perfuse young and old Tg AD mice (Heinzer et al., 2006; Krucker, Lang, & Meyer, 2006; Meyer et al., 2007; Meyer, Ulmann-Schuler, Staufenbiel, & Krucker, 2008). After perfusion and time for th e resins to polymerize, chemical removal of the surrounding tissue was performed. These resins that were used have minimal shrinkage, leaving high quality re plicas of the lume ns of even the smallest capillaries. VCC allows for 3-dimensional, very high resolution examination of the microvasculatures morphology under scanni ng electron microscopy, which can even determine endothelial cell imprints and show distinction between arterial versus veinal vessels within the vascular tree (Heinzer et al., 2008; Weiger, Lametschwandtner, & Stockmayer, 1986). The authors found that am yloid deposits begin to accumulate on the capillary vessels at very early ages in the AD mice, in shapes desc ribed as pompoms or cubes of A species, as determined by mass spectro metry analysis, and in doing so, the microvessels begin to crimp and become o ccluded. These pompoms or cubes are not found in normal age-matched control mice, and also would not be detectable by conventional histochemistry, since conventiona l histochemistry does not retain the 3D architecture of the entire cerebral vasculature and capillary vessels. As the AD mice age and plaques became large enough to view by conventional histochemistry, VCC showed that the vasculature becomes deformed, trunc ated, or occluded surrounding the amyloid plaques, forming significant holes in the vasc ular system (Figure 1) (Beckmann et al., 2003; Meyer et al., 2008). There also was extensive increased vascular remodeling around the truncated ends of the vessels, as the vessels were studded with knobs,

PAGE 29

19budding, and other abnormal protrusions, in an apparent attempt to circumvent the amyloid plaques. However, the intrinsic nature of A itself is anti-angiogenic and accelerates the senescence of endothelial cells at very ea rly stages of vascular development (Donnini et al., 2010), possibly by mechanisms, such as the direct binding of A to the receptor for VEGF (Patel et al., 2010). Indeed, a ten amino acid peptide with A itself, has been recently reported to inhib it tumorigenesis via an ti-angiogenic effects (Paris et al., 2010). Thus, alt hough the vasculature attempts to circumvent the amyloid plaques, it is prevented by the intrinsic anti-angiogen ic nature of A and only after physical enzymatic dissol ution, degradation, or phagocytic removal of A would the vascular remodeling have a chance to be su ccessful and restore cerebral blood flow. Downs Sydrome, Aneuploidy, Cell Cycle, and Alzheimers Disease. Downs Syndrome (DS) individuals invariably deve lop Alzheimers disease by their third or fourth decade of life (Epstein, 1990; Glenner & Wong, 1984; Olson & Shaw, 1969). DS individuals are born with three copies of Chromosome 21 in every cell because of meiotic chromosome mis-segregation (Lejeune, Gau tier, & Turpin, 1959; Mrak & Griffin, 2004), and the gene encoding amyloid precursor pr otein (APP) is located on Chromosome 21. Genetic mutations in APP have been found to result in FAD (Goate et al., 1991; Naruse et al., 1991; Tanzi et al., 1991) and it has been thought that DS individuals should overexpress APP throughout their life more than non-DS individuals. DS individuals begin accumulating fibrillar A deposition in their brains by age of adolescence (Lemere et al., 1996; Mrak & Griffin, 2004; Rumble et al., 1989). A sp ecific case of a DS 78 year old woman, who had only partial trisomy 21 w ith only two alleles of APP and who died without any clinical or AD pa thology, showed that extra e xpression of APP is necessary

PAGE 30

20for AD onset in DS patients, but not for development of DS (Mrak & Griffin, 2004; Prasher et al., 1998). However, extra APP e xpression does not completely account for the onset of AD in non-DS individuals. For inst ance, autopsy of deceased individuals from the Nun Study (Davis, Schmitt, Wekstein, & Ma rkesbery, 1999; Gold et al., 2000; Riley, Snowdon, & Markesbery, 2002), showed that ther e were subjects with high levels of cerebral amyloidosis but who had no documented cognitive impairment, indicative of AD, before their death. Moreover, in the ha lted AN1792 immunotherapy clinical trial, follow-up autopsy of some individuals who had received anti-A therapy and who had died with progressive dementia, were found to be almost devoid of cerebral amyloid deposition (Holmes et al., 2008). Furthermore, DS fetal tissues and cultured primary fibroblasts do not show an overexpression of APP, arguing against a gene dosaging effect for APP (Argellati et al., 2006; Cheon et al., 2003; Engidawork et al ., 2001; Farrer et al., 1997). On the other hand, duplication of the APP locus on Chromosome 21(Sleegers et al., 2006), along with just 5-12 adjacent gene s in the centomeric Down syndrome critical region (Fuentes et al., 1995; Rahmani et al ., 1989), has been reported in families with autosomal-dominant early-onset AD, which coincided with cerebral amyloid angiopathy (CAA) (Rovelet-Lecrux et al., 2006). Thus, these seemingly-conflicting observations actually suggest that there may be other fact ors contained within the genes of the extra Chromosome 21 that also pred ispose DS individuals to AD, especially since some DS individuals develop onset of AD dementia by as early as their thirties. Besides APP, there are about 323 other genes expressed on Chromosome 21 (Antonarakis, Lyle, Dermitzakis, Reymond, & Deutsch, 2004),and some such as S100B, Dual-specificity tryrosine (Y)-phosphorylation Regulated Kina se 1A (DYRK1A) and Downs Syndrome

PAGE 31

21Candidate Region-1(DSCR1, also known as RCAN1 ), have been described to contribute to pathology consistent with AD. S100B is an astrocyte-secreted cyto kine, which stimulates phosphoinositide hydrolysis by phospholipase C in order to mob ilize intracellular calcium stores (Barger & Van Eldik, 1992; Griffin et al., 1989). Th ese actions are neurotrophic to neurons, promoting neurite outgrowth, and to glia, promoting proliferati on and differentiation (Selinfreund, Barger, Welsh, & Van Eldi k, 1990). However, lifelong overexpression of S100B in DS individuals may actually cause a decrease in any trophic actions. For instance, transgenic mice that overexpre ss human S100B have increased hippocampal dendrites when they are young, but this dendrit ic density decreases throughout the age of the mice, and young S100B mice also show behavi oral and learning defects that may also be analogous to DS developmental abnormalities (Whitaker-Azmitia et al., 1997). S100B has been reported to have levels much more than 1.5 times as expected in DS brains than for normal brains, throughout the entire life of the DS individual (Griffin et al., 1998). These levels correlate with chronic overexpre ssion of Interleukin-1 (IL-1) (Griffin et al., 1989), and S100B regulates the expression of IL-1 in neurons by transcription factor, Sp1, and in microglia and astroc ytes by transcription factor, NF B (nuclear factor kappalight-chain-enhancer of activated B cells) (L. Liu, Li, Van Eldik, Griffin, & Barger, 2005). IL-1 has been shown to induce A production (Forloni, Demicheli, Giorgi, Bendotti, & Angeretti, 1992; Goldgaber et al., 1989), and in turn, S100B and A induce IL-1 (Barger & Harmon, 1997; L. Liu et al., 2005), forming a feed-forward mechanism that can contribute to AD pathogenesis, through Tau-related and other neuroinflammatory actions.

PAGE 32

22The kinase DYRK1A has also been shown to directly affect AD pathogenesis. For instance, DYRK1A has been shown to interact with and directly phosphorylate tau and APP in immortalized H19-7 hippocampal progenitor cells (J. Park, Yang, Yoon, & Chung, 2007). It is known that DYRK1A mRNA levels in the hippocampus are significantly elevated in patients with AD. In the brain of AD Tg mice, DYRK1A mRNA levels were found to be upregulat ed along with an increase in A deposition. In neuroblastoma cells overexpressing tau, it was shown that A induces an increase in the DYRK1A transcript, which also leads to tau phosphorylation at Thr212 (R. Kimura et al., 2007). Another study has shown that Tg mice that over-express human DYRK1A protein have elevated phospho-Tau at serine -202 and serine-204, which are two hyperphosphorylated Tau residues that are indicated in the fo rmation of neurofibrillary tangles (NFTs) (Ryoo et al., 2007). This group al so demonstrated in mammalian cells that APP is phosphorylated at Thr668 by DYRK1A and that phospho-APP, A and DYRK1A are elevated in the transgenic mice and in DS brains (Ryoo et al., 2008). Taken together, DYRK1A over-expression induces a feed-forward mechanism with increased A deposition, which then induces additiona l DYRK1A expression, resulting in more A deposition and phosphorylation of both APP and Tau at sites consistent with AD pathogenesis. However, DYRK1As involvement with A and Tau may be only a portion of its role in AD pathogenesis. In a recent pub lication by Baek et. al., DYRK1A and DSCR1, another gene mapped to Chromosome 21, were both shown to significantly inhibit angiogenesis and provide compelling reason fo r why DS individuals do not develop solid tumors (Hasle, Clemmensen, & Mikkelse n, 2000), since all solid tumors require

PAGE 33

23neovascularization to grow larger than 2mm, invade tissues, and metastasize (Folkman, 1992). Both DSCR1 and DYRK1A suppress the VEGF-calcineurin angiogenic pathway. This pathway is activated by VEGF binding to its receptor (VEGFR), which then causes an intracellular cascade of events in which the transcription factor, Nuclear Factor of Activated T-cells, cytoplasmic 2 (NFATc2) is dephosphorylated and activated and translocates to the nucleus to induce C ox-2 expression (Hernandez et al., 2001). Cox-2 expression and activity is also induced by various stimuli, including hypoxia (Schmedtje, Ji, Liu, DuBois, & Runge, 1997), TNF (Jones, Carlton, McIntyre, Zimmerman, & Prescott, 1993), basic fibroblas t growth factor (Kage et al., 1999), IL-1 (Karim, Habib, Levy-Toledano, & Maclouf 1996), and LPS (Inoue, Yokoyama, Hara, Tone, & Tanabe, 1995). As mentioned above Cox-2 is only expressed in reactive microglia by LPS, and these other stimuli w ould only affect neuronal Cox-2 expression, especially in AD-vulnerable pyramidal neur ons (Hoozemans et al., 2001; Hoozemans et al., 2002). Prostaglandin E2 (PGE2), the ma jor end product from Cox-2 activity, is a stimulator of angiogenesis (Form & Auerbach, 1983) and works by inducing the up-regulation of the tr anscription factor H ypoxia-inducible Factor-1 (HIF-1 ) (X. H. Liu et al., 2002), which can then bind to pr omoter/enhancer elements of VEGF and other hypoxia-sensitive genes (G. L. Wang, Jiang, Rue, & Semenza, 1995). In the study of Baek et al., the authors report that just one extra allelic copy of DSCR1 and DYRK1A are sufficient to preven t tumor growth and that this tumor inhibition is directly related to DSCR 1 and DYRK1A suppression of angiogenesis through the VEGF-calicineuri n pathway (Baek et al., 2009 ). One mechanism by which DSCR1 could inhibit angiogenesis may be by binding to Raf-1 (Cho, Abe, Kim, & Sato,

PAGE 34

242005), putatively causing disruption of the retinoblastoma (Rb)-Raf-1interaction, and preventing Raf-1 from phosphorylating and inactivating Rb, which when Rb is inactivated, is involved in cell proliferati on and VEGF-mediated angiogenesis (Dasgupta et al., 2004). While the supe rnumerary expression of DY RK1A and DSCR1 may prevent tumorigenesis, the resultant reduction in cerebral blood fl ow throughout life may also predispose DS individuals to the deficient neurodevelopment observed in all DS individuals. DS individuals ha ve smaller brains than normal and also have less density of neurons. Their neurons have ramified dendritic trees, but the number and complexity of them does not progress through age, causi ng the neurons to be described from histochemistry as having a tree in winter type of appearance (Takashima, Becker, Armstrong, & Chan, 1981; Takashima, Iida, Mito, & Arima, 1994). The vascular and nervous systems ar e intricately associated throughout neurodevelopment, with the sa me classical axonal-guidance proteins, such as netrins, ephrins, semaphorins, and slits, also being used as the cues to direct the cerebral blood vessel growth down predestined tracks (Carmeliet, 2003; Carmeliet & Tessier-Lavigne, 2005; Weinstein, 2005; Zacchigna, Lambrechts & Carmeliet, 2008). Both nerves and blood vessels grow together to their ultimat e destination, and VEGF regulates neuronal patterning of some types of neurons (Sch warz et al., 2004). One could thus hypothesize that the reduced cerebral bl ood flow from birth may contribute to the developmental cognitive deficits seen in DS individuals, as well as contributing to the later onset of severe AD pathogenesis, by both anti-angi ogenic and amyloidogenic mechanisms. In support of this hypothesis, an epidemiologi cal study in 1987 of 14 DS individuals, 46 AD patients, and 114 age-matched controls showed that DS patients have a similar reduction

PAGE 35

25in cerebral blood flow throughout their brai ns as do AD patients (Melamed, Mildworf, Sharav, Belenky, & Wertman, 1987). It is similarly noteworthy that people with the ApoE4 genotype, who are at very high risk for developing AD, have widespread decline in regional cerebral blood flow over time a nd before they develop onset of dementia (Thambisetty, Beason-Held, An, Kraut, & Resnick, 2010). Furthermore, it is plausible that the anti-angiogenic effects from Cox-2 i nhibition (Hyde & Missailidis, 2009; Raut et al., 2004; L. Wang, Chen, Xie, He, & Bai, 2008) may indeed be an underlying factor for many of the failures of the NSAID clinical tria ls against AD, as reve rsal of AD would not only require removal of cerebral amyloidos is, but also would s ubsequently require neovascularization to correct the truncated and occluded microvasculature where the plaques were located (Meyer et al., 2008). Besides the greater than 1.5 times overexpression from ge ne loading of the APP, S100B, DYRK1A, and DSCR1 genes described above, there are other factors within trisomy 21 cells that may also contribute to AD pathogenesis, such as the involvement of kinesins and dyneins, which play roles in the chromosomal duplication and missegregation during mitosis and meiosis, as well as in axonal transport in mature neurons. It has been hypothesized that in normal individuals, defective mitosis, likely due to microtubule (MT) dysfunction, may generate the accumulation of trisomy 21 cells in their bodies throughout life, and within the br ain it could contribute to the development of AD, just as trisomy 21 invariably pred isposes DS individuals to AD (Potter, 1991). Many studies have confirmed that trisomy 21 mosaicism may indeed contribute to AD pathogenesis, as trisomy 21 and other aneupl oid cells have been found in primary skin fibroblast cultures of both familial (genetically predispositioned to develop AD early in

PAGE 36

26life) AD patients and sporadic (occurring la ter in life) AD patien ts (Geller & Potter, 1999), as well as trisomy 21 being found in pe ripheral leukocytes, buc cal cells, and brain neurons of sporadic AD patients (Iourov, Vo rsanova, Liehr, & Yurov, 2009; Migliore et al., 1999; Mosch et al., 2007; Thomas & Fenech, 2008; Yang, Geldmacher, & Herrup, 2001). Mothers, who have a DS child before th e age of 35, have a five times greater risk of developing dementia, with trisomy 21 mosaicism being found in their lymphocytes, than that of control mothers (Migliore et al., 2006; Schupf, Kape ll, Lee, Ottman, & Mayeux, 1994). Sporadic AD patients, who develop AD in mid-life, have been found to have between 1-10% trisomy 21 mosaicism, suggesting that even minimal amounts of aneuploidic mechanisms are sufficient to induce their early AD ons et (Puri, Zhang, & Singh, 1994; Ringman, Rao, Lu, & Cederbaum, 2008; Rowe, Ridler, & Gibberd, 1989). It can also be predicted under this ch romosome mis-segregation/MT hypothesis for AD that the same genes that are res ponsible for familial AD onset when mutated might also participate in the cell cycle, mitotic machinery, and contribute to trisomy 21 mosaicism. Indeed it has been shown that po lymorphisms in PS-1 cause an increased risk of both AD onset and propensity to have DS children (Higuchi, Muramatsu, Matsushita, Arai, & Sasaki, 1996; Lucarelli et al., 2004; Petersen et al., 2000; Wragg, Hutton, & Talbot, 1996). It has also been shown th at endogenous PS-1, APP, their proteolytic products, and their interacting proteins are intr icately involved in th e mitotic process and cell cycle, being localized at the nuclear enve lope, centrosomes, or kinetochores and also being involved in microtubul ar transport (Honda et al., 2000; Johnsingh et al., 2000; N. Kimura et al., 2001; J. Li, Xu, Zhou, Ma, & Po tter, 1997; Nizzari et al., 2007; Tezapsidis, Merz, Merz, & Hong, 2003; Zimmermann et al., 1988; Zitnik, Wang, Martin, & Hu,

PAGE 37

272007). Aberrant incomplete neuronal re-entry of the cell cycle has been implicated in the neurodegeneration of AD pathogenesis (Ogawa et al., 2003; Varvel et al., 2008; Vincent, Rosado, & Davies, 1996), as evidenced by about 3% tetraploid neurons and about 30% aneuploid cells (between 2n and 4n) in late -stage AD patients (Mos ch et al., 2007; Yang et al., 2001). It is also kn own that APP and Tau are hyperphosphorylated during mitosis and contribute to paired helic al filaments, the precursors of NFTs (Pope et al., 1994; Preuss, Doring, Illenberger, & Mandelkow, 1995; Suzuki et al., 1994). As mentioned above, trisomic overexpre ssion of S100B induces chronic IL-1 expression (Griffin et al., 1989), which in turn induces the expression of alpha-1-antichymotrypsin (ACT), an acute phase serine protease inhibitor, which promotes polymerization of A (Abraham, Shirahama, & Potter, 1990; Das & Potter, 1995), Tau hyperphosphorylation, and neuronal apoptosis (Padmanabhan et al., 2006). A recent study examining transgenic (Tg) mice with FAD mutant forms of APP or PS-1 showed that aneuploidy is signi ficantly induced in both the sp leen and in the brain of the mice compared to wild-type age-matched cont rols, and that these findings are confirmed in transfected mammalian cells. Moreover, th e authors showed that administration of either of the prevalent A species (1-40 and 1-42), and only to normal cells containing APP and Tau, induce rapid chromosome mis-se gregation and aneuploidy, highlighting a mechanism by which A disrupts MT function and induc es more aneuploidy. Thus DS individuals, who are predisposed to accumulation of cerebral A deposition as they age, will have more aneuploidy and MT defects, which will affect neurogenesis and neuronal function, and which will also contribute in a feed-forward mechanism for additional

PAGE 38

28amyloidosis, aneugenesis, and progressive dementia (Granic, Padmanabhan, Norden, & Potter, 2010). Conclusions and Research Theory. The above-described feed-forward mechanisms of A to propagate itself through DYRK1 As up-regulation and vice versa, for A to disrupt MT function and result in mo re aneuploidic and trisomic 21 cells, for these effects to reduce cerebral blood fl ow and prevent neovascularization through DSCR1 and DYRK1A, and for the resultant up-regulated and self-propagating S100BIL-1-ACT-A neuroinflammatory A polymerization pathway, which all work in combination to deposit extracellular amyloi d plaque, to induce in traneuronal tauopathy, and to cause cerebral microvascular occlus ions and dystrophic ne urites surrounding the amyloid plaques, portrays the insidious but pr ogressive nature of Alzheimers disease. Thus in theory, to inhibit and reverse AD pathology, there must first be simultaneous strategies to remove the cerebral amyloidos is and to provide neuroprotection to the micro-penumbras that surround the plaques a nd occluded vessels. Then after removal of amyloid, there must be strategies to imme diately induce neovas cularization in the infarcted areas where the plaques were locate d and to also induce neuritogenesis and/or neurogenesis to rebuild the neural and vascul ar networks in order to restore cognitive function. The following chapters report on the investigation of a bi ological therapeutic, whose human analogue has been safely used for two decades in leukopenic patients, and which has actions that attack all of these defects just described in th e preceding theory. The next chapter will first outline the ra tionale, investigative plan, and working hypotheses, and the following chapters continue with the results and conclusions of the experiments. These experiments shed li ght on the inverse relationship between

PAGE 39

29Rheumatoid arthritis and Alzheimers disease, and putatively indicates a therapeutic for AD which quickly removes amyloid deposit ion in aged transgenic AD mice by two different routes of administration, which qui ckly reverses cognitive impairment following daily peripheral subcutaneous administration, while also increasing synaptic area and microglial density, and which has been reported to be neuroprotective in the penumbras of ischemic infarcts, to indu ce cerebral neovascularization af ter ischemic infarct, and to stimulate the proliferation and differentiation of neural stem cells, which are known to migrate to sites of cerebral injury.

PAGE 40

Figure 1. Extensive Cerebrovascular Damage in AD. Artistic rendition of scanning electron microscopy results (Mey er et al., 2008) in which sp ecialized polurethane resin was perfused into aged APP23 mice, and in which the resultant corrosion casting reveals microvessel distortions, occlusions, and trunc ations in the cerebrovasulature that surrounds the area which contained an amyl oid plaque, that was chemically removed during the VCC processing of the tissue. 30

PAGE 41

31References Abraham, C. R. (2001). Reactive astrocytes and alpha1-antichymotrypsin in Alzheimer's disease. Neurobiol Aging, 22 (6), 931-936. Abraham, C. R., Selkoe, D. J., & Potter, H. (1988). Immunochemical identification of the serine protease inhibitor alpha 1-antic hymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell, 52 (4), 487-501. Abraham, C. R., Shirahama, T., & Potter, H. (1990). Alpha 1-antichymotrypsin is associated solely with amyloid deposits containing the beta-protein. Amyloid and cell localization of alpha 1-antichymotrypsin. Neurobiol Aging, 11 (2), 123-129. Ahmed, R. R., Holler, C. J., We bb, R. L., Li, F., Beckett, T. L., & Murphy, M. P. (2010). BACE1 and BACE2 enzymatic activiti es in Alzheimer's disease. J Neurochem, 112(4), 1045-1053. Aisen, P. S., Schafer, K. A., Grundman, M., Pf eiffer, E., Sano, M., Davis, K. L., et al. (2003). Effects of rofecoxib or naprox en vs placebo on Alzheimer disease progression: a randomized controlled trial. Jama, 289(21), 2819-2826. Aisen, P. S., Schmeidler, J., & Pasinetti, G. M. (2002). Randomized pilot study of nimesulide treatment in Alzheimer's disease. Neurology, 58 (7), 1050-1054. Alzheimer's_Association. (2010). 2010 Alzh eimer's Disease Facts and Figures. Retrieved 6-15-2010, from http://www.alz.org/documents_c ustom/report_alzfactsfigures2010.pdf Andersen, K., Launer, L. J., Ott, A., Hoes, A. W., Breteler, M. M., & Hofman, A. (1995). Do nonsteroidal anti-inflammatory drugs decrease the risk for Alzheimer's disease? The Rotterdam Study. Neurology, 45 (8), 1441-1445. Antonarakis, S. E., Lyle, R., Dermitzakis E. T., Reymond, A., & Deutsch, S. (2004). Chromosome 21 and down syndrome: fr om genomics to pathophysiology. Nat Rev Genet, 5 (10), 725-738. Argellati, F., Massone, S., d'Abramo, C., Mari nari, U. M., Pronzato, M. A., Domenicotti, C., et al. (2006). Evidence against the overexpression of APP in Down syndrome. IUBMB Life, 58(2), 103-106. Arima, K., Murayama, S., Oyanagi, S., Akashi, T., & Inose, T. (1992). Presenile dementia with progressive supranuclear palsy tangles and Pick bodies: an unusual degenerative disorder involving the cere bral cortex, cerebral nuclei, and brain stem nuclei. Acta Neuropathol, 84 (2), 128-134. Baek, K. H., Zaslavsky, A., Lynch, R. C., Britt, C., Okada, Y., Siarey, R. J., et al. (2009). Down's syndrome suppression of tumour gr owth and the role of the calcineurin inhibitor DSCR1. Nature, 459(7250), 1126-1130. Baker, M., Kwok, J. B., Kucera, S., Crook, R ., Farrer, M., Houlden, H., et al. (1997). Localization of frontotemporal dementia with parkinsonism in an Australian kindred to chromosome 17q21-22. Ann Neurol, 42(5), 794-798. Bales, K. R., Verina, T., Cummins, D. J., D u, Y., Dodel, R. C., Sa ura, J., et al. (1999). Apolipoprotein E is essential for am yloid deposition in the APP(V717F) transgenic mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A, 96(26), 15233-15238.

PAGE 42

32Barger, S. W., & Harmon, A. D. (1997). Mi croglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature, 388(6645), 878881. Barger, S. W., & Van Eldik, L. J. (1992). S100 beta stimulates calciu m fluxes in glial and neuronal cells. J Biol Chem, 267 (14), 9689-9694. Basi, G., Frigon, N., Barbour, R., Doan, T ., Gordon, G., McConlogue L., et al. (2003). Antagonistic effects of be ta-site amyloid precursor protein-cleaving enzymes 1 and 2 on beta-amyloid peptide production in cells. J Biol Chem, 278 (34), 3151231520. Beard, C. M., Kokman, E., & Kurland, L. T. (1991). Rheumatoid arthritis and susceptibility to Alzheimer's disease. Lancet, 337(8754), 1426. Beckmann, N., Schuler, A., Mueggler, T., Meye r, E. P., Wiederhold, K. H., Staufenbiel, M., et al. (2003). Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer's disease. J Neurosci, 23 (24), 8453-8459. Buee, L., Hof, P. R., Bouras, C., Delacourte, A ., Perl, D. P., Morrison, J. H., et al. (1994). Pathological alterations of the cerebral microvasculature in Alzheimer's disease and related dementing disorders. Acta Neuropathol, 87 (5), 469-480. Butterfield, D. A., Abdul, H. M., Opii, W., Newm an, S. F., Joshi, G., Ansari, M. A., et al. (2006). Pin1 in Alzheimer's disease. J Neurochem, 98 (6), 1697-1706. Carmeliet, P. (2003). Blood vessels and nerv es: common signals, pathways and diseases. Nat Rev Genet, 4 (9), 710-720. Carmeliet, P., & Tessier-Lavigne, M. (2005) Common mechanisms of nerve and blood vessel wiring. Nature, 436(7048), 193-200. Chartier-Harlin, M. C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., et al. (1991). Early-onset Alzheimer's diseas e caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature, 353 (6347), 844-846. Cheon, M. S., Kim, S. H., Yaspo, M. L., Bl asi, F., Aoki, Y., Melen, K., et al. (2003). Protein levels of genes encoded on ch romosome 21 in fetal Down syndrome brain: challenging the gene dos age effect hypothesis (Part I). Amino Acids, 24(12), 111-117. Cho, Y. J., Abe, M., Kim, S. Y., & Sato, Y. (2005). Raf-1 is a binding partner of DSCR1. Arch Biochem Biophys, 439(1), 121-128. Chui, D. H., Tabira, T., Izumi, S., Koya, G., & Ogata, J. (1994). Decreased beta-amyloid and increased abnormal Tau deposition in th e brain of aged patients with leprosy. Am J Pathol, 145 (4), 771-775. Chung, S. H. (2009). Aberrant phosphorylati on in the pathogenesis of Alzheimer's disease. BMB Rep, 42(8), 467-474. Claudio, L. (1996). Ultrastructural features of the blood-brain barri er in biopsy tissue from Alzheimer's disease patients. Acta Neuropathol, 91 (1), 6-14. Das, S., & Potter, H. (1995). Expression of the Alzheimer amyloid-promoting factor antichymotrypsin is induced in human astrocytes by IL-1. Neuron, 14(2), 447456. Dasgupta, P., Sun, J., Wang, S., Fusaro, G., Betts, V., Padmanabhan, J., et al. (2004). Disruption of the Rb--Raf-1 interaction i nhibits tumor growth and angiogenesis. Mol Cell Biol, 24 (21), 9527-9541.

PAGE 43

33Dastur, D. K., & Razzak, Z. A. (1971). Dege neration and regeneration in teased nerve fibres. I. Leprous neuritis. Acta Neuropathol, 18 (4), 286-298. Davis, D. G., Schmitt, F. A., Wekstein, D. R., & Markesbery, W. R. (1999). Alzheimer neuropathologic alterations in ag ed cognitively normal subjects. J Neuropathol Exp Neurol, 58(4), 376-388. De Jong, G. I., Farkas, E., Stienstra, C. M., Plass, J. R., Keijser, J. N., de la Torre, J. C., et al. (1999). Cerebral hypoperfusion yields capillary damage in the hippocampal CA1 area that correlates with spatial memory impairment. Neuroscience, 91 (1), 203-210. de la Torre, J. C. (2004). Alzheimer's diseas e is a vasocognopathy: a new term to describe its nature. Neurol Res, 26 (5), 517-524. de la Torre, J. C. (2009). Cerebrovascular and cardiovasc ular pathology in Alzheimer's disease. Int Rev Neurobiol, 84 35-48. de la Torre, J. C. (2010). Vascular risk factor detec tion and control may prevent Alzheimer's disease. Ageing Res Rev de la Torre, J. C., & Stefano, G. B. ( 2000). Evidence that Alzheimer's disease is a microvascular disorder: the role of constitutive nitric oxide. Brain Res Brain Res Rev, 34(3), 119-136. De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., et al. (1998). Deficiency of presenilin-1 inhib its the normal cleavage of amyloid precursor protein. Nature, 391(6665), 387-390. Delisle, M. B., Murrell, J. R., Richardson, R., Tr ofatter, J. A., Rascol, O., Soulages, X., et al. (1999). A mutation at codon 279 (N279K) in exon 10 of the Tau gene causes a tauopathy with dementia and supranuclear palsy. Acta Neuropathol, 98 (1), 62-77. Dewji, N. N., & Singer, S. J. (1996). Speci fic transcellular binding between membrane proteins crucial to Alzheimer disease. Proc Natl Acad Sci U S A, 93 (22), 1257512580. Donnini, S., Solito, R., Cetti, E., Corti, F., Gi achetti, A., Carra, S., et al. (2010). A{beta} peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. Faseb J Duskin, A., & Eisenberg, R. A. The role of antibodies in inflammatory arthritis. Immunol Rev, 233(1), 112-125. Endoh, M., Kunishita, T., & Tabira, T. (1999). No effect of anti-leprosy drugs in the prevention of Alzheimer's disease and beta-amyloid neurotoxicity. J Neurol Sci, 165(1), 28-30. Engidawork, E., Baiic, N., Fountoulakis, M., Dierssen, M., Greber-Platzer, S., & Lubec, G. (2001). Beta-amyloid precursor protein, ETS-2 and collagen alpha 1 (VI) chain precursor, encoded on chromosome 21, are not overexpressed in fetal Down syndrome: further evidence against gene dosage effect. J Neural Transm Suppl (61), 335-346. Epstein, C. J. (1990). The consequences of chromosome imbalance. Am J Med Genet Suppl, 7, 31-37. Farkas, E., De Jong, G. I., Apro, E., De Vos, R. A., Steur, E. N., & Luiten, P. G. (2000). Similar ultrastructural brea kdown of cerebrocortical cap illaries in Alzheimer's disease, Parkinson's disease, and ex perimental hypertension. What is the functional link? Ann N Y Acad Sci, 903, 72-82.

PAGE 44

34Farkas, E., De Vos, R. A., Jansen Steur, E. N., & Luiten, P. G. (2000). Are Alzheimer's disease, hypertension, and cereb rocapillary damage related? Neurobiol Aging, 21(2), 235-243. Farrer, M. J., Crayton, L., Davies, G. E., O liver, C., Powell, J., Holland, A. J., et al. (1997). Allelic variabil ity in D21S11, but not in APP or APOE, is associated with cognitive decline in Down syndrome. Neuroreport, 8(7), 1645-1649. Farzan, M., Schnitzler, C. E., Vasilieva, N., Leung, D., & Choe, H. (2000). BACE2, a beta -secretase homolog, cleaves at the be ta site and within the amyloid-beta region of the amyloid-be ta precursor protein. Proc Natl Acad Sci U S A, 97 (17), 9712-9717. Fidani, L., Rooke, K., Chartier-H arlin, M. C., Hughes, D., Tanzi, R., Mullan, M., et al. (1992). Screening for mutations in the ope n reading frame and promoter of the beta-amyloid precursor protein gene in familial Alzheimer's disease: identification of a further family with APP717 Val-->Ile. Hum Mol Genet, 1 (3), 165-168. Folkman, J. (1992). The role of angiogenesis in tumor growth. Semin Cancer Biol, 3 (2), 65-71. Forloni, G., Demicheli, F., Gior gi, S., Bendotti, C., & Angeretti, N. (1992). Expression of amyloid precursor protein mRNAs in endothelial, neuronal and glial cells: modulation by interleukin-1. Brain Res Mol Brain Res, 16 (1-2), 128-134. Form, D. M., & Auerbach, R. (1983). PGE2 and angiogenesis. Proc Soc Exp Biol Med, 172(2), 214-218. Fuentes, J. J., Pritchard, M. A., Planas, A. M., Bosch, A., Ferrer, I., & Estivill, X. (1995). A new human gene from the Down syndr ome critical region encodes a prolinerich protein highly expressed in fetal brain and heart. Hum Mol Genet, 4(10), 1935-1944. Furuzawa-Carballeda, J., Vargas-Rojas, M. I., & Cabral, A. R. (2007). Autoimmune inflammation from the Th17 perspective. Autoimmun Rev, 6 (3), 169-175. Galloway, P. G. (1988). Antigenic characteristic s of neurofibrillary tangles in progressive supranuclear palsy. Neurosci Lett, 91 (2), 148-153. Geller, L. N., & Potter, H. (1999). Ch romosome missegregation and trisomy 21 mosaicism in Alzheimer's disease. Neurobiol Dis, 6(3), 167-179. Glenner, G. G., & Wong, C. W. (1984). Alzheimer's di sease and Down's syndrome: sharing of a unique cerebrovasc ular amyloid fibril protein. Biochem Biophys Res Commun, 122(3), 1131-1135. Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., et al. (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature, 349(6311), 704-706. Gold, G., Bouras, C., Kovari, E., Canuto, A ., Glaria, B. G., Malky, A., et al. (2000). Clinical validity of Braak neuropath ological staging in the oldest-old. Acta Neuropathol, 99 (5), 579-582; discussion 583-574. Goldgaber, D., Harris, H. W., Hla, T., Maciag, T., Donnelly, R. J., Jacobsen, J. S., et al. (1989). Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells. Proc Natl Acad Sci U S A, 86 (19), 7606-7610. Goldgaber, D., Lerman, M. I., McBride, W. O., Saffiotti, U., & Gajdusek, D. C. (1987). Isolation, characteri zation, and chromosomal locali zation of human brain cDNA

PAGE 45

35clones coding for the precursor of the am yloid of brain in Alzheimer's disease, Down's syndrome and aging. J Neural Transm Suppl, 24 23-28. Goto, M., Kimura, T., Hagio, S., Ueda, K., Kitajima, S., Tokunaga, H., et al. (1995). Neuropathological analysis of demen tia in a Japanese leprosarium. Dementia, 6(3), 157-161. Graeber, M. B., Kosel, S., Egensperger, R., Banati, R. B., Muller, U., Bise, K., et al. (1997). Rediscovery of the case described by Alois Alzheimer in 1911: historical, histological and molecular genetic analysis. Neurogenetics, 1(1), 73-80. Graeber, M. B., Kosel, S., Grasbon-Frodl, E ., Moller, H. J., & Mehraein, P. (1998). Histopathology and APOE genotype of th e first Alzheimer disease patient, Auguste D. Neurogenetics, 1 (3), 223-228. Granic, A., Padmanabhan, J., Norden, M., & Potter, H. (2010). Alzheimer Abeta peptide induces chromosome mis-segregation and aneuploidy, including trisomy 21: requirement for tau and APP. Mol Biol Cell, 21 (4), 511-520. Griffin, W. S., Sheng, J. G., McKenzie, J. E., Royston, M. C., Gentleman, S. M., Brumback, R. A., et al. (1998). Life-l ong overexpression of S 100beta in Down's syndrome: implications fo r Alzheimer pathogenesis. Neurobiol Aging, 19 (5), 401405. Griffin, W. S., Stanley, L. C ., Ling, C., White, L., MacLeod, V., Perrot, L. J., et al. (1989). Brain interleukin 1 and S-100 i mmunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci U S A, 86 (19), 7611-7615. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., Zaidi, M. S., & Wisniewski, H. M. (1986). Microtubule-associated protein tau. A compone nt of Alzheimer paired helical filaments. J Biol Chem, 261 (13), 6084-6089. Grundke-Iqbal, I., Iqbal, K., Tung, Y. C., Quin lan, M., Wisniewski, H. M., & Binder, L. I. (1986). Abnormal phosphorylation of th e microtubule-associated protein tau (tau) in Alzheimer cy toskeletal pathology. Proc Natl Acad Sci U S A, 83(13), 4913-4917. Harrington, L. E., Hatton, R. D., Mangan, P. R., Turner, H., Murphy, T. L., Murphy, K. M., et al. (2005). Interleuki n 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol, 6 (11), 1123-1132. Hasegawa, H., Nishi, S., Ito, S., Saeki, T., Kuroda, T., Kimura, H ., et al. (1996). High prevalence of serum apolipoprotein E4 isopr otein in rheumatoid arthritis patients with amyloidosis. Arthritis Rheum, 39 (10), 1728-1732. Hasle, H., Clemmensen, I. H., & Mikkelse n, M. (2000). Risks of leukaemia and solid tumours in individuals with Down's syndrome. Lancet, 355(9199), 165-169. Hauw, J. J., Verny, M., Delaere, P., Cervera, P., He, Y., & Duyckaerts, C. (1990). Constant neurofibrilla ry changes in the neocortex in progressive supranuclear palsy. Basic differences with Alzheimer's disease and aging. Neurosci Lett, 119(2), 182-186. Heinzer, S., Krucker, T., Stampanoni, M., Abel a, R., Meyer, E. P ., Schuler, A., et al. (2006). Hierarchical microimaging for multiscale analysis of large vascular networks. Neuroimage, 32(2), 626-636. Heinzer, S., Kuhn, G., Krucker, T., Meyer, E., Ulmann-Schuler, A., Stampanoni, M., et al. (2008). Novel three-dimensional analys is tool for vascul ar trees indicates

PAGE 46

36complete micro-networks, not single capil laries, as the angiogenic endpoint in mice overexpressing human VEGF(165) in the brain. Neuroimage, 39 (4), 15491558. Heneka, M. T., Sastre, M., Dumitrescu-Ozimek, L., Hanke, A., Dewachter, I., Kuiperi, C., et al. (2005). Acute treatment with the PPARgamma agonist pioglitazone and ibuprofen reduces glial inflammation and Abeta1-42 levels in APPV717I transgenic mice. Brain, 128(Pt 6), 1442-1453. Hernandez, G. L., Volpert, O. V., Iniguez, M. A., Lorenzo, E., Martinez-Martinez, S., Grau, R., et al. (2001). Selective inhibition of vascular endothelial growth factormediated angiogenesis by cyclosporin A: ro les of the nuclear factor of activated T cells and cyclooxygenase 2. J Exp Med, 193 (5), 607-620. Higuchi, S., Muramatsu, T., Matsushita, S., Ar ai, H., & Sasaki, H. (1996). Presenilin-1 polymorphism and Alzheimer's disease. Lancet, 347(9009), 1186. Ho, A., & Sudhof, T. C. (2004). Binding of Fspondin to amyloid-beta precursor protein: a candidate amyloid-beta pr ecursor protein ligand that modulates amyloid-beta precursor protein cleavage. Proc Natl Acad Sci U S A, 101 (8), 2548-2553. Holmes, C., Boche, D., Wilkinson, D., Yadega rfar, G., Hopkins, V., Bayer, A., et al. (2008). Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet, 372(9634), 216-223. Honda, T., Nihonmatsu, N., Yasutake, K., Ohtake, A., Sato, K., Tanaka, S., et al. (2000). Familial Alzheimer's disease-associated mutations block translocation of fulllength presenilin 1 to the nuclear envelope. Neurosci Res, 37 (2), 101-111. Hoozemans, J. J., Rozemuller, A. J., Janssen, I., De Groot, C. J., Veerhuis, R., & Eikelenboom, P. (2001). Cyclooxygenase expr ession in microglia and neurons in Alzheimer's disease and control brain. Acta Neuropathol, 101 (1), 2-8. Hoozemans, J. J., Veerhuis, R., Janssen, I., van Elk, E. J., Rozemuller, A. J., & Eikelenboom, P. (2002). The role of cyclo-oxygenase 1 and 2 activity in prostaglandin E(2) secretion by cultured human adult microglia: implications for Alzheimer's disease. Brain Res, 951 (2), 218-226. Hopper, M. W., & Vogel, F. S. (1976). The limbic system in Alzheimer's disease. A neuropathologic investigation. Am J Pathol, 85 (1), 1-20. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C., et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci, 14 (6), 419-427. Hussain, I., Powell, D. J., Howlett, D. R., Chapman, G. A., Gilmour, L., Murdock, P. R., et al. (2000). ASP1 (BACE2) cleaves the amyloid precursor pr otein at the betasecretase site. Mol Cell Neurosci, 16 (5), 609-619. Hyde, C. A., & Missailidis, S. (2009). Inhibi tion of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis. Int Immunopharmacol, 9(6), 701-715. Inoue, H., Yokoyama, C., Hara, S., Tone, Y ., & Tanabe, T. (1995). Transcriptional regulation of human prostaglandinendoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expr ession site and cAMP response element. J Biol Chem, 270 (42), 24965-24971.

PAGE 47

37Iourov, I. Y., Vorsanova, S. G., Liehr, T., & Yurov, Y. B. (2009). Aneuploidy in the normal, Alzheimer's disease and ataxia-telangiectasia brain: differential expression and pathological meaning. Neurobiol Dis, 34 (2), 212-220. Jacobs, J. M., Shetty, V. P., & Antia, N. H. (1987). Myelin changes in leprous neuropathy. Acta Neuropathol, 74 (1), 75-80. Jantzen, P. T., Connor, K. E., DiCarlo, G., Wen k, G. L., Wallace, J. L., Rojiani, A. M., et al. (2002). Microglial activa tion and beta -amyloid deposit reduction caused by a nitric oxide-releasing nonsteroidal antiinflammatory drug in amyloid precursor protein plus presenilin-1 transgenic mice. J Neurosci, 22(6), 2246-2254. Jenkinson, M. L., Bliss, M. R., Brain, A. T., & Scott, D. L. (1989). Rheumatoid arthritis and senile dementia of the Alzheimer's type. Br J Rheumatol, 28 (1), 86-88. Job, C. K. (1971). Pathology of peripheral nerv e lesions in lepromat ous leprosy--a light and electron microscopic study. Int J Lepr Other Mycobact Dis, 39 (2), 251-268. Johnsingh, A. A., Johnston, J. M., Merz, G., X u, J., Kotula, L., Jacobsen, J. S., et al. (2000). Altered binding of mutated presenilin with cytoskeleton-interacting proteins. FEBS Lett, 465 (1), 53-58. Jones, D. A., Carlton, D. P., McIntyre, T. M., Zimmerman, G. A., & Prescott, S. M. (1993). Molecular cloning of human prostaglandin endopero xide synthase type II and demonstration of expressi on in response to cytokines. J Biol Chem, 268 (12), 9049-9054. Kage, K., Fujita, N., Oh-hara, T., Ogata, E., Fujita, T., & Tsuruo, T. (1999). Basic fibroblast growth factor induces cyclooxygena se-2 expression in endothelial cells derived from bone. Biochem Biophys Res Commun, 254 (1), 259-263. Kalaria, R. N., & Hedera, P. (1995). Di fferential degeneration of the cerebral microvasculature in Alzheimer's disease. Neuroreport, 6(3), 477-480. Kalsheker, N. A. (1996). Alpha 1-antichymotrypsin. Int J Biochem Cell Biol, 28 (9), 961964. Karim, S., Habib, A., Levy-Toledano, S., & Maclouf, J. (1996). Cyclooxygenase-1 and -2 of endothelial cells util ize exogenous or endogenous arachidonic acid for transcellular production of thromboxane. J Biol Chem, 271 (20), 12042-12048. Karsenty, G. (1999). The genetic transformation of bone biology. Genes Dev, 13 (23), 3037-3051. Kaufmann, W. E., Andreasson, K. I., Isak son, P. C., & Worley, P. F. (1997). Cyclooxygenases and the central nervous system. Prostaglandins, 54(3), 601-624. Kertesz, A., Kawarai, T., Rogaeva, E., St Ge orge-Hyslop, P., Poorkaj, P., Bird, T. D., et al. (2000). Familial frontotemporal dementia with ubiquitin-positive, tau-negative inclusions. Neurology, 54 (4), 818-827. Kimura, N., Nakamura, S. I., Honda, T., Takashima, A., Nakayama, H., Ono, F., et al. (2001). Age-related changes in the locali zation of presenilin-1 in cynomolgus monkey brain. Brain Res, 922 (1), 30-41. Kimura, R., Kamino, K., Yamamoto, M., Nuripa A., Kida, T., Kazui, H., et al. (2007). The DYRK1A gene, encoded in chromosome 21 Down syndrome critical region, bridges between beta-amyloid producti on and tau phosphorylation in Alzheimer disease. Hum Mol Genet, 16(1), 15-23.

PAGE 48

38Krucker, T., Lang, A., & Meyer, E. P. ( 2006). New polyurethane-based material for vascular corrosion casting with improve d physical and imaging characteristics. Microsc Res Tech, 69 (2), 138-147. Ksiezak-Reding, H., Morgan, K., Mattiace, L. A., Davies, P., Liu, W. K., Yen, S. H., et al. (1994). Ultrastructure and biochemical composition of paired helical filaments in corticobasal degeneration. Am J Pathol, 145 (6), 1496-1508. Kukar, T., Murphy, M. P., Eriksen, J. L., Sa gi, S. A., Weggen, S., Smith, T. E., et al. (2005). Diverse compounds mimic Alzh eimer disease-causing mutations by augmenting Abeta42 production. Nat Med, 11 (5), 545-550. Lee, K. J., Moussa, C. E., Lee, Y., Sung, Y., Howell, B. W., Turner, R. S., et al. (2010). Beta amyloid-independent role of amyloid precursor protein in generation and maintenance of dendritic spines. Neuroscience Lee, V. M., Balin, B. J., Otvos, L., Jr., & Trojanowski, J. Q. (1991). A68: a major subunit of paired helical filaments and derivatized forms of normal Tau. Science, 251(4994), 675-678. Lejeune, J., Gautier, M., & Turpin, R. ( 1959). [Study of somatic chromosomes from 9 mongoloid children.]. C R Hebd Seances Acad Sci, 248 (11), 1721-1722. Lemere, C. A., Blusztajn, J. K., Yamaguchi, H., Wisniewski, T., Saido, T. C., & Selkoe, D. J. (1996). Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol Dis, 3(1), 16-32. Li, G., Yin, H., & Kuret, J. (2004). Casein ki nase 1 delta phosphorylat es tau and disrupts its binding to microtubules. J Biol Chem, 279 (16), 15938-15945. Li, J., Xu, M., Zhou, H., Ma, J., & Potter, H. (1997). Alzheimer presen ilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role in chromosome segregation. Cell, 90 (5), 917-927. Li, Y. M., Xu, M., Lai, M. T., Huang, Q., Cast ro, J. L., DiMuzio-Mower, J., et al. (2000). Photoactivated gamma-secretase inhibitors directed to the activ e site covalently label presenilin 1. Nature, 405(6787), 689-694. Lim, G. P., Yang, F., Chu, T., Chen, P., Beec h, W., Teter, B., et al. (2000). Ibuprofen suppresses plaque pathology and inflamma tion in a mouse model for Alzheimer's disease. J Neurosci, 20 (15), 5709-5714. Liu, F., Liang, Z., Shi, J., Yin, D., El-Akka d, E., Grundke-Iqbal, I., et al. (2006). PKA modulates GSK-3betaand cdk5-catalyzed phosphorylation of tau in siteand kinase-specific manners. FEBS Lett, 580(26), 6269-6274. Liu, L., Li, Y., Van Eldik, L. J., Griffin, W. S., & Barger, S. W. (2005). S100B-induced microglial and neuronal IL-1 expression is mediated by cell type-specific transcription factors. J Neurochem, 92 (3), 546-553. Liu, X. H., Kirschenbaum, A., Lu, M., Yao, S., Dosoretz, A., Holland, J. F., et al. (2002). Prostaglandin E2 induces hypoxia-inducib le factor-1alpha stabilization and nuclear localization in a human prostate cancer cell line. J Biol Chem, 277 (51), 50081-50086. Lorenzo, A., Yuan, M., Zhang, Z., Paganetti, P. A., Sturchler-Pierrat, C., Staufenbiel, M., et al. (2000). Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease. Nat Neurosci, 3 (5), 460-464.

PAGE 49

39Lucarelli, P., Piciullo, A., Palmarino, M., Verd ecchia, M., Saccucci, P., Arpino, C., et al. (2004). Association between presenilin-1 -48C/T polymorphism and Down's syndrome. Neurosci Lett, 367(1), 88-91. Lucca, U., Tettamanti, M., Forloni, G., & Spagnoli, A. (1994). Nonsteroidal antiinflammatory drug use in Alzheimer's disease. Biol Psychiatry, 36 (12), 854856. Ma, J., Brewer, H. B., Jr., & Potter, H. (1996). Alzheimer A beta neurotoxicity: promotion by antichymotrypsin, ApoE4; inhibition by A beta-related peptides. Neurobiol Aging, 17(5), 773-780. Ma, J., Yee, A., Brewer, H. B., Jr., Das, S ., & Potter, H. (1994). Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature, 372(6501), 92-94. Mancardi, G. L., Perdelli, F., Rivano, C., Leona rdi, A., & Bugiani, O. (1980). Thickening of the basement membrane of cortical capillaries in Alzheimer's disease. Acta Neuropathol, 49 (1), 79-83. Matsumoto, T., & Tsurumoto, T. (2002). Inappropriate serum levels of IGF-I and IGFBP3 in patients with rheumatoid arthritis. Rheumatology (Oxford), 41 (3), 352-353. Maury, C. P., Liljestrom, M., Tiitinen, S., Laiho, K., Kaarela, K., & Ehnholm, C. (2001). Apolipoprotein E phenotypes in rheumatoid arthritis with or without amyloidosis. Amyloid, 8 (4), 270-273. Mcgeer, P. L., Harada N., Kimura H., McGeer E.G., Schulzer M. (1992). Prevalence of dementia amongst elderly Japanese with le prosy: apparent eff ect of chronic drug therapy. Dementia, 3, 146-1149. McGeer, P. L., Klegeris, A., Walker, D. G., Yasuhara, O., & McGeer, E. G. (1994). Pathological proteins in senile plaques. Tohoku J Exp Med, 174 (3), 269-277. McGeer, P. L., McGeer, E., Rogers, J., & Si bley, J. (1990). Anti-inflammatory drugs and Alzheimer disease. Lancet, 335 (8696), 1037. McGeer, P. L., Rogers, J., & McGeer, E. G. (2006). Inflammation, anti-inflammatory agents and Alzheimer disease: the last 12 years. J Alzheimers Dis, 9 (3 Suppl), 271-276. McGeer, P. L., Schulzer, M., & McGeer, E. G. (1996). Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies. Neurology, 47 (2), 425-432. Meinert, C. L., McCaffrey, L. D., & Breitn er, J. C. (2009). Alzheimer's Disease Antiinflammatory Prevention Trial: desi gn, methods, and baseline results. Alzheimers Dement, 5(2), 93-104. Melamed, E., Mildworf, B., Sharav, T., Belenky, L., & Wertman, E. (1987). Regional cerebral blood flow in Down's syndrome. Ann Neurol, 22(2), 275-278. Meyer, E. P., Beer, G. M., Lang, A., Manestar, M., Krucker, T., Meier, S., et al. (2007). Polyurethane elastomer: a new material for the visual ization of cadaveric blood vessels. Clin Anat, 20 (4), 448-454. Meyer, E. P., Ulmann-Schuler, A., Staufe nbiel, M., & Krucker, T. (2008). Altered morphology and 3D architecture of brai n vasculature in a mouse model for Alzheimer's disease. Proc Natl Acad Sci U S A, 105 (9), 3587-3592.

PAGE 50

40Migliore, L., Boni, G., Bernardini, R., Trippi, F., Colognato, R., Fontan a, I., et al. (2006). Susceptibility to chromosome malsegrega tion in lymphocytes of women who had a Down syndrome child in young age. Neurobiol Aging, 27(5), 710-716. Migliore, L., Botto, N., Scarpato, R., Petrozzi L., Cipriani, G., & Bonuccelli, U. (1999). Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients. Cytogenet Cell Genet, 87 (1-2), 41-46. Mori, H., Nishimura, M., Namba, Y., & Od a, M. (1994). Corticobasal degeneration: a disease with widespread a ppearance of abnormal tau a nd neurofibrillary tangles, and its relation to progressi ve supranuclear palsy. Acta Neuropathol, 88 (2), 113121. Mosch, B., Morawski, M., Mittag, A., Le nz, D., Tarnok, A., & Arendt, T. (2007). Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease. J Neurosci, 27 (26), 6859-6867. Mrak, R. E., & Griffin, W. S. (2004). Trisomy 21 and the brain. J Neuropathol Exp Neurol, 63(7), 679-685. Murakami, M., Iwai, S., Hiratsuka, S., Yamauc hi, M., Nakamura, K., Iwakura, Y., et al. (2006). Signaling of vascular endothelial gr owth factor receptor-1 tyrosine kinase promotes rheumatoid arthritis throug h activation of monocytes/macrophages. Blood, 108(6), 1849-1856. Myllykangas-Luosujarvi, R., & Isomaki, H. ( 1994). Alzheimer's disease and rheumatoid arthritis. Br J Rheumatol, 33 (5), 501-502. Nalbandian, A., Crispin, J. C., & Tsokos, G. C. (2009). Interleukin-17 and systemic lupus erythematosus: current concepts. Clin Exp Immunol, 157 (2), 209-215. Naruse, S., Igarashi, S., Kobayashi, H., Aoki K., Inuzuka, T., Kane ko, K., et al. (1991). Mis-sense mutation Val----Ile in exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer's disease. Lancet, 337(8747), 978-979. Nasreddine, Z. S., Loginov, M., Clark, L. N., Lamarche, J., Miller, B. L., Lamontagne, A., et al. (1999). From genotype to ph enotype: a clinical pathological, and biochemical investigation of frontotempor al dementia and parkinsonism (FTDP17) caused by the P301L tau mutation. Ann Neurol, 45(6), 704-715. Nilsson, L. N., Bales, K. R., DiCarlo, G., Go rdon, M. N., Morgan, D., Paul, S. M., et al. (2001). Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease. J Neurosci, 21 (5), 14441451. Nizzari, M., Venezia, V., Repetto, E., Caorsi, V., Magrassi, R., Gagliani, M. C., et al. (2007). Amyloid precursor protein and Pr esenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling. J Biol Chem, 282 (18), 13833-13844. O'Banion, M. K., Sadowski, H. B., Winn, V., & Young, D. A. (1991). A serumand glucocorticoid-regulated 4-kilobase mR NA encodes a cyclooxygenase-related protein. J Biol Chem, 266 (34), 23261-23267. Ogawa, O., Zhu, X., Lee, H. G., Raina, A., Obrenovich, M. E., Bowser, R., et al. (2003). Ectopic localization of p hosphorylated histone H3 in Alzheimer's disease: a mitotic catastrophe? Acta Neuropathol, 105 (5), 524-528. Olson, M. I., & Shaw, C. M. (1969). Presen ile dementia and Alzheimer's disease in mongolism. Brain, 92(1), 147-156.

PAGE 51

41Padmanabhan, J., Levy, M., Dickson, D. W., & Potter, H. (2006). Alpha1antichymotrypsin, an inflammatory protei n overexpressed in Alzheimer's disease brain, induces tau phosphorylation in neurons. Brain, 129(Pt 11), 3020-3034. Paris, D., Patel, N., Ganey, N. J., Laporte, V., Quadros, A., & Mullan, M. J. (2010). AntiTumoral Activity of a Short Decapeptide Fragment of the Alzheimer's Abeta Peptide. Int J Pept Res Ther, 16 (1), 23-30. Park, H., Li, Z., Yang, X. O., Chang, S. H., Nurieva, R., Wang, Y. H., et al. (2005). A distinct lineage of CD4 T cells re gulates tissue infl ammation by producing interleukin 17. Nat Immunol, 6 (11), 1133-1141. Park, J., Yang, E. J., Yoon, J. H., & Chung, K. C. (2007). Dyrk1A overexpression in immortalized hippocampal cells produces the neuropathological features of Down syndrome. Mol Cell Neurosci, 36 (2), 270-279. Patel, N. S., Mathura, V. S., Bachmeier, C., Beaulieu-Abdelahad, D., Laporte, V., Weeks, O., et al. (2010). Alzheimer's beta-amylo id peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2. J Neurochem, 112(1), 66-76. Pei, J. J., Braak, H., An, W. L., Winblad, B., Cowburn, R. F., Iqbal, K., et al. (2002). Upregulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degenera tion in Alzheimer's disease. Brain Res Mol Brain Res, 109 (1-2), 45-55. Perlmutter, L. S., Myers, M. A., & Barron, E. (1994). Vascular basement membrane components and the lesions of Alzheimer's disease: light and electron microscopic analyses. Microsc Res Tech, 28 (3), 204-215. Petersen, M. B., Karadima, G., Samaritaki, M., Avramopoulos, D., Vassilopoulos, D., & Mikkelsen, M. (2000). Association betw een presenilin-1 polymorphism and maternal meiosis II errors in Down syndrome. Am J Med Genet, 93(5), 366-372. Poorkaj, P., Bird, T. D., Wijsman, E., Neme ns, E., Garruto, R. M., Anderson, L., et al. (1998). Tau is a candidate gene for ch romosome 17 frontotemporal dementia. Ann Neurol, 43(6), 815-825. Pope, W. B., Lambert, M. P., Leypold, B., Se upaul, R., Sletten, L., Krafft, G., et al. (1994). Microtubule-associated protein ta u is hyperphosphorylated during mitosis in the human neuroblastoma cell line SH-SY5Y. Exp Neurol, 126 (2), 185-194. Potter, H. (1991). Review and hypothesis: Alzheimer disease and Down syndrome-chromosome 21 nondisjunction may underlie both disorders. Am J Hum Genet, 48(6), 1192-1200. Potter, H., Wefes, I. M., & Nilsson, L. N. (2001). The inflammation-induced pathological chaperones ACT and apo-E are necessary catalysts of Alzheimer amyloid formation. Neurobiol Aging, 22 (6), 923-930. Prasher, V. P., Farrer, M. J., Kessling, A. M., Fisher, E. M., West, R. J., Barber, P. C., et al. (1998). Molecular mapping of Alzheime r-type dementia in Down's syndrome. Ann Neurol, 43(3), 380-383. Preuss, U., Doring, F., Illenberger, S., & Ma ndelkow, E. M. (1995). Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells. Mol Biol Cell, 6 (10), 1397-1410. Puri, B. K., Zhang, Z., & Singh, I. (1994). SPECT in adult mosaic Down's syndrome with early dementia. Clin Nucl Med, 19 (11), 989-991.

PAGE 52

42Rahmani, Z., Blouin, J. L., Creau-Goldberg, N., Watkins, P. C., Mattei, J. F., Poissonnier, M., et al. (1989). Critical role of th e D21S55 region on chromosome 21 in the pathogenesis of Down syndrome. Proc Natl Acad Sci U S A, 86 (15), 5958-5962. Raut, C. P., Nawrocki, S., Lashinger, L. M., Davis, D. W., Kha nbolooki, S., Xiong, H., et al. (2004). Celecoxib inhibits angiogenesis by inducing endothel ial cell apoptosis in human pancreatic tumor xenografts. Cancer Biol Ther, 3(12), 1217-1224. Reines, S. A., Block, G. A., Morris, J. C., Liu, G., Nessly, M. L., Lines, C. R., et al. (2004). Rofecoxib: no effect on Alzheimer's disease in a 1-year, randomized, blinded, controlled study. Neurology, 62 (1), 66-71. Rich, J. B., Rasmusson, D. X., Folstein, M. F., Carson, K. A., Kawas, C., & Brandt, J. (1995). Nonsteroidal anti-inflammator y drugs in Alzheimer's disease. Neurology, 45(1), 51-55. Riley, K. P., Snowdon, D. A., & Markesbery, W. R. (2002). Alzheimer's neurofibrillary pathology and the spectrum of cognitive function: findings from the Nun Study. Ann Neurol, 51(5), 567-577. Ringman, J. M., Rao, P. N., Lu, P. H., & Ce derbaum, S. (2008). Mosaicism for trisomy 21 in a patient with young-onset dementia : a case report and brief literature review. Arch Neurol, 65 (3), 412-415. Rogers, J., Kirby, L. C., Hempelman, S. R., Berry, D. L., McGeer, P. L., Kaszniak, A. W., et al. (1993). Clinical trial of indomethacin in Alzheimer's disease. Neurology, 43 (8), 1609-1611. Rovelet-Lecrux, A., Hannequin, D., Raux, G., Le Meur, N., Laquerriere, A., Vital, A., et al. (2006). APP locus duplication cause s autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy. Nat Genet, 38 (1), 24-26. Rowe, I. F., Ridler, M. A., & Gibberd, F. B. (1989). Presenile dementia associated with mosaic trisomy 21 in a patient with a Down syndrome child. Lancet, 2(8656), 229. Rumble, B., Retallack, R., Hilbich, C., Simms G., Multhaup, G., Martins, R., et al. (1989). Amyloid A4 protei n and its precursor in Down's syndrome and Alzheimer's disease. N Engl J Med, 320(22), 1446-1452. Ryoo, S. R., Cho, H. J., Lee, H. W., Jeong, H. K., Radnaabazar, C., Kim, Y. S., et al. (2008). Dual-specificity tyrosine(Y)phosphorylation regulated kinase 1Amediated phosphorylation of amyloid precurs or protein: evidence for a functional link between Down syndrome and Alzheimer's disease. J Neurochem, 104 (5), 1333-1344. Ryoo, S. R., Jeong, H. K., Radnaabazar, C., Yoo, J. J., Cho, H. J., Lee, H. W., et al. (2007). DYRK1A-mediated hyperphosphoryl ation of Tau. A functional link between Down syndrome and Alzheimer disease. J Biol Chem, 282 (48), 3485034857. Sarkar, S., Tesmer, L. A., Hindnavis, V., Endr es, J. L., & Fox, D. A. (2007). Interleukin17 as a molecular target in immune-m ediated arthritis: immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin-4. Arthritis Rheum, 56(1), 89-100. Save, M. P., Shetty, V. P., Shetty, K. T., & Antia, N. H. (2004). Alterations in neurofilament protein(s) in human leprous nerves: morphology,

PAGE 53

43immunohistochemistry and Wester n immunoblot correlative study. Neuropathol Appl Neurobiol, 30(6), 635-650. Scharf, S., Mander, A., Ugoni, A., Vajda, F., & Christophidis, N. (1999). A double-blind, placebo-controlled trial of diclofenac/ misoprostol in Alzheimer's disease. Neurology, 53 (1), 197-201. Scheibel, A. B. (1987). Alterations of the cereb ral capillary bed in the senile dementia of Alzheimer. Ital J Neurol Sci, 8 (5), 457-463. Scheibel, A. B., Duong, T. H., & Tomiyasu, U. (1987). Denervation microangiopathy in senile dementia, Alzheimer type. Alzheimer Dis Assoc Disord, 1 (1), 19-37. Schmedtje, J. F., Jr., Ji, Y. S., Liu, W. L., DuBois, R. N., & Runge, M. S. (1997). Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem, 272 (1), 601-608. Schneider, A., & Mandelkow, E. (2008). Tau-based treatment strategies in neurodegenerative diseases. Neurotherapeutics, 5 (3), 443-457. Schupf, N., Kapell, D., Lee, J. H., Ottman, R., & Mayeux, R. (1994). Increased risk of Alzheimer's disease in mothers of adults with Down's syndrome. Lancet, 344(8919), 353-356. Schwarz, Q., Gu, C., Fujisawa, H., Sabelko, K ., Gertsenstein, M., Nagy, A., et al. (2004). Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve. Genes Dev, 18(22), 2822-2834. Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins, W., et al. (1994). Pharmacological and biochemical demonstr ation of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A, 91 (25), 12013-12017. Selinfreund, R. H., Barger, S. W., Welsh, M. J., & Van Eldik, L. J. (1990). Antisense inhibition of glial S100 beta production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation. J Cell Biol, 111(5 Pt 1), 20212028. Shah, S., Lee, S. F., Tabuchi, K., Hao, Y. H., Yu, C., LaPlant, Q., et al. (2005). Nicastrin functions as a gamma-secretase-substrate receptor. Cell, 122 (3), 435-447. Singh, T. J., Grundke-Iqbal, I., & Iqbal, K. (1995). Phosphorylation of tau protein by casein kinase-1 converts it to an abnormal Alzheimer-like state. J Neurochem, 64(3), 1420-1423. Singh, T. J., Grundke-Iqbal, I., McDonald, B ., & Iqbal, K. (1994). Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases. Mol Cell Biochem, 131 (2), 181-189. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., et al. (1999). Purification and cloni ng of amyloid precursor protein beta-secretase from human brain. Nature, 402(6761), 537-540. Sisodia, S. S., & Price, D. L. (1995). Role of the beta-amyloid protein in Alzheimer's disease. Faseb J, 9 (5), 366-370. Sleegers, K., Brouwers, N., Gijselinck, I., Th euns, J., Goossens, D., Wauters, J., et al. (2006). APP duplication is sufficient to cau se early onset Alzheimer's dementia with cerebral amyloid angiopathy. Brain, 129(Pt 11), 2977-2983.

PAGE 54

44Slomnicki, L. P., & Lesniak, W. (2008). A putative role of the Amyloid Precursor Protein Intracellular Domain (AICD) in transcription. Acta Neurobiol Exp (Wars), 68 (2), 219-228. Spillantini, M. G., Yoshida, H., Rizzini, C., La ntos, P. L., Khan, N., Rossor, M. N., et al. (2000). A novel tau mutation (N296N) in familial dementia with swollen achromatic neurons and corticobasal inclusion bodies. Ann Neurol, 48(6), 939943. St George-Hyslop, P. H., Haines, J. L., Fa rrer, L. A., Polinsky, R., Van Broeckhoven, C., Goate, A., et al. (1990). Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder. FAD Collaborative Study Group. Nature, 347(6289), 194-197. Strittmatter, W. J., Weisgraber, K. H., Hu ang, D. Y., Dong, L. M., Salvesen, G. S., Pericak-Vance, M., et al. ( 1993). Binding of human apoli poprotein E to synthetic amyloid beta peptide: isoform-specific e ffects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci U S A, 90 (17), 8098-8102. Strobel, G. (2009, 21 July 2009). Vienna: New Genes, Anyone? ICAD Saves Best for Last. Retrieved 7-1-2010, from http://www.alzforum.org/new/ detail.asp?id=2197#breitner Sun, X., Wang, Y., Qing, H., Christensen, M. A., Liu, Y., Zhou, W., et al. (2005). Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. Faseb J, 19 (7), 739-749. Suzuki, T., Oishi, M., Marshak, D. R., Czernik, A. J., Nairn, A. C., & Greengard, P. (1994). Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. Embo J, 13 (5), 1114-1122. Swift, T. R. (1974). Peripheral nerve involve ment in leprosy: qua ntitative histologic aspects. Acta Neuropathol, 29 (1), 1-8. Takashima, S., Becker, L. E., Armstrong, D. L., & Chan, F. (1981). Abnormal neuronal development in the visual cortex of the human fetus and infant with down's syndrome. A quantitative and qualitative Golgi study. Brain Res, 225 (1), 1-21. Takashima, S., Iida, K., Mito, T., & Arim a, M. (1994). Dendritic and histochemical development and ageing in patients with Down's syndrome. J Intellect Disabil Res, 38 ( Pt 3) 265-273. Tanaka, S., Nakamura, S., Ueda, K., Kameyama M., Shiojiri, S., Takahashi, Y., et al. (1988). Three types of amyloid protein precursor mRNA in hu man brain: their differential expression in Alzheimer's disease. Biochem Biophys Res Commun, 157(2), 472-479. Tanaka, S., Shiojiri, S., Takahashi, Y., K itaguchi, N., Ito, H., Kameyama, M., et al. (1989). Tissue-specific expression of th ree types of beta-protein precursor mRNA: enhancement of protease inhibitor-h arboring types in Alzheimer's disease brain. Biochem Biophys Res Commun, 165 (3), 1406-1414. Tanzi, R. E., George-Hyslop, P. S., & Guse lla, J. F. (1991). Molecular genetics of Alzheimer disease amyloid. J Biol Chem, 266 (31), 20579-20582. Tezapsidis, N., Merz, P. A., Merz, G., & H ong, H. (2003). Microtubular interactions of presenilin direct kinesis of Ab eta peptide and its precursors. Faseb J, 17(10), 1322-1324.

PAGE 55

45Thakker, P., Leach, M. W., Kuang, W., Benoit, S. E., Leonard, J. P., & Marusic, S. (2007). IL-23 is critical in the induction but not in the effector phase of experimental autoimmune encephalomyelitis. J Immunol, 178 (4), 2589-2598. Thambisetty, M., Beason-Held, L., An, Y., Kraut, M. A., & Resnick, S. M. (2010). APOE epsilon4 genotype and longitudinal changes in cerebral blood flow in normal aging. Arch Neurol, 67(1), 93-98. Thomas, P., & Fenech, M. ( 2008). Chromosome 17 and 21 ane uploidy in buccal cells is increased with ageing and in Alzheimer's disease. Mutagenesis, 23(1), 57-65. Trojanowski, J. Q., Mawal-Dewan, M., Schmidt, M. L., Martin, J., & Lee, V. M. (1993). Localization of the mitogen activated prot ein kinase ERK2 in Alzheimer's disease neurofibrillary ta ngles and senile plaque neurites. Brain Res, 618 (2), 333-337. Varvel, N. H., Bhaskar, K., Patil, A. R., Pimplikar, S. W., Herrup, K., & Lamb, B. T. (2008). Abeta oligomers induce neuronal cell cycle events in Alzheimer's disease. J Neurosci, 28 (43), 10786-10793. Vassar, R., Bennett, B. D., Babu-Khan, S., Ka hn, S., Mendiaz, E. A., Denis, P., et al. (1999). Beta-secretase cleavage of Alzhei mer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286 (5440), 735-741. Vincent, I., Rosado, M., & Davies, P. (1996). Mitotic mechanisms in Alzheimer's disease? J Cell Biol, 132 (3), 413-425. Vlad, S. C., Miller, D. R., Kowall, N. W., & Felson, D. T. (2008). Protective effects of NSAIDs on the development of Alzheimer disease. Neurology, 70 (19), 16721677. Waaler, E. (1940). On the occurrence of a f actor in human serum activating the specific agglutination of sheep red corpuscles. Acta Pathologica et Microbiologica Scandinavica 17 172-188. Wang, G. L., Jiang, B. H., Rue, E. A., & Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-heli x-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A, 92 (12), 5510-5514. Wang, L., Chen, W., Xie, X., He, Y., & Bai, X. (2008). Celecoxib i nhibits tumor growth and angiogenesis in an orthotopic im plantation tumor model of human colon cancer. Exp Oncol, 30(1), 42-51. Wang, P., Yang, G., Mosier, D. R., Chang, P., Zaidi, T., Gong, Y. D., et al. (2005). Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2. J Neurosci, 25 (5), 1219-1225. Weiger, T., Lametschwandtner, A., & Stockm ayer, P. (1986). Technical parameters of plastics (Mercox CL-2B a nd various methylmethacrylates) used in scanning electron microscopy of vascular corrosion casts. Scan Electron Microsc(Pt 1), 243-252. Weinstein, B. M. (2005). Vessels and nerves: marching to the same tune. Cell, 120(3), 299-302. Whitaker-Azmitia, P. M., Wingate, M., Borella, A., Gerlai, R., Roder, J., & Azmitia, E. C. (1997). Transgenic mice overexpressing the neurotrophic f actor S-100 beta show neuronal cytoskeletal and behavior al signs of altere d aging processes: implications for Alzheimer's disease and Down's syndrome. Brain Res, 776 (1-2), 51-60.

PAGE 56

46Wisniewski, T., & Frangione, B. (1992). Apo lipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett, 135(2), 235-238. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Di ehl, T. S., Kimberly, W. T., & Selkoe, D. J. (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature, 398(6727), 513-517. Woodard, J. S. (1966). Alzheimer' s disease in late adult life. Am J Pathol, 49 (6), 11571169. Wragg, M., Hutton, M., & Talbot, C. (1996). Genetic association between intronic polymorphism in presenilin-1 gene and late-onset Alzheimer's disease. Alzheimer's Disease Collaborative Group. Lancet, 347 (9000), 509-512. Wynn, T. A. (2005). T(H)-17: a gi ant step from T(H)1 and T(H)2. Nat Immunol, 6 (11), 1069-1070. Yan, Q., Zhang, J., Liu, H., Babu-Khan, S., Vassar, R., Biere, A. L., et al. (2003). Antiinflammatory drug therapy alters beta-a myloid processing and deposition in an animal model of Alzheimer's disease. J Neurosci, 23 (20), 7504-7509. Yan, R., Bienkowski, M. J., Shuck, M. E., Miao H., Tory, M. C., Pauley, A. M., et al. (1999). Membrane-anchored aspartyl prot ease with Alzheimer's disease betasecretase activity. Nature, 402 (6761), 533-537. Yan, R., Munzner, J. B., Shuck, M. E., & Bi enkowski, M. J. (2001). BACE2 functions as an alternative alpha-s ecretase in cells. J Biol Chem, 276 (36), 34019-34027. Yang, Y., Geldmacher, D. S., & Herrup, K. (2001). DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci, 21 (8), 2661-2668. Yasojima, K., Schwab, C., McGeer, E. G., & McGeer, P. L. (1999). Distribution of cyclooxygenase-1 and cyclooxygenase-2 mR NAs and proteins in human brain and peripheral organs. Brain Res, 830 (2), 226-236. Yermakova, A. V., Rollins, J., Callahan, L. M., Rogers, J., & O'Banion, M. K. (1999). Cyclooxygenase-1 in human Alzheimer and cont rol brain: quantitative analysis of expression by microglia a nd CA3 hippocampal neurons. J Neuropathol Exp Neurol, 58(11), 1135-1146. Zacchigna, S., Lambrechts, D., & Carmeliet, P. (2008). Neurovascular signalling defects in neurodegeneration. Nat Rev Neurosci, 9 (3), 169-181. Zarow, C., Barron, E., Chui, H. C., & Perl mutter, L. S. (1997). Vascular basement membrane pathology and Alzheimer's disease. Ann N Y Acad Sci, 826 147-160. Zhao, G., Liu, Z., Ilagan, M. X., & Kopan, R. (2010). Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyl oid precursor protein in the absence of nicastrin. J Neurosci, 30 (5), 1648-1656. Zimmermann, K., Herget, T., Salbaum, J. M., Schubert, W., Hilbich, C., Cramer, M., et al. (1988). Localization of the putative precu rsor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle. Embo J, 7 (2), 367-372. Zitnik, G., Wang, L., Martin, G. M., & H u, Q. (2007). Localizations of endogenous APP/APP-proteolytic produc ts are consistent with microtubular transport. J Mol Neurosci, 31 (1), 59-68.

PAGE 57

47 CHAPTER 2 RESEARCH RATIONALE Introduction In the period leading up to 2006, our inte rest in the role of inflammation in AD led us to think that perhaps RA protected against AD not because of the use of NSAIDs but because of some intrinsic anti-inflammatory or possibly inflammatory factor released during RA. This interest came to a head as a consequence of NSAID clinical trial failures in AD patients and we began to look again at the inverse relationship of RA and AD. If indeed, the NSAIDs are not responsible for preventing AD, a neurodegenerative disorder, then there must be some intrinsic characteris tic(s) of RA pathogenesis itself, a peripheral disorder, that has the capability to traverse the body and reach the brain, where it would exert its protective effects. Since RA is a hi ghly-inflammatory autoimmune disease, both aspects of the immune system, innate and ad aptive, could potentially be acting against AD, including not only the various cells of the immune system, but also through their secreted products. This chapter will examine th ese cellular characterist ics of RA that may impact AD pathogenesis and outline the research plan to inves tigate RA as a negative risk factor of AD from a non-NSAID perspective. Secreted Factors in RA and AD Many of the factors (Chapter 1) that ar e up-regulated in RA pathogenesis have also been reported to have ci rcumstantial relationships in AD pathogenesis. For instance, Macrophage Migration Inhibitory Factor (MIF) has been found to have the ability to act as an A -binding protein (Flex et al., 2004; Las huel et al., 2005), and is absolutely necessary for the progression of RA, as MIFdeficient mice do not develop experimental

PAGE 58

48 arthritis and anti-MIF neutra lizing antibodies ameliorate RA in mouse models (Onodera et al., 2007; Popa et al., 2006). MIF has also recently been shown to be up-regulated in the brains of transgenic AD mice, and in th e cerebrospinal fluid of MCI and AD patients, and it is proposed to contribute to the ne uroinflammation of AD, since MIF is a known cytokine that elicits prod uction of other inflammatory mediators, such as TNF IL-6, and IFN (Bacher et al., 2010; Bryan et al ., 2008; Popp et al., 2009). In fact, in vitro studies show that murine and human neuron al cell lines are protected from A -induced cellular toxicity by Iso-1, a small molecule inhibitor of MIF (Bacher et al., 2010). On the other hand, MIFs functions have not all been elucidat ed, as a recent paper reports that MIF is actually expressed by astrocytes in the CNS and is involved in hippocampal cell proliferation. In this study, it was found that MIF is expressed in the subgranular zone of the dentate gyrus within neurogenic stem cells, cells undergoing proliferation, and in newly proliferated cells, but not in mature neurons, microglia, or oligodendrocytes. MIF antagonism by Iso-1 and in MIF knockout mice resulted in behavioral dysfunction and impaired hippocampus-dependent memory (Conboy et al., 2010). Monocyte Chemoattractant Protein-1, (MCP-1, also known as CCL2: chemokine C-C motif ligand 2), IL-6, and IL-8 have also been reported to be up-regulated in both RA and AD (Ishizuka et al., 1997; Sokolova et al., 2009), but contrasting studies have reported that while MCP-1 is elevated in MC I patients, its expression is lost as MCI patients progress to AD (Dawson, Miltz, Mir, & Wiessner, 2003; Galimberti et al., 2006). Another study reports elevated levels of osteoprotegerin (OPG) in AD and Vascular Dementia patients, proposes a role for OPG in microglia function and phagocytosis of A plaques, and suggests that OPG could be a bi omarker for these diseases (Emanuele et al.,

PAGE 59

49 2004).TRAF6 (TNF receptor-associated factor 6), also known as E3 ubiquitin ligase, is involved in the ubiquination of Tau, as show n in TRAF6-knock out mice (Babu, Geetha, & Wooten, 2005). TRAF6 is also activated, along with NF B, after proteolysis of membrane receptors, p75NTR and neurotroph in receptor homolog NRH2 (Kanning et al., 2003). Like APP and Notch, p75NTR is a ligand for -secretase intramembrane cleavage after -secretase extracellular domain cleavage. The -secretase that is expressed in RA for the cleavage of RANKL and the T lymphocyte chemokine, soluble CXCL16, is ADAM10. ADAM10 is found abundantly expressed by RA macrophages expressing the chemokine, CXCL16, which is involved in e ffector/memory T cell function (van der Voort et al., 2005), and anti-TNF therapy for RA down-regulates this CXCL16 expression (van Lieshout et al., 2007). In the brain, ADAM10, along with PS1, works to cleave N-cadherin following NMDA activation, and this activity has been found to be inhibited in an AD mouse model (Uemur a et al., 2007). Furthermore, ADAM10 overexpression has been shown to decrease A plaque formation and prevent cognitive deficits in another AD murine model (Fahre nholz & Postina, 2006; Postina et al., 2004). There are also angiogenic factors contributi ng to the pannus growth in RA that are associated with AD pathogenesis, such as In sulin-Like Growth Factor 1 (IGF-1), which is upregulated in RA synovial flui d, but is reduced in the AD brain, and has been associated with cognitive deficits (Rivera et al., 2005; Svensson et al., 2006). IGF-1 levels are similarly decreased in patients with Diabetes Type II, a positive risk factor for AD onset, and it is reported that systemic administration of IGF-1 in diabetic ra ts prevents cognitive deficits (Lupien, Bluhm, & Ishii, 2003, 2006). VEGFs relationship in AD pathology is still not completely clear and has been debate d in the literature. A lthough it is reported to

PAGE 60

50 bind A and to be found associated with amyloid plaques, reports also suggest VEGF to have neuroprotective activity (Moser & Humpel, 2005; S. P. Yang, Kwon, Gho, & Chae, 2005). VEGF upregulation and endothelia l activation are found in AD brain, even though cerebrovascular degeneration occurs and correlates with A deposition (Miao et al., 2005; Schultheiss et al., 2006). A recent study investigating cardiovascular disease in PS2/APP mice, reported that administrati on of IGF-1 improved cognitive function, reduced VEGF levels, reduced endothelial cell proliferation, and thus normalized vascular density (Lopez-Lopez, Dietrich, Metzger, Loetscher, & Torres-Aleman, 2007). Although many of these above-mentioned infl ammatory mediators participate in the chronic neuroinflammation that contributes to AD pathogenesis, if they are also up-regulated in RA pathogenesis, it would not be expected that these specific factors are the reason for the inverse relationship between RA and AD. On the other hand, if they are deficient in AD, but are up-regulated in RA they might provide insight into RAs protective effect against AD. However, they would also have to be secreted in high enough concentrations to be ab le to circulate through the vasculature, reach the brain, cross the Blood Brain Barrier (BBB), and initia te a set of protectiv e events against AD onset. These actions would be very improbabl e without affecting a ll other organ systems in the process, which would have been reported in all RA patients. Since this is not the case, the peripheral secretion of inflammatory mediators may not be the most likely reason for RA and ADs inverse relationship. No netheless, they cannot all be completely ignored in this comparison, since they are secreted by th e leukocytes of RA, which do have the ability to traverse the circulator y system, diapedese across the BBB, and could secrete these factors into the brains parenchyma to elicit protective effects. Of all the

PAGE 61

51 above-mentioned factors, a couple that are de ficient in AD, but are upregulated in RA, are IGF-1 and ADAM10, and these do have the potential to provide benefit to AD pathogenesis if they can localize into the brain parenchyma. However, they are secondary to the RA-induced leukocytes, which must secret e them into the brain, and thus the primary focus for elucidating RAs negative risk factor against AD should be on the leukocytes themselves. Leukocytes in RA and AD Although up-regulated leukocytes in RA c ould potentially enter into the brain and inhibit development of AD pathology and/or neuronal dysfunction, lymphocytic infiltrates into AD patient brains have not been repor ted. The lack of lymphocyte infiltration suggests th at RA-induced proliferation and activation of the innate immune system might be responsible for preven ting AD pathology in RA patients. Evidence supporting the innate immune systems role in AD pathogenesis show that bone marrowderived microglia play a critical role in restricting amyloid deposition, and indeed, microglia activation and many associated receptors and enzymes, such as CD36, scavenger receptor A, receptor for advanced glycation end products, neprilysin, insulysin, and matrix metalloproteinases, decline with ag e as risk of AD pathol ogy increases (J. El Khoury et al., 2007; Hickman, Allison, & El Khoury, 2008; Simard, Soulet, Gowing, Julien, & Rivest, 2006). Further evidence imp licating the role of the innate immune system in AD are that complement proteins are highly up-regulated in AD brain, and that inhibition of C3 convertase significantly in creases amyloid pathology in AD mice (WyssCoray et al., 2002). A chronic upregulation of complement proteins in AD suggests that innate leukocytes need to also be increased to phagocytically remove the complement-

PAGE 62

52 bound complexes, via the complement receptors th at they contain on th eir surface. Both the complement system and the ability for peripheral phagocytes to enter the brain represent two broad immunologica l areas of active research th at show many implications in the pathogenesis of AD, and which will be discussed further in the following sections. Complement System and its Involvement in AD Overview. The complement system is a cascade of about 30 cell bound or circulating proteins that aid in the clearan ce of immune-stimulatory molecules, whether contained on pathogens, on endoge nous cells, or secreted within the body. As part of the innate immune system, the complement system is a powerful amplifier and mediator of inflammation, and functions to opsonize antigens for phagocytos is, to release chemotactic molecules for attracting phagocytes, to lyse me mbranes of cells via the membrane attack complex (MAC), to aggregate antigenic proteins, to induce nave B lymphocyte activation, and to neutrali ze viral activity (Carroll, 2008; Jayasekera, Moseman, & Carroll, 2007; Roitt IM, 1998). The complement sy stem is activated in three biochemical pathways, known as the classical (since it wa s first identified), the lectin, and the alternative pathways (see Figure 2 for an overview of the complement cascade) Classical Pathway. The classical complement pathway is usually activated by either IgM or IgG binding to an antigen, which then triggers complement component, C1q, to bind to the first conserved regions of these immunoglobulins. Clq can also bind independent of antibodies to DNA, RNA, C-reactive protein, serum amyloid P, LPS (from Gram bacteria), funga l/viral membranes, and in AD, it has been shown to bind directly to amyloid plaques (Afagh, Cu mmings, Cribbs, Cotman, & Tenner, 1996; Eikelenboom, Hack, Rozemuller, & Stam, 1989; Jiang, Burdick, Glabe, Cotman, &

PAGE 63

53 Tenner, 1994; Rogers et al., 1992; Tacn et-Delorme, Chevallier, & Arlaud, 2001; Veerhuis, Janssen, Hack, & Eikelenboom, 1996; Zanjani et al., 2005). C1q binding causes a conformational change in itself, wh ich activates two molecules of the serine protease, Clr, to cleave and ac tivate two molecules of anothe r serine protease, Cls. This process results in the formati on of the C1 complex (C1q-C1r2-C1s2), which then acts to cleave complement components C4 and C2 into C4a, C4b, C2a, and C2b. The binding of C4b and C2a form the C3-convertase (C 4b2a) of the classical pathway. Although the regulatory protein for C1 complex, C1-inhibitor, is normally produced in the brain and is the only known inhibitor of C1 r and C1s, it has been found in human AD brains to be cleaved and this reacted form is lo calized to dystrophic neurites and neuropil threads (Walker, Yasuhara, Patston, McGeer, & McGeer, 1995). C1-inhibitor has also been found to be slightly elevated only in the regions of prominent AD pathology, such as the entorhinal cortex, hippocampus, medialtemporal gyrus and medialfrontal gyrus, but this slight over-express ion is unable to inhibit complement activation (Yasojima, McGeer, & McGeer, 1999). Of particul ar interest is the finding that the N-terminal 1-11 region of the A 1-42 peptide binds to C1q and activates the classical complement cascade in the absence of C1-inhibitor and at a physiological ratio of up to 8:0 of C1-inhibitor:C1 (Tacnet-Delor me et al., 2001). This finding might explain a relationship between both ACT (Ma, Brewer & Potter, 1996; Ma, Yee, Brewer, Das, & Potter, 1994; Potter, Wefes, & Nilsson, 2001) and the inhibition of the complement cascade to exacerbate cerebral amyloid deposition (Wyss-Coray et al., 2002).

PAGE 64

54 Lectin Pathway. The lectin complement pathway is similar to the classical pathway, except that ficolins and mannose-bind ing lectin (MBL) are the opsonins, which then activate the MBL-associated serine proteases (MASP)-1 and -2, in a manner analagous to the C1r and C1s, respectively. The ficolinor MBL-MASP1,2 complex then splits C4 and C2, resulting in the same C3-convertase, C4b-2a. MBL, similar in structure to Clq (J. Lu, Wiedemann, Timpl, & Rei d, 1993)and ficolins (types L, H, or M in humans) are pattern-recognition molecules, with MBL binding mannose (J. H. Lu, Thiel, Wiedemann, Timpl, & Reid, 1990), N-acetylglucosamine (GlcNAc, a component of Gram + bacterial cell walls), a nd some forms of LPS (Neth et al., 2000), and with ficolins binding various N-acetylated saccharides, in cluding GlcNAc (Matsushita, 2009). Ficolins have not been implicated in AD, but H-Ficolin has been shown to be highly expressed by human astrocytic glioma cell lines (Kuraya, Matsushita, Endo, Thiel, & Fujita, 2003). On the other hand, while serum levels of MBL ha ve been found to be associated with the cerebrovasculature at similar levels in both AD patients and normal controls, MBL is reduced in the cerebrospinal fluid of AD pa tients as compared to controls, suggesting increased MBL consumption is associated with complement activation in AD patients (Lanzrein et al., 1998). Alternative Pathway. The alternative complement pathway is activated independent of C4 and C2 and initiates in a process called C3 tickover, which is the spontaneous reaction between C3 and water to break C3s internal thioester bond and form iC3 (C3-H2O), which induces a conformational change that allows the serum protein, complement Factor B, to bind and form the iC3B complex. This iC3B complex allows, in the presence of Magnesium (Mg2+), for Factor D, a serine protease also known

PAGE 65

55 as adipsin, which is released into the blood by adipocytes, to cleave Factor B into Ba and Bb, leaving the fluid-phase al ternative complement pathway C3-convertase (iC3Bb). This activated iC3Bb can then cleav e circulating C3 into C3a a nd C3b, but fluid-phase iC3Bb is liable for proteolytic degr adation unless it comes in cont act with a surface to bind, in which case another serum protein, Factor P (also known as properdin), will bind and stabilize the complex, forming iC3Bb-P, which is now a surface-bound C3-convertase capable of cleaving many molecules of C3 and forming a positive feedback amplification loop (Roitt IM, 1998). It was initially thought that only the classical comple ment pathway was involved in AD, and not the alternative pathway (McG eer, Akiyama, Itagaki, & McGeer, 1989). However, it has recently been shown that the alternative pathway components, Ba and Bb, as well as the regulatory protein, Fact or H, are indeed increased in AD brain compared to controls (Strohmeyer, Ramirez, Cole, Mueller, & Rogers, 2002; Strohmeyer, Shen, & Rogers, 2000). Furthermore, the Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), which present with neuropathological similarities as AD, such as neurodegeneration, NFTs, parenchymal and vascular cerebral amyloidosis, and associated gliosis, have positive immunohistochemical staining for activated complement component s of both the classi cal and alternative pathways associated with their pathology (Rostagno et al., 2002). C3-Convertase. Both the surface-bound C4b2a C3-convertase from the classical and lectin pathways and the iC3Bb-P C3-c onvertase work to amplify the production of the opsonin C3b and release the chemotaxin C3a. In this amplification loop, newly cleaved C3b can covalently bind a surface and intiate the same alternative pathway with

PAGE 66

56 Factor B binding, then C3bB getting cleav ed by Factor D in the presence of Mg2+, and leaving a new surface-bound C3-convertase, C3bBb which continues the amplification loop (Roitt IM, 1998). As with any feed-forward mechanism, ther e needs to be regulatory proteins to inhibit uncontrolled amplific ation, and if left unchecke d, there would be complete depletion of complement component C3 (whi ch is analogous to the effects of cobra venom in the body (Janssen et al., 2009)). Some regulation occurs by the released soluble C3b being quickly hydrolyzed a nd inactivated by water, unless it comes into contact with a surface that it can covalently bind first. Also, the fluid-phase iC3bBb undergoes rapid dissociation, by the binding of soluble Fact or H. Surface-bound C3b can be cleaved by Factor I, using Factor H as a cofactor, to produce surface-bound inactive C3b, which can then undergo additional cleavage by Factor I to split inactive C 3b into C3c and C3dg. This last step to produce C3c and C3dg occurs with the help of Complement Receptor 1 (CR1, also known as CD35) and with Membra ne Cofactor of Proteolysis (MCP, also known as CD46). Both CR1 and MCP are membrane-bound and work in the disassociation of the classi cal C3-convertase, C4b2a. A nother regulator, C4-binding protein (C4BP), cleaves activated C4 before it can act on C2 to inhi bit the formation of the classical C3-convertase, as well as being another cofactor for Factor I. The final known human regulator of C3-convertase is surface-bound Decay Accelerating Factor (DAF, also known as CD55) which competes with Factor B on the cell surface to inhibit the C3bBb complex, as well as inducing th e disassociation of Bb and C2a from already-formed C3-convertases (Roitt IM, 1998). In mice, the widely-expressed regulator on most cell membranes, Complement recepto r-1related gene/protein Y (Crry), functions

PAGE 67

57 as the structural analog of both DAF a nd MCP, and is the main inhibitor of C3-convertase in mice, blocking both th e classical and alternative pathways. C3 and AD. Many of the components and regu lators in the formation of C3-Convertase have been implicated in the pathogenesis of AD. Amyloid plaques have been examined by electron immuno-microscopy and found to contain C3b, C3c, C3d, C4, as well as C1q on the amyloid fibrils, but not elsewhere such as normal or degenerative neurites, NFTs, and glial cells (Eikelenboom & Stam, 1982; Ishii & Haga, 1984). Furthermore, A was shown to actually induce th e production of C3 in primary microglial cultures (Haga, Ikeda, Sato, & Is hii, 1993), and as already mentioned, the experimental blockage of C3-convertase, by the overexpression of soluble Crry (sCrry), resulted in increased amyloid deposition in transgenic AD mice. Conversely, it was also shown that increased C3 production resulted in decreased amyloidosis (Wyss-Coray et al., 2002). The C4BP regulator has also been found local ized to amyloid plaques, to directly bind A to bind to apoptotic and necrotic cells, but not to bind to viable astrocytes, neurons, or oligodendrocytes (Trouw et al ., 2008). Whether C4BP has a protective or deleterious effect in AD is not known. Of similar association with AD is Factor D (Adipsin), which is upregulated when -Secretase is inhibited (S earfoss et al., 2003), and thus may play a role in FAD with PS mutati ons, or since it is highly expressed in obese individuals, may contribute to Type II Diabetes (Flier, Cook, Usher, & Spiegelman, 1987), a positive risk factor for AD. Also of unknown relevancy to AD pathogenesis is Factor H, mentioned above to be upregulated in late onset AD (Strohmeyer et al., 2002), and which was reported to correlate in a bundance with disease prognosis (Hye et al.,

PAGE 68

58 2006). However, polymorphisms in Factor H were not found to be related to late onset AD (Hamilton et al., 2007). One regulator of C3 that does have si gnificant involvement with AD pathogenesis is Complement Receptor-1 (CR1, also known as CD35). CR1 is located on B lymphocytes, glomerular epithelial cells, follicular dendritic cells, monocytes/macrophages, neutrophils, and espe cially on erythrocytes (Lambert et al., 2009; Roitt IM, 1998). While CR1s actions to inhibit the formation of C3-convertase was described above and is certainly impor tant in preventing excessive C3-convertase activity, CR1s major role in the pathogene sis of AD may be peripheral, instead of parenchymal, since it is most abundantly expr essed on erythrocytes. CR1s major ligands are C3b, iC3b, C4b, and C1q, which are all capable of binding A as described above, and aggregated A can bind C3b and activate C3s pr oduction (Kuo et al., 2000; Rogers et al., 2006; Webster, Bradt, Rogers, & Cooper, 1997) A recent 2009 large genome-wide association study (GWAS) of 6010 AD patients and 8625 control individuals from France, Belgium, Finland, Italy, and Spain found that the genes for CR1 and CLU (clusterin, also known as ApoJ, anot her complement regulator described below) were confirmed as risk loci for AD, in additi on to the other known su sceptibility locus of ApoE (Lambert et al., 2009). An earlier st udy looking at the amount of erythrocyte-bound A within the blood of AD, MCI, and non-demented (ND) individuals, showed that AD patients had the least amount of bound A and that MCI patients had more bound than AD but significantly less than ND individuals (Rogers et al., 2006), suggesting that C3-dependent adherence of A to erythrocytes may provide for its clearance from the bloodstream (Kuo et al., 2000; Zhou, Fonseca, Pisalyaput, & Tenner, 2008). Ligand

PAGE 69

59 bound to erythrocytic CR1 is removed via the CR1g receptors on liver Kupffer macrophages (Helmy et al., 2006; Wiesmann et al., 2006), and thus provide a mechanism for peripheral clearance of parenc hymal-to-blood transport of A C3 Receptors. There are 4 known receptors (CR1, CR2, CR3, and CR4) that bind the three surface-bound C3 products (C 3b, iC3b, and C3dg). CR1, just described above, can bind to C3b and iC3b. CR2 (a lso known as CD21), expressed on B lymphocytes, follicular dendritic cells, and some epithelial cells binds iC3b and C3dg and functions in roles independent of AD pathogenesis. However, CR3 (also known as CD11b/CD18) and CR4 (also known as CD11c /CD18) are expressed on monocytic lineage innate leukocytes a nd microglia, and they both bind C3b and iC3b, and can function to promote phagocytosis and removal of complement-bound A complexes (Choucair-Jaafar et al., 2010). An increase in the amount of these phagocytic receptors could presumably aid in the clearance of am yloid plaque from the brain. The phagocytes containing these receptors,will be discussed further in the section below, Leukocyte Recruitment in the Brain. Formation of the Membrane Attack Complex (Figure 2) After the formation of either C3-convertase, complement component C3 is cleaved into C3a and C3b. C3a is released into the fluid phase and C3b remains to bind with either C3-convertase to form the two C5-convertases, C4b2a3b and iC3BbC3b -P, which then can cleave complement component C5 into C5a and C5b. As with C3a, C5a is also released into the fluid phase and C5b remains with the complex in the progression of the full complement cascade to the Membrane Attack Complex (MAC), which forms a hydrophobic pore through the cellular membrane upon which the complement is attached, usually re sulting in lysis of

PAGE 70

60 the cell. The components C6 through C9 bind stepwise and non-enzy matically, with the binding of C7 beginning the hydrophobic intera ctions within the membrane. Up to fourteen C9 components may bind in the forma tion of the MAC. As with the prior steps of the complement cascade, the formation of the MAC is also ti ghtly regulated. The three known regulators of the complement cascade after C5-Convert ase are Vitronectin (also known as the S-protein), Protectin (also known as CD59 ), and Clusterin (CLU, also known as ApoJ). Regulation of the MAC. Vitronectin Vitronectin is a plasma protein that acts to inhibit the formation of the MAC in either of two ways. It can pr event the C5b-7 complex from binding the membrane, resulting in soluble C5b-7 (sC5b-7) which is lytically-inactive, or it can prevent the polymerization of C9 (Podack, Kolb, & Muller-Eberhard, 1977; Podack & Muller-Eberhard, 1978; Sheehan, Morris, Pussel l, & Charlesworth, 1995). Vitronectin is a multifunctional glycoprotein that has other roles besides complement regulation, such as cellular adhesion, coagulat ion, and fibrinolysis ( Reviewed in (Preissner, 1989)). Vitronectin has also been shown to be invol ved in the migration of smooth muscle cells through the vitronectin receptor, V3 integrin (Brown, Lundgren, Nordt, & Fujii, 1994), and to play an essential role in vascular remodeling of human microvessel endothelial cells (Salasznyk et al., 2007; WilcoxAdelman, Wilkins-Port, & McKeown-Longo, 2000). Thus, Vitronectin may be responsible for participating in the aberrant vascularization around amyloid plaques, as was illustrated by the V CC of Tg AD mice in Figure 1 (Chapter 1). Vitron ectin has also been found w ithin AD amyloid plaques and thoughout the body in other amyloid pathologies, such as in deposits associated with age-

PAGE 71

61 related macular degeneration, atheroslcerosis, systemic amyloidosis, and deposits within the glomeruli. Furthermore, Vitronectin was recently repor ted to readily form soluble amyloid oligomers and fibrils itself, although these actions are not known to be relevant in vivo (Shin et al., 2008). Thus the exact ro les of Vitronectin in AD pathogenesis are still unclear. Protectin Protectin (also known as CD59) is a membrane-bound protein that inhibits the final step in MAC formati on, by binding to C8 and preventing the polymerization of the C9 subunits. CD59 is c onstitutively expressed at low levels in neurons and oligodendrocytes and it is sligh tly upregulated during complement activation (McGeer et al., 1991). Using CD59 knockout mi ce (Holt et al., 2001), CD59 has been shown to play a protective role against neurodegeneration following traumatic brain injury (Stahel et al., 2009), a nd thus CD59 might also be ex pected to be neuroprotective against dementia pugilistica. Another recent report has also shown that CD59 is neuroprotective after cerebral ischemia (Harhausen et al., 2010). However in AD, the expression of CD59 has been reported to be deficient, and that this deficiency may contribute to AD pathogenesis (L. B. Yang, Li, Meri, Rogers, & Shen, 2000; Yasojima et al., 1999). Clusterin Clusterin (also known as ApoJ), like vitronectin, is a plasma protein that has multifunctional roles in the CNS. Clusterin acts, along with vitronectin, to inhibit the insertion of the MAC into th e cell membrane, by binding to the C7 and -subunits of C8 and C9 and resulting in a lytically-inactive clusterinand vitronectin-bound soluble MAC (SMAC) (T schopp, Chonn, Hertig, & French, 1993). Systemically, SMAC has been described as pa rt of circulaing im une complexes, which

PAGE 72

62 have been found in renal tissue deposits and implicated in SLE pathogenesis (Chauhan & Moore, 2006; Murphy, Davies, Morrow, & d'Ap ice, 1989). In the CNS, Clusterin was first demonstrated in 1990 to be increased in the hippocampus of AD patients, and to be expressed within pyramidal neurons a nd some non-pyramidal neurons of the hippocampus and entorhinal cortex (May et al ., 1990). It was proposed that clusterin may be involved in the neuroprotection from complement-mediated cell lysis (May & Finch, 1992). As mentioned above, Clusterins gene wa s also found in the GWAS to be a major risk locus for the developm ent of AD (Lambert et al ., 2009). Clusterin, protectin, vitronectin, and C5-9 compone nts have all been detected colocalized with dystrophic neurites and NFTs in AD and DS (McGeer et al., 1989; McGeer, Kawamata, & Walker, 1992; Stoltzner et al., 2000; Zanjani et al., 2005), but of these, only clusterin and vitronectin were found to intensely stain amyloid plaques, and clusterin had only scattered staining with NFTs (McGeer et al ., 1992). In the study by Yasuhara et al. (1994) of the tauopathy, Picks disease, virtua lly all of the Pick bodi es, contained within the dentate fascia, hippocampal pyramidal la yers, entorhinal cortex, and affected neocortices were strongly immunopositive for MAC and protectin, but were weakly stained for vitronectin, and did not stain at all for clusterin. However, clusterin did strongly stain pyramidal neurons in the parahippocampal gyrus, including ballooned ones, as well as in some scattered neurons of the affected cortical areas (Yasuhara, Aimi, McGeer, & McGeer, 1994). Taken together, these studies show that Protectin, which has insufficient levels in AD, can be found in ar eas of extreme tau pathologies, while the soluble vitronectin and clus terin MAC inhibitors are de ficiently expressed, although vitronectin and clusterin do strongly associ ate with amyloid plaques, which are not

PAGE 73

63 colocalized with NFTs in AD pathology. T hus, it could be hypothesized that in AD, clusterin and vitronectin are being sequestered by A deposits, and resultingly, they are not in sufficient concentrations around the areas of NFTs where the MAC may be contributing to neurodegeneration. Futher support for this h ypothesis comes from examini ng Clusterin (ApoJ) in its other major role within the brain. Cl usterin and ApoE are the most abundant apoliproteins in the CNS (May & Finch, 1992; Roheim, Carey, Forte, & Vega, 1979). Both clusterin and ApoE bind to A and are found in amyloi d plaques (Calero et al., 2000; May et al., 1990), as well as contributing to the fibri llogenesis, neurotoxicity, and deposition of A (DeMattos et al., 2002; Potter et al., 2001). However, these apolipoproteins are also thought to help clear A from the brain across the BBB, especially A 1-42 (Bell et al., 2007; DeMattos et al., 2004; Holtzman, 2004; Holtzman et al., 1999; Lambert & Amouyel, 2007). Clusteri n is the main protein carrier for A in biological fluids (Calero et al., 2000) and its multiligand receptor, low-density lipoprotein receptor-related protein-2 (LRP2, also known as Megalin) is expre ssed at the BBB and at the blood-CSF barrier in the choroid plexus (Chun, Wang, Pasinet ti, Finch, & Zlokovic, 1999; Hammad, Ranganathan, Loukinova, Tw al, & Argraves, 1997; Zlokovic et al., 1996). The recent study by Bell et al. ( 2007) showed that soluble, free A 1-42 is cleared at the BBB at a rate of about 1.9-fold lower than soluble, free A 1-40, that LRP2 is required by Clusterin for efflux acr oss the BBB, and that A1-42 binding to Clusterin accelerated A s clearance across the BBB by over 80 percent (Bell et al., 2007). Another recent study by Carro et al. (2005), using two Tg AD mouse models and other murine models, reported that LRP2 is involved in the transp ort of exercise-induced IGF-1(Carro, Nunez,

PAGE 74

64 Busiguina, & Torres-Aleman, 2000) from the bl ood and into the brain. The authors used viral-directed overexpression, as well as RNA interference of LRP2, to show that IGF-1 is induced systemically from regular physical exercise (on a Letica treadmill for 1h/day at 17m/min for 4 weeks at 5d/week), that circ ulating IGF-1 induces the clearance of A from the brain through LRP2, that tau hyper phosphorylation and cogniti ve deficits were subsequently prevented by these actions, a nd that LRP2 expression is decreased in normal aged animals (Carro, Spuch, Trej o, Antequera, & Torres-Aleman, 2005). Thus in consideration of both of Cluste rins main roles in the CNS, it could be hypothesized that Clusterins abi lity to transport soluble-free A across the BBB through LRP2 may decrease with age-related declining LRP2 expression, while allowing parenchymal A deposition to occur, in which Clus terin is sequestered and may even participate in this amyloid deposition, and which subsequently causes Clusterin to be deficiently expressed homogeneously thr oughout the brain and unable to prevent MAC from acting on NFT-prone neurons. Proteolytic Fragments of Complement. The formation of the two C3-convertases yields three different secreted fluid-phase molecules: C2b, C4a, and Ba. In the classical and leptin pathways, surface -bound C4b allows C2 to bind in the presence of Mg2+, in which C2 is then cleaved by activat ed C1s or MASP-2, respectively, to form a surface-bound C4b2a C3-convertase and a rele ased 30 kDa N-terminal C2b fragment into the fluid phase (Krishnan, Xu, Macon, Vo lanakis, & Narayana, 2009). The exact role of C2b in the fluid phase is not complete ly known, but one study has reported C2b to enhance vascular permeability and lead to angioedema (Strang et al., 1988). Since complement activation is induced by amyloid in AD pathogenesis, C2b production would

PAGE 75

65 be expected to also be increased, but whether C2b contributes to any cerebrovascular dysfunction in AD is unknown. Similarly, any in volvement of C4a in AD pathogenesis has not been reported. However, C4a may have future utility as a biomarker for Multiple Sclerosis (MS) prognosis (Ingram, Hakobyan, Robertson, & Morgan, 2010), for rare central nervous system (CNS) vasculitis (Langlois, Sharon, & Gawryl, 1989), and for other autoimmune diseases, such as Systemic Lupus Erythematosis (SLE) (Rupert et al., 2002). The role of the alternative pathway component, Ba, in AD pathogenesis is also not clear, but it has been reported to be significantly up-regulated in AD brain (Strohmeyer et al., 2002). The released fluid phase anaphylotoxin s, C3a and C5a, from the C3and C5-convertases, are involved in the activa tion and chemotaxis of myeloid-lineage leukocytes, through their surface-bound receptors C3aR and C5aR, respectively. Although both C3a and C5a have similar functi onal type roles, they have distinct biological activities within the brain. For example, C3a has been shown to be neuroprotective against N-Methyl-D-Asparta te (NMDA)-induced excitotoxicity (van Beek et al., 2001), to induce human microglia l cells to produce nerve growth factor (NGF) (Heese, Hock, & Otten, 1998), and to ha ve a protective anti-inflammatory effect in the CNS after LPS-induced septic shoc k (Boos, Szalai, & Barnum, 2005), while C5a was reported to participate in septic pathogenesis (Ger ard, 2003; Guo & Ward, 2005). Furthermore, C3aR and C5aR have varied e xpression after acute focal cerebral ischemia. Both receptors are constitutively expre ssed on neurons and glial cells, but after experimental permanent middle cerebral artery (MCA) occlusion, they had induced expression on the endothelial cells of blood vesse ls within the ischemic cortex at 6 hours

PAGE 76

66 and 2 days postocclusion, as well as incr eased expression on myeloid cells and reactive astrocytes one week later (Van Beek et al ., 2000). However, a similar follow-up study found that only C5aR is highly elevated at 3 hours after acute MCA, while C3aR is reduced at 3 hours post-MCA to only 25 percent of the expression in control sham-operated animals. Interestingly, at 24 hours post-MCA, C3aR expression had recovered and was increased similarly to th e study by Van Beek et al.(2000), and both C5aR and C3aR had peaked to their highest levels of the study at 24 hours (Barnum et al., 2002). Other studies have shown that inhibiting both C3aR and C5aR 6 hours post intracerebral hemmorhage can be protective (Garrett et al., 2009; Rynkowski et al., 2009), and that complement activation contribute s to injury in human s in acute ischemic stroke (Szeplaki et al., 2009). While acute ischemia is a much di fferent pathology than the chronic neurodegeneration of AD, it is also suggested in AD that C3a, C5a, and their respective receptors may have similar differential roles, which may either be neuroprotective or deleterious. Amyloid plaques in AD have l ong been reported to contain C1q, C4, and C3 fragments, but are devoid of C3a, indicating an activated complement cascade in which C3a has been released into the fluid pha se (Eikelenboom et al., 1989). As already mentioned, blockage of C3-convertase exacerb ates amyloidosis, and that increasing C3 production reduces amyloidosis (Wyss-Coray et al., 2002). Conversel y, the findings that the MAC is co-localized with dystrophic neurites and NFTs in human AD patients (Itagaki, Akiyama, Saito, & McGeer, 1994), th at blockage of C5aR in transgenic AD mice reduces amyloidosis and improves behavi or (Fonseca et al., 2009), and that C5aR is up-regulated in the microglia surrounding amyloid plaques (Ager et al., 2010), suggests

PAGE 77

67 that progression from C3-convertase to C5-c onvertase is a critical tipping point or theshold in the pathogenesis of AD, with the complement cascade going from a neuroprotective role to that of a deleterious role. Conclusion. The complement cascade plays a significant role in the pathogenesis of AD at almost every step of the process. Complement activation has been shown to be necessary for the clearance of amyloid from the brain, especially by C1q binding to A at a site that is known to promote amyloid deposition by ACT involvement and also by the activation of C3-Convertase. On the other hand, a dysregulated complement system, from C5 through MAC insertion, appears to be dele terious and contribute to neurodegeneration at areas of tau pathology. The recent findings, reported at ICAD in 2009, shed light on both of these components of the complement cascade. These findings of CR1 and CLU should not be surprising consid ering the vast cerebrovascular damage found in AD, as demonstrated from VCC studies and which is illustrated in Figure 1 (Chapter 1). This damage begins with accumulation of A (pompoms and cubes) on the parenchymal surfaces of the microvessels, which induces some luminal crimpage at first, presumably from the deficient transport of A across the BBB (age-dependent decrease in LRP2) or from the lack of receptors to bind and clear complement-bound A both within the parenchyma (by CR3 and CR4) and in the va sculature (by CR1, CR3, and CR4). These effects result in a feed-forward mechanism from age-related reductions in cerebral blood flow, further defects in vasculature, decreased expression of necessary A clearance receptors, and inversely with increased amyloidosis and subsequent tau hyperphosphorylation. As the occlusions contin ue to narrow and inhibit more cerebral perfusion, the vessels eventually pinch o ff and become completely occluded and

PAGE 78

68 truncated around a growing parenchymal anti -angiogenic amyloid plaque, which could essentially be described as progressive microvasculature ischemia and infarct. Leukocyte Recruitment in the Brain Bone Marrow-Derived Cells (BMDCs) in the CNS. Although the developmental origin of microglia has been debated (Altman, 1994; Theele & Streit, 1993) as to whether microglia are derived fr om neuroepithelial ce lls (Kitamura, Miyake, & Fujita, 1984; Lewis, 1968; Neuhaus & Fedoroff, 1994) or from hematopoietic monocytic cells (Eglitis & Mezey, 1997; Ling & Wong, 1993; Pe rry & Gordon, 1988), it is now generally-accepted that BMDCs can cross the BBB and become functional cells within the CNS One study that has demons trated this phenomenon transplanted wildtype male bone marrow into recipient PU.1null female mice (Reviewed in (Mezey & Chandross, 2000)). PU.1 is a transcription fa ctor expressed exclusiv ely in hematopoietic lineage cells, and mice with a homozygous muta tion in PU.1s gene are born extremely leukopenic, with an inability to produce new macrophages, neutrophils, mast cells, osteoclasts, or B/T lymphocytes, and they die within 48 hours from severe septicemia (McKercher et al., 1996; Tondravi et al., 1997) These mice can be rescued to normal by intraperitoneal administration of wild-type bone marrow. Using these mice, Mezey et al. observed Y chromosome-containi ng cells thoughout both the wh ite and gray matter of the brain, having phenotypes of astrocytes (GFAP immunopositivity) and neurons (NeuN-immunopositive nuclei within neuronspecific enolase immunopositive cells). There was an increased accumulation of these Y chromosome-containing cells in the choroid plexus, the ependyma of the ventricu lar system, and in the subarachnoid space and up to 5% of all cells evenly distributed thoughout the areas of the brain involved in

PAGE 79

69 AD pathogenesis, including the cerebral cortex, hypothalamus, hippocampus, amygdala, periaqueductal gray, and striatum (Mezey, Chan dross, Harta, Maki, & McKercher, 2000). Another study that was reported simultaneou sly with Mezey et al., administered bone marrow, which was harvested from 8to 10-week-old transgenic mice and which ubiquitously expressed green flourescent protein (GFP+), into isogenic age-matched lethally-irradiated mice, and allowed the recipient mice to age for 1 to 6 months (Brazelton, Rossi, Keshet, & Blau, 2000). The authors found upon subsequent examination of the mice brains, that signifi cant amounts of GFP+ neurons and microglia populated the brain throughout the CNS, such as in the hippocampus, cerebral cortex, cerebellum, and olfactory bulb, a specific area of the brain where neuronal regeneration is frequently ongoing (Hinds, 1968; Lois & Alvarez-Buylla, 1994; Luskin, 1993). Since these two studies, many other investigations have used these techniques to study the roles of bone marrow-derived cells within the brain and CNS and under various disease and experimental conditions (Beck et al., 2003; Beers et al., 2006 ; Boissonneault et al., 2009; Corti et al., 2004; Hess et al., 2002; Keshet et al., 2007; Malm et al., 2005; Priller et al., 2001; Sanchez-Ramos et al., 2009; Simard & Rivest, 2004). BMDCs and AD. One such study of GFP+ bone marrow transplantation into recipient lethally-irradiated Tg AD mice, aime d to investigate whether the microglia that decorate amyloid plaques are bone marrowderived or whether the amyloid plaques sequester the resident microglia already in the brain (Malm et al., 2005). The authors transplanted GFP+ bone marrow into young 2.5month-old Tg mice (Study I), which had not yet acquired amyloidosis, and into old 21or 25-month-old Tg mi ce (Studies II and III respectively), which already had establishe d amyloid deposition. In Study I, the mice

PAGE 80

70 were aged 26 weeks post-transplantation of GFP+ bone marrow and subsequent examination of their brains revealed that the number of engrafted GFP+ cells were significantly higher in the brains of Tg AD mice, comp ared to age-matched wild-type control mice, and that about 6% of the total GFP+ cells found within the brains were associated with amyloid plaques. In Study II of 21-month-old Tg mice and age-matched controls, which received GFP+ bone marrow and were aged 14 weeks post-transplantation, examination of the brai ns did not reveal any difference in GFP+ bone marrow-derived microglia between the Tg and wild type groups and only 3% of the GFP+ microglia, that were found within the Tg AD brain parenchymas, were associated with amyloid plaque. However, when comparing BMDC recruitment between the young and old Tg mice, there was a 17-fold higher number of GFP+ micr oglia surrounding the plaques in Study I than were found in Study II, suggesting that there is significant BMDC recruitment into the brain as amyloid is bei ng deposited, but not afte r plaque deposition is already established. To examine the established amyloid plaques further, in Study III, 25-month-old Tg AD mice were transplanted with GFP+ bone marrow, allowed to age for 16 weeks, and then LPS was injected ipsi laterally into the righ t hippocampi and saline control injected contralaterally. One week pos t-injections, the mice were sacrificed and examined, which revealed that the LPS-inje cted hippocampi had a 6-fold increase in GFP+ microglia (as distinguished by CD11b immunopositivity), a 5-fold increase of these GFP+ microglia associated with the amyloid plaques, and a significant 24% reduction in the total A burden. The authors concluded th at the infiltration of BMDCs contribute to the microglial reaction in AD, and that local inflammation can recruit BMDCs which reduce amyloid burden.

PAGE 81

71 A similar study followed that examined the actual role of th e BMDCs in relation to amyloid plaques (Simard et al., 2006). Transplantation of GFP+ bone marrow into 2-month-old lethally-irradiated Tg AD mice, and then the mice aged for 2 to 7 months post-transplantation, also showed that GFP+ microglia associated with amyloid plaques, that the average amyloid plaque area gradua lly increased each month up to 6 months of age, and that at 6 months, bone marrow-derived GFP+ plaque-associated microglia were significantly increased. However, at 9 months, the average amyloid plaque area had dramatically increased, while the number of plaque-associated GFP+ microglia had modestly decreased. Although this microglial decrease was just briefly mentioned by the authors, we took notice and hypothesized that the rapid increase in A deposition might be due to age-related depressed hematopoiesis and that strategies to increase BMDCs might inhibit or reverse amyloidosis. The au thors further work s upports this hypothesis, in which they had created a new transgenic animal model (denoted here as PS/APP/TK mice), in which a Tg AD mouse line expressed a mutant thymidine ki nase (TK) protein under the control of the CD11b promoter, such that when the mice were treated with ganciclovir, every cell expressing TK (monocytic lineage cells only) would be eliminated during cellular division. Since global administration of ganc iclovir in these mice would cause severe leukopenia, and anemic or septic mortality after chr onic administration for 10 days, the authors continuously infused ganc iclovir for 28 days intracerebroventicularly via commercial osmotic alzet pumps into th e 15to 24week-old PS/APP/TK mice. The authors demonstrated that if tr eated with ganciclovir, these mice at 6 months of age have significantly increased amyloid deposition. Furthermore, confocal microscopy of the brain tissue from the GFP+ bone marrow-transp lanted mice at 6 months of age, showed

PAGE 82

72 that in the GFP+ bone marrow-derived micr oglia, and not the resident microglia, A was present and had been phagocytosed. Taken together the authors concluded that it is the bone marrow-derived microglia which are able to remove amyloid deposition via a cell-specific phagocytic mechanism, and that depletion of these BMDCs will exaccerbate AD pathology. In further support of the notion that BMDC recruitm ent into the brain could ameliorate AD pathogenesis comes from a st udy that inhibited the migration of BMDCs into the brains of Tg AD mice (J. El K houry et al., 2007). In this study, the authors generated APP mice that were deficient in the chemokine receptor, CCR2. CCR2 has been shown to be involved in mononuclear phagocyte infiltration in to the brain of MS patients (Izikson, Klein, Charo, Weiner, & Luster, 2000), and to be required for macrophage infiltration into damaged areas of the hippocampus following axonal injury (Babcock, Kuziel, Rivest, & Owens, 2003). CCR2 binds to its ligand Chemokine C-C motif Ligand 2 (CCL2, also known as MCP-1) (Charo & Peters, 2003), and as mentioned above, CCL2 has been reported to be upregul ated in AD brain (Ishizuka et al., 1997; Sokolova et al., 2009), but expression may be lost as AD pathogenesis increases (Dawson et al., 2003; Galimberti et al., 2006). CCL2 ha s been shown to be produced by microglial and astrocytic cultures (J. B. El Khoury et al., 2003; Smits et al., 2002), and that neutralizing antibodies against CCL2 inhibit A -stimulated chemotaxis of microglia and monocytes (J. B. El Khoury et al., 2003). In another study by El Khoury et al. (2007), the authors demonstrated that Tg AD mice, wh ich were deficient in CCR2, had significant exacerbation of amyloidosis and premature mo rtality, and that these effects correlated

PAGE 83

73 with expression of CCR2 gene dosage. The au thors concluded that increased microglial density may indeed play a protective role against AD by promoting the clearance of A Research Plan The notion that NSAIDs may not account for RA being a negative risk factor against AD, and that secreted factors within RA pathogenesis itself were also unlikely to explain this phenomenon alone, we hypothesi zed that the RA up-regulated leukocytes, especially those of the innate immune system may potentially provide beneficial effects against AD and provide insight into this inverse relati onship between RA and AD. A significant amount of literature regarding di fferent components of the innate immune system lent credence to this idea, from ex tensive involvement of the complement system in AD, to the critical requirement of BMDCs to infiltrate the brain and affect AD pathology. It was also highly consistent with the literature that increasing the cellular components of the innate immune system may overcome age-related depressed hematopoiesis and also provi de therapeutic benefit. Thus, to investigate the interactions be tween the innate immune system and AD, we studied the effects on AD pathology of three colony-stimulating factors (M-CSF, G-CSF, and GM-CSF), which are up-regulated during RA pathogene sis and that drive much of the leukocytosis involved in RA pannus formation (Kawaji, Yokomura, Kikuchi, Somoto, & Shirai, 1995; Nakamura et al., 2000; Olszewski et al., 2001; Xu, Firestein, Taetle, Kaushansky, & Zvaifler, 1989) These CSFs enhance the survival and function of their respective leukocytes and drive their prolifera tion and differentiation from myeloid lineage precurs ors. GM-CSF induces dendritic cells, macrophages, and granulocytes (neutrophils, basophils, a nd eosinophils), while M-CSF and G-CSF

PAGE 84

74 respectively induce the macrophage and gra nulocyte subsets of the innate immune system. These innate cells have the ability to diapedese from the circulatory system and to differentiate further into various specia lized immune cells within organs (microglia, Langerhans cells, etc.). Specifically, we hypot hesized that the activ ation/differentiation of innate immune system cells by colony s timulating factors released during RA might lead to phagocytosis/rem oval of the pathogenic lesi ons of AD and to cognitive improvement. GM-CSF and G-CSF are also known to be involved in erythropoiesis, and GM-CSF and erythropoietin act synergistically in the maturation and proliferation of the burst-forming and colony-forming erythroi d units to the no rmoblast stage of erythropoiesis (Emerson et al., 1985; Wu, Liu, Jaenisch, & Lodish, 1995). As noted above, circulating A binds to complement opsonins in an antibody-independent fashion, and these opsonized particles bind to the complement receptor, CR1, on erythrocytes and subsequently to CR1g on liver-resident kupffer macrophages (Helmy et al., 2006; Rogers et al., 2006). Thus these CSFs could provide function in both the pe ripheral clearance of A in neovascularization and resultant in creased cerebral blood flow, and in bone marrow-derived microglial activity because th ey are involved in the proliferation, differentiation, and maintenance of most inna te leukocytes involved in these functions (see Figure 3 for overview of the hematopoietic system). The following chapters detail the resu lts on cerebral amyl oid deposition from intrahippocampal administration of the three CSFs into aged transgenic AD mice, as well as the behavior and pathological effects fr om daily subcutaneous administration of GM-CSF, the most active of the CSFs in the hippocampal injections.

PAGE 85

Figure 2. Overview of the Complement System. Many regulatory proteins of the complement system are implicated in AD and a ll are highlighted here in red text. Figure adapted and redrawn from (Tegla et al., 2009). 75

PAGE 86

Figure 3. Overview of the Hematopoietic System. G-CSF acts primarily to induce granulocytic cells from myel oid precursor cells, while MCSF upregulates macrophages. Although not indicated here, M-CSF also i nduces Myeloid-Derived Suppressor Cells. GM-CSF induces cellular populations of each of these innate hematopoietic cells. 76

PAGE 87

77 References Afagh, A., Cummings, B. J., Cribbs, D. H., Cotman, C. W., & Tenner, A. J. (1996). Localization and cell association of C1q in Alzheimer's disease brain. Exp Neurol, 138(1), 22-32. Ager, R. R., Fonseca, M. I., Chu, S. H., Sa nderson, S. D., Taylor, S. M., Woodruff, T. M., et al. (2010). Microglial C5aR (CD 88) expression correlates with amyloidbeta deposition in murine mode ls of Alzheimer's disease. J Neurochem, 113 (2), 389-401. Altman, J. (1994). Microglia emerge from the fog. Trends Neurosci, 17 (2), 47-49. Babcock, A. A., Kuziel, W. A., Rivest, S., & Owens, T. (2003). Chemokine expression by glial cells directs leuk ocytes to sites of axonal injury in the CNS. J Neurosci, 23(21), 7922-7930. Babu, J. R., Geetha, T., & Wooten, M. W. (2005). Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation. J Neurochem, 94 (1), 192-203. Bacher, M., Deuster, O., Aljabari, B., Egensper ger, R., Neff, F., Jessen, F., et al. (2010). The role of macrophage migration inhib itory factor in Alzheimer's disease. Mol Med, 16(3-4), 116-121. Barnum, S. R., Ames, R. S., Maycox, P. R., Ha dingham, S. J., Meakin, J., Harrison, D., et al. (2002). Expression of the complement C3a and C5a receptors after permanent focal ischemia: An alternative interpretation. Glia, 38(2), 169-173. Beck, H., Voswinckel, R., Wagner, S., Ziegelhoeffer, T., Heil, M., Helisch, A., et al. (2003). Participation of bone marrow-derive d cells in long-term repair processes after experimental stroke. J Cereb Blood Flow Metab, 23 (6), 709-717. Beers, D. R., Henkel, J. S., Xiao, Q., Zha o, W., Wang, J., Yen, A. A., et al. (2006). Wildtype microglia extend survival in PU .1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A, 103 (43), 16021-16026. Bell, R. D., Sagare, A. P., Friedman, A. E., Bedi, G. S., Holtzman, D. M., Deane, R., et al. (2007). Transport pathways for clearan ce of human Alzheimer's amyloid betapeptide and apolipoproteins E and J in the mouse central nervous system. J Cereb Blood Flow Metab, 27(5), 909-918. Boissonneault, V., Filali, M., Lessard, M., Relton, J., W ong, G., & Rivest, S. (2009). Powerful beneficial effect s of macrophage colony-stimulating factor on {beta}amyloid deposition and cognitive impairment in Alzheimer's disease. Brain Boos, L., Szalai, A. J., & Barnum, S. R. (2005). C3a expressed in the central nervous system protects against LPS-induced shock. Neurosci Lett, 387(2), 68-71. Brazelton, T. R., Rossi, F. M., Keshet, G. I., & Blau, H. M. (2000). From marrow to brain: expression of neurona l phenotypes in adult mice. Science, 290 (5497), 1775-1779. Brown, S. L., Lundgren, C. H., Nordt, T., & Fu jii, S. (1994). Stimula tion of migration of human aortic smooth muscle cells by vitron ectin: implications for atherosclerosis. Cardiovasc Res, 28(12), 1815-1820. Bryan, K. J., Zhu, X., Harris, P. L., Perry, G ., Castellani, R. J., Smith, M. A., et al. (2008). Expression of CD74 is increased in neurofibrillary tangles in Alzheimer's disease. Mol Neurodegener, 3 13.

PAGE 88

78 Calero, M., Rostagno, A., Matsubara, E., Zlokovi c, B., Frangione, B., & Ghiso, J. (2000). Apolipoprotein J (clusterin) and Alzheimer's disease. Microsc Res Tech, 50 (4), 305-315. Carro, E., Nunez, A., Busiguina, S., & TorresAleman, I. (2000). Circulating insulin-like growth factor I mediates eff ects of exercise on the brain. J Neurosci, 20 (8), 29262933. Carro, E., Spuch, C., Trejo, J. L., Antequera D., & Torres-Aleman, I. (2005). Choroid plexus megalin is involved in neuroprote ction by serum insulinlike growth factor I. J Neurosci, 25 (47), 10884-10893. Carroll, M. C. (2008). Comple ment and humoral immunity. Vaccine, 26 Suppl 8 I28-33. Charo, I. F., & Peters, W. (2003). Chemokine receptor 2 (CCR2) in atherosclerosis, infectious diseases, and regul ation of T-cell polarization. Microcirculation, 10(34), 259-264. Chauhan, A. K., & Moore, T. L. (2006). Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulatin g immune complexes (CIC). Clin Exp Immunol, 145 (3), 398-406. Choucair-Jaafar, N., Laporte, V., Levy, R., Poindron, P., Lombard, Y., & Gies, J. P. (2010). Complement receptor 3 (CD11b/CD18) is implicated in the elimination of beta-amyloid peptides. Fundam Clin Pharmacol Chun, J. T., Wang, L., Pasinetti, G. M., Finch, C. E., & Zlokovic, B. V. (1999). Glycoprotein 330/megalin (LRP-2) has lo w prevalence as mRNA and protein in brain microvessels and choroid plexus. Exp Neurol, 157 (1), 194-201. Conboy, L., Varea, E., Castro, J. E., Sakouhi-Oue rtatani, H., Calandra, T., Lashuel, H. A., et al. (2010). Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell pro liferation and in anxiety, depression, and me mory-related behaviors. Mol Psychiatry Corti, S., Locatelli, F., Donadoni, C., Guglieri M., Papadimitriou, D., Strazzer, S., et al. (2004). Wild-type bone marrow cells ame liorate the phenotype of SOD1-G93A ALS mice and contribute to CNS, h eart and skeletal muscle tissues. Brain, 127(Pt 11), 2518-2532. Dawson, J., Miltz, W., Mir, A. K., & Wiessner, C. (2003). Targeting monocyte chemoattractant protein-1 signalling in disease. Expert Opin Ther Targets, 7 (1), 35-48. DeMattos, R. B., Cirrito, J. R., Parsadanian, M ., May, P. C., O'Dell, M. A., Taylor, J. W., et al. (2004). ApoE and clusterin coop eratively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo. Neuron, 41(2), 193-202. DeMattos, R. B., O'Dell M, A., Parsadanian, M., Taylor, J. W., Harmony, J. A., Bales, K. R., et al. (2002). Clusterin promotes amyl oid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A, 99(16), 10843-10848. Eglitis, M. A., & Mezey, E. (1997). Hematopoie tic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci U S A, 94 (8), 40804085.

PAGE 89

79 Eikelenboom, P., Hack, C. E., Rozemuller, J. M., & Stam, F. C. (1989). Complement activation in amyloid plaque s in Alzheimer's dementia. Virchows Arch B Cell Pathol Incl Mol Pathol, 56 (4), 259-262. Eikelenboom, P., & Stam, F. C. (1982). Im munoglobulins and complement factors in senile plaques. An immunoperoxidase study. Acta Neuropathol, 57(2-3), 239-242. El Khoury, J., Toft, M., Hickman, S. E., Mean s, T. K., Terada, K., Geula, C., et al. (2007). Ccr2 deficiency impairs mi croglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med, 13 (4), 432-438. El Khoury, J. B., Moore, K. J., Means, T. K., Leung, J., Terada, K., Toft, M., et al. (2003). CD36 mediates the innate ho st response to beta-amyloid. J Exp Med, 197(12), 1657-1666. Emanuele, E., Peros, E., Scioli, G. A., D'A ngelo, A., Olivieri, C., Montagna, L., et al. (2004). Plasma osteoprotegerin as a biochemical marker for vascular dementia and Alzheimer's disease. Int J Mol Med, 13 (6), 849-853. Emerson, S. G., Sieff, C. A., Wang, E. A., Wong, G. G., Clark, S. C., & Nathan, D. G. (1985). Purification of fetal hematopoietic progenitors and demonstration of recombinant multipotential colony-stimulating activity. J Clin Invest, 76 (3), 12861290. Fahrenholz, F., & Postina, R. (2006). Alpha -secretase activation--an approach to Alzheimer's disease therapy. Neurodegener Dis, 3 (4-5), 255-261. Flex, A., Pola, R., Serricchio, M., Gaetani, E., Proia, A. S., Di Gior gio, A., et al. (2004). Polymorphisms of the macrophage inhibito ry factor and C-reactive protein genes in subjects with Alzheimer's dementia. Dement Geriatr Cogn Disord, 18 (3-4), 261-264. Flier, J. S., Cook, K. S., Usher, P., & Sp iegelman, B. M. (1987). Severely impaired adipsin expression in genetic and acquired obesity. Science, 237 (4813), 405-408. Fonseca, M. I., Ager, R. R., Chu, S. H., Yazan, O., Sanderson, S. D., LaFerla, F. M., et al. (2009). Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease. J Immunol, 183(2), 1375-1383. Galimberti, D., Fenoglio, C., Lovati, C., Venturel li, E., Guidi, I., Corra B., et al. (2006). Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer's disease. Neurobiol Aging, 27 (12), 1763-1768. Garrett, M. C., Otten, M. L., Star ke, R. M., Komotar, R. J., Ma gotti, P., Lambris, J. D., et al. (2009). Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage. Brain Res, 1298 171-177. Gerard, C. (2003). Complement C5a in the sepsis syndrome--too much of a good thing? N Engl J Med, 348 (2), 167-169. Guo, R. F., & Ward, P. A. (2005). Role of C5a in inflammatory responses. Annu Rev Immunol, 23, 821-852. Haga, S., Ikeda, K., Sato, M., & Ishii, T. (1993). Synthetic Alzh eimer amyloid beta/A4 peptides enhance production of compleme nt C3 component by cultured microglial cells. Brain Res, 601 (1-2), 88-94. Hamilton, G., Proitsi, P., Williams, J., O'Donovan, M., Owen, M., Powell, J., et al. (2007). Complement factor H Y402H polymor phism is not associated with lateonset Alzheimer's disease. Neuromolecular Med, 9 (4), 331-334.

PAGE 90

80 Hammad, S. M., Ranganathan, S., Loukinova, E., Twal, W. O., & Argraves, W. S. (1997). Interaction of apolipoprotein Jamyloid beta-peptide complex with low density lipoprotein receptor-related prot ein-2/megalin. A mechanism to prevent pathological accumulation of amyloid beta-peptide. J Biol Chem, 272 (30), 1864418649. Harhausen, D., Khojasteh, U., Stahel, P. F., Mo rgan, B. P., Nietfeld, W., Dirnagl, U., et al. (2010). Membrane attack complex i nhibitor CD59a prot ects against focal cerebral ischemia in mice. J Neuroinflammation, 7 15. Heese, K., Hock, C., & Otten, U. (1998). In flammatory signals induce neurotrophin expression in human microglial cells. J Neurochem, 70 (2), 699-707. Helmy, K. Y., Katschke, K. J., Jr., Gorgani, N. N., Kljavin, N. M., Elliott, J. M., Diehl, L., et al. (2006). CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell, 124(5), 915-927. Hess, D. C., Hill, W. D., Martin-Studdard, A ., Carroll, J., Brailer, J., & Carothers, J. (2002). Bone marrow as a source of endot helial cells and Ne uN-expressing cells After stroke. Stroke, 33(5), 1362-1368. Hickman, S. E., Allison, E. K., & El K houry, J. (2008). Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci, 28 (33), 8354-8360. Hinds, J. W. (1968). Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. J Comp Neurol, 134 (3), 287-304. Holt, D. S., Botto, M., Bygrave, A. E., Hanna, S. M., Walport, M. J., & Morgan, B. P. (2001). Targeted deletion of the CD59 gene causes spontane ous intravascular hemolysis and hemoglobinuria. Blood, 98 (2), 442-449. Holtzman, D. M. (2004). In vivo effects of ApoE and clusterin on amyloid-beta metabolism and neuropathology. J Mol Neurosci, 23(3), 247-254. Holtzman, D. M., Bales, K. R., Wu, S., Bhat, P., Parsadanian, M., Fagan, A. M., et al. (1999). Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease. J Clin Invest, 103 (6), R15-R21. Hye, A., Lynham, S., Thambisetty, M., Causevic M., Campbell, J., Byers, H. L., et al. (2006). Proteome-based plasma biomarkers for Alzheimer's disease. Brain, 129(Pt 11), 3042-3050. Ingram, G., Hakobyan, S., Robertson, N. P., & Morgan, B. P. (2010). Elevated plasma C4a levels in multiple sclerosis correlate with disease activity. J Neuroimmunol, 223(1-2), 124-127. Ishii, T., & Haga, S. (1984). Immuno-electro n-microscopic localization of complements in amyloid fibrils of senile plaques. Acta Neuropathol, 63 (4), 296-300. Ishizuka, K., Kimura, T., Igata-yi, R., Kats uragi, S., Takamatsu, J., & Miyakawa, T. (1997). Identification of monocyte chemoattr actant protein-1 in senile plaques and reactive microglia of Alzheimer's disease. Psychiatry Clin Neurosci, 51 (3), 135138. Itagaki, S., Akiyama, H., Sa ito, H., & McGeer, P. L. (1994) Ultrastructura l localization of complement membrane attack comple x (MAC)-like immunoreactivity in brains of patients with Alzheimer's disease. Brain Res, 645 (1-2), 78-84.

PAGE 91

81 Izikson, L., Klein, R. S., Charo, I. F., Weiner, H. L., & Luster, A. D. (2000). Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J Exp Med, 192(7), 1075-1080. Janssen, B. J., Gomes, L., Koning, R. I., Svergun, D. I., Koster, A. J., Fritzinger, D. C., et al. (2009). Insights into complement conve rtase formation based on the structure of the factor B-cobra venom factor complex. Embo J, 28(16), 2469-2478. Jayasekera, J. P., Moseman, E. A., & Carroll, M. C. (2007). Natural antibody and complement mediate neutralization of in fluenza virus in the absence of prior immunity. J Virol, 81(7), 3487-3494. Jiang, H., Burdick, D., Glabe, C. G., Cotma n, C. W., & Tenner, A. J. (1994). betaAmyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain. J Immunol, 152 (10), 5050-5059. Kanning, K. C., Hudson, M., Amieux, P. S., W iley, J. C., Bothwell, M., & Schecterson, L. C. (2003). Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-terminal fr agments with signaling capability. J Neurosci, 23(13), 5425-5436. Kawaji, H., Yokomura, K., Kikuchi, K., So moto, Y., & Shirai, Y. (1995). [Macrophage colony-stimulating factor in patie nts with rheumatoid arthritis]. Nippon Ika Daigaku Zasshi, 62 (3), 260-270. Keshet, G. I., Tolwani, R. J., Trejo, A., Kraft, P., Doyonnas, R., Clayberger, C., et al. (2007). Increased host neuronal survival and motor function in BMT Parkinsonian mice: involvement of immunosuppression. J Comp Neurol, 504 (6), 690-701. Kitamura, T., Miyake, T., & Fujita, S. (1984). Genesis of resting microglia in the gray matter of mouse hippocampus. J Comp Neurol, 226 (3), 421-433. Krishnan, V., Xu, Y., Macon, K., Volanakis, J. E., & Narayana, S. V. (2009). The structure of C2b, a fragment of comple ment component C2 produced during C3 convertase formation. Acta Crystallogr D Biol Crystallogr, 65(Pt 3), 266-274. Kuo, Y. M., Kokjohn, T. A., Kalback, W., Luehrs, D., Galasko, D. R., Chevallier, N., et al. (2000). Amyloid-beta peptid es interact with plasma proteins and erythrocytes: implications for their quantitation in plasma. Biochem Biophys Res Commun, 268(3), 750-756. Kuraya, M., Matsushita, M., Endo, Y., Thiel, S., & Fujita, T. (2003). Expression of Hficolin/Hakata antigen, mannose-binding le ctin-associated serine protease (MASP)-1 and MASP-3 by huma n glioma cell line T98G. Int Immunol, 15 (1), 109-117. Lambert, J. C., & Amouyel, P. (2007). Gene tic heterogeneity of Alzheimer's disease: complexity and advances. Psychoneuroendocrinology, 32 Suppl 1 S62-70. Lambert, J. C., Heath, S., Even, G., Campi on, D., Sleegers, K., Hiltunen, M., et al. (2009). Genome-wide association study id entifies variants at CLU and CR1 associated with Alzheimer's disease. Nat Genet, 41 (10), 1094-1099. Langlois, P. F., Sharon, G. E., & Gawryl, M. S. (1989). Plasma concentrations of complement-activation complexes correlate with disease activity in patients diagnosed with isolated central nervous system vasculitis. J Allergy Clin Immunol, 83(1), 11-16.

PAGE 92

82 Lanzrein, A. S., Jobst, K. A., Thiel, S., Jensen ius, J. C., Sim, R. B., Perry, V. H., et al. (1998). Mannan-binding lectin in human serum, cerebrospinal fluid and brain tissue and its role in Alzheimer's disease. Neuroreport, 9(7), 1491-1495. Lashuel, H. A., Aljabari, B., Sigurdsson, E. M., Metz, C. N., Leng, L., Callaway, D. J., et al. (2005). Amyloid fibril formation by m acrophage migration inhibitory factor. Biochem Biophys Res Commun, 338 (2), 973-980. Lewis, P. D. (1968). The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia. Brain, 91(4), 721-736. Ling, E. A., & Wong, W. C. (1993). The origin and nature of ramified and amoeboid microglia: a historical revi ew and current concepts. Glia, 7(1), 9-18. Lois, C., & Alvarez-Buylla, A. (1994). Longdistance neuronal migration in the adult mammalian brain. Science, 264 (5162), 1145-1148. Lopez-Lopez, C., Dietrich, M. O., Metzger, F., Loetscher, H., & Torres-Aleman, I. (2007). Disturbed cross talk between in sulin-like growth factor I and AMPactivated protein kinase as a possible cause of vascular dysfunction in the amyloid precursor protein/presenilin 2 mouse model of Alzheimer's disease. J Neurosci, 27(4), 824-831. Lu, J., Wiedemann, H., Timpl, R., & Reid, K. B. (1993). Similarity in structure between C1q and the collectins as judged by electron microscopy. Behring Inst Mitt (93), 616. Lu, J. H., Thiel, S., Wiedemann, H., Timpl, R., & Reid, K. B. (1990). Binding of the pentamer/hexamer forms of mannan-bindi ng protein to zymosan activates the proenzyme C1r2C1s2 complex, of the clas sical pathway of complement, without involvement of C1q. J Immunol, 144 (6), 2287-2294. Lupien, S. B., Bluhm, E. J., & Ishii, D. N. (2003). Systemic insulin-like growth factor-I administration prevents cognitive impair ment in diabetic rats, and brain IGF regulates learning/memory in normal adult rats. J Neurosci Res, 74 (4), 512-523. Lupien, S. B., Bluhm, E. J., & Ishii, D. N. (2006). Effect of IGF-I on DNA, RNA, and protein loss associated with brain atrophy and impaired learning in diabetic rats. Neurobiol Dis, 21(3), 487-495. Luskin, M. B. (1993). Restricted prolifera tion and migration of postnatally generated neurons derived from the fo rebrain subventricular zone. Neuron, 11(1), 173-189. Ma, J., Brewer, H. B., Jr., & Potter, H. (1996). Alzheimer A beta neurotoxicity: promotion by antichymotrypsin, ApoE4; inhibition by A beta-related peptides. Neurobiol Aging, 17(5), 773-780. Ma, J., Yee, A., Brewer, H. B., Jr., Das, S ., & Potter, H. (1994). Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature, 372(6501), 92-94. Malm, T. M., Koistinaho, M., Parepalo, M., Vatanen, T., Ooka, A., Karlsson, S., et al. (2005). Bone-marrow-derived cells contri bute to the recruitm ent of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol Dis, 18 (1), 134-142. Matsushita, M. (2009). Ficolins: complement-activating lectins involved in innate immunity. J Innate Immun, 2 (1), 24-32. May, P. C., & Finch, C. E. (1992). Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration. Trends Neurosci, 15 (10), 391-396.

PAGE 93

83 May, P. C., Lampert-Etchells, M., Johnson, S. A ., Poirier, J., Masters, J. N., & Finch, C. E. (1990). Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat. Neuron, 5(6), 831-839. McGeer, P. L., Akiyama, H., Itagaki, S., & McGeer, E. G. (1989). Activation of the classical complement pathway in br ain tissue of Alzheimer patients. Neurosci Lett, 107(1-3), 341-346. McGeer, P. L., Kawamata, T., & Walker, D. G. (1992). Distribution of clusterin in Alzheimer brain tissue. Brain Res, 579 (2), 337-341. McGeer, P. L., Walker, D. G., Akiyama, H., Ka wamata, T., Guan, A. L., Parker, C. J., et al. (1991). Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain. Brain Res, 544 (2), 315-319. McKercher, S. R., Torbett, B. E., Anders on, K. L., Henkel, G. W., Vestal, D. J., Baribault, H., et al. (1996). Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. Embo J, 15(20), 5647-5658. Mezey, E., & Chandross, K. J. (2000). Bone marrow: a possible alternative source of cells in the adult nervous system. Eur J Pharmacol, 405 (1-3), 297-302. Mezey, E., Chandross, K. J., Harta, G., Maki R. A., & McKercher, S. R. (2000). Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 290 (5497), 1779-1782. Miao, J., Xu, F., Davis, J., Otte-Holler, I., Verbeek, M. M., & Van Nostrand, W. E. (2005). Cerebral microvascul ar amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein. Am J Pathol, 167 (2), 505515. Moser, K. V., & Humpel, C. (2005). Vascular endothelial growth factor counteracts NMDA-induced cell death of adult choliner gic neurons in rat basal nucleus of Meynert. Brain Res Bull, 65 (2), 125-131. Murphy, B. F., Davies, D. J., Morrow, W., & d'Apice, A. J. (1989). Localization of terminal complement components S-pr otein and SP-40,40 in renal biopsies. Pathology, 21 (4), 275-278. Nakamura, H., Ueki, Y., Sakito, S., Matsumot o, K., Yano, M., Miyake, S., et al. (2000). High serum and synovial fluid granulocyt e colony stimulating factor (G-CSF) concentrations in patients with rheumatoid arthritis. Clin Exp Rheumatol, 18 (6), 713-718. Neth, O., Jack, D. L., Dodds, A. W., Holzel H., Klein, N. J., & Turner, M. W. (2000). Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun, 68 (2), 688-693. Neuhaus, J., & Fedoroff, S. (1994). Developm ent of microglia in mouse neopallial cell cultures. Glia, 11(1), 11-17. Olszewski, W. L., Pazdur, J., Kubasiewicz, E ., Zaleska, M., Cooke, C. J., & Miller, N. E. (2001). Lymph draining from foot joints in rheumatoid arthritis provides insight into local cytokine and chemokine pr oduction and transport to lymph nodes. Arthritis Rheum, 44(3), 541-549. Onodera, S., Ohshima, S., Tohyama, H., Yasuda K., Nishihira, J., Iwakura, Y., et al. (2007). A novel DNA vaccine targeting macr ophage migration inhibitory factor

PAGE 94

84 protects joints from inflammation and dest ruction in murine m odels of arthritis. Arthritis Rheum, 56(2), 521-530. Perry, V. H., & Gordon, S. (1988). Macrophage s and microglia in the nervous system. Trends Neurosci, 11 (6), 273-277. Podack, E. R., Kolb, W. P., & Muller-Ebe rhard, H. J. (1977). The SC5b-7 complex: formation, isolation, properties, and subunit composition. J Immunol, 119 (6), 2024-2029. Podack, E. R., & Muller-Eberhard, H. J. (1978). Binding of desoxycholate, phosphatidylcholine vesicles, lipoprotein a nd of the S-protein to complexes of terminal complement components. J Immunol, 121 (3), 1025-1030. Popa, C., van Lieshout, A. W., Roelofs, M. F., Geurts-Moespot, A., van Riel, P. L., Calandra, T., et al. (2006). MIF production by dendritic cells is differentially regulated by Toll-like receptors and in creased during rheumatoid arthritis. Cytokine, 36 (1-2), 51-56. Popp, J., Bacher, M., Kolsch, H., Noelker, C ., Deuster, O., Dodel, R., et al. (2009). Macrophage migration inhibitory factor in mild cognitive impairment and Alzheimer's disease. J Psychiatr Res, 43 (8), 749-753. Postina, R., Schroeder, A., Dewachter, I., B ohl, J., Schmitt, U., Kojro, E., et al. (2004). A disintegrin-metalloproteinase preven ts amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest, 113(10), 1456-1464. Potter, H., Wefes, I. M., & Nilsson, L. N. (2001). The inflammation-induced pathological chaperones ACT and apo-E are necessary catalysts of Alzheimer amyloid formation. Neurobiol Aging, 22 (6), 923-930. Preissner, K. T. (1989). The role of vitr onectin as multifunctional regulator in the hemostatic and immune systems. Blut, 59 (5), 419-431. Priller, J., Persons, D. A., Klett, F. F., Ke mpermann, G., Kreutzberg, G. W., & Dirnagl, U. (2001). Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo. J Cell Biol, 155 (5), 733-738. Rivera, E. J., Goldin, A., Fulmer, N., Tavares, R., Wands, J. R., & de la Monte, S. M. (2005). Insulin and insulin-like growth f actor expression and function deteriorate with progression of Alzheimer's disease: link to brain reductions in acetylcholine. J Alzheimers Dis, 8 (3), 247-268. Rogers, J., Li, R., Mastroeni, D., Grover, A., Leonard, B., Ahern, G., et al. (2006). Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes. Neurobiol Aging, 27(12), 1733-1739. Rogers, J., Schultz, J., Brachova, L., Lue, L. F., Webster, S., Bradt, B., et al. (1992). Complement activation and beta-amyloid-m ediated neurotoxicity in Alzheimer's disease. Res Immunol, 143 (6), 624-630. Roheim, P. S., Carey, M., Forte, T., & Vega, G. L. (1979). Apolipoproteins in human cerebrospinal fluid. Proc Natl Acad Sci U S A, 76(9), 4646-4649. Roitt IM, B. J., Male DK. (1998). Immunology (5th ed.). London, UK: Mosby International Ltd. Rostagno, A., Revesz, T., Lashley, T., Tomi dokoro, Y., Magnotti, L., Braendgaard, H., et al. (2002). Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease. J Biol Chem, 277 (51), 49782-49790.

PAGE 95

85 Rupert, K. L., Moulds, J. M., Yang, Y., Arnett, F. C., Warren, R. W., Reveille, J. D., et al. (2002). The molecular basis of complete complement C4A and C4B deficiencies in a systemic lupus erythe matosus patient with homozygous C4A and C4B mutant genes. J Immunol, 169 (3), 1570-1578. Rynkowski, M. A., Kim, G. H., Garrett, M. C., Zacharia, B. E., Otten, M. L., Sosunov, S. A., et al. (2009). C3a receptor antagonist attenuates brain injury after intracerebral hemorrhage. J Cereb Blood Flow Metab, 29 (1), 98-107. Salasznyk, R. M., Zappala, M., Zheng, M., Yu, L., Wilkins-Port, C., & McKeownLongo, P. J. (2007). The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells. Matrix Biol, 26(5), 359-370. Sanchez-Ramos, J., Song, S., Sava, V., Catlow, B., Lin, X., Mori, T., et al. (2009). Granulocyte colony stimulating factor decreases brain amyloid burden and reverses cognitive impairment in Alzheimer's mice. Neuroscience, 163 (1), 55-72. Schultheiss, C., Blechert, B., Gaertner, F. C., Dr ecoll, E., Mueller, J., Weber, G. F., et al. (2006). In vivo characterization of endothelial cell activation in a transgenic mouse model of Alzheimer's disease. Angiogenesis, 9 (2), 59-65. Searfoss, G. H., Jordan, W. H., Calligaro, D. O., Galbreath, E. J., Schirtzinger, L. M., Berridge, B. R., et al. (2003). Adipsin, a bi omarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor. J Biol Chem, 278 (46), 46107-46116. Sheehan, M., Morris, C. A., Pussell, B. A., & Charlesworth, J. A. (1995). Complement inhibition by human vitronectin invo lves non-heparin binding domains. Clin Exp Immunol, 101(1), 136-141. Shin, T. M., Isas, J. M., Hsieh, C. L., Kayed, R., Glabe, C. G., Langen, R., et al. (2008). Formation of soluble amyloid olig omers and amyloid fibrils by the multifunctional protein vitronectin. Mol Neurodegener, 3 16. Simard, A. R., & Rivest, S. (2004). Bone ma rrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J, 18(9), 998-1000. Simard, A. R., Soulet, D., Gowing, G., Julie n, J. P., & Rivest, S. (2006). Bone marrowderived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron, 49 (4), 489-502. Smits, H. A., Rijsmus, A., van Loon, J. H., Wat, J. W., Verhoef, J., Boven, L. A., et al. (2002). Amyloid-beta-induced chemokine production in primary human macrophages and astrocytes. J Neuroimmunol, 127 (1-2), 160-168. Sokolova, A., Hill, M. D., Rahimi, F., Warden, L. A., Halliday, G. M., & Shepherd, C. E. (2009). Monocyte chemoattractant protein1 plays a dominant role in the chronic inflammation observed in Alzheimer's disease. Brain Pathol, 19 (3), 392-398. Stahel, P. F., Flierl, M. A., Morgan, B. P., Persigehl, I., Stoll, C., Conrad, C., et al. (2009). Absence of the complement re gulatory molecule CD59a leads to exacerbated neuropathology after trau matic brain injury in mice. J Neuroinflammation, 6, 2. Stoltzner, S. E., Grenfell, T. J., Mori, C., Wisniewski, K. E., Wisniewski, T. M., Selkoe, D. J., et al. (2000). Temporal accrual of complement proteins in amyloid plaques in Down's syndrome with Alzheimer's disease. Am J Pathol, 156 (2), 489-499.

PAGE 96

86 Strang, C. J., Cholin, S., Spragg, J., Davis, A. E., 3rd, Schneeberger, E. E., Donaldson, V. H., et al. (1988). Angioedema induced by a peptide derived from complement component C2. J Exp Med, 168(5), 1685-1698. Strohmeyer, R., Ramirez, M., Cole, G. J., Mu eller, K., & Rogers, J. (2002). Association of factor H of the alterna tive pathway of complement with agrin and complement receptor 3 in the Alzheimer's disease brain. J Neuroimmunol, 131 (1-2), 135-146. Strohmeyer, R., Shen, Y., & Rogers, J. ( 2000). Detection of comp lement alternative pathway mRNA and proteins in the Alzheimer's disease brain. Brain Res Mol Brain Res, 81 (1-2), 7-18. Svensson, J., Diez, M., Engel, J., Wass, C., Tivesten, A., Jansson, J. O., et al. (2006). Endocrine, liver-derived IGF -I is of importance for spatial learning and memory in old mice. J Endocrinol, 189 (3), 617-627. Szeplaki, G., Szegedi, R., Hirschberg, K., Gomb os, T., Varga, L., Karadi, I., et al. (2009). Strong complement activation after acute ischemic stroke is associated with unfavorable outcomes. Atherosclerosis, 204(1), 315-320. Tacnet-Delorme, P., Chevallier, S., & Arlaud, G. J. (2001). Beta-amyloid fibrils activate the C1 complex of complement under physiological conditions: evidence for a binding site for A beta on the C1q globular regions. J Immunol, 167 (11), 63746381. Tegla, C. A., Cudrici, C., Rus, V., Ito, T., Vlaicu, S., Singh, A., et al. (2009). Neuroprotective effects of the comp lement terminal pathway during demyelination: implications for oligodendrocyte survival. J Neuroimmunol, 213(1-2), 3-11. Theele, D. P., & Streit, W. J. (1993) A chronicle of mi croglial ontogeny. Glia, 7(1), 5-8. Tondravi, M. M., McKercher, S. R., Anders on, K., Erdmann, J. M., Quiroz, M., Maki, R., et al. (1997). Osteopetrosis in mice lack ing haematopoietic transcription factor PU.1. Nature, 386(6620), 81-84. Trouw, L. A., Nielsen, H. M., Minthon, L., L ondos, E., Landberg, G., Veerhuis, R., et al. (2008). C4b-binding protein in Alzheimer's disease: binding to Abeta1-42 and to dead cells. Mol Immunol, 45(13), 3649-3660. Tschopp, J., Chonn, A., Hertig, S., & French, L. E. (1993). Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9. J Immunol, 151 (4), 2159-2165. Uemura, K., Kuzuya, A., Aoyagi, N., Ando, K., Shimozono, Y., Ninomiya, H., et al. (2007). Amyloid beta inhi bits ectodomain shedding of N-cadherin via downregulation of cell-surface NMDA receptor. Neuroscience, 145(1), 5-10. Van Beek, J., Bernaudin, M., Petit, E., Gasque, P., Nouvelot, A., MacKenzie, E. T., et al. (2000). Expression of receptors for co mplement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Exp Neurol, 161 (1), 373-382. van Beek, J., Nicole, O., Ali, C., Ischenko, A., MacKenzie, E. T., Buisson, A., et al. (2001). Complement anaphylatoxin C3a is selectively protective against NMDAinduced neuronal cell death. Neuroreport, 12(2), 289-293. van der Voort, R., van Lieshout, A. W., Toone n, L. W., Sloetjes, A. W., van den Berg, W. B., Figdor, C. G., et al. (2005). Elevated CXCL16 expression by synovial

PAGE 97

87 macrophages recruits memory T cells into rheumatoid joints. Arthritis Rheum, 52(5), 1381-1391. van Lieshout, A. W., Popa, C., Meyer-Wentrup, F., Lemmers, H. L., Stalenhoef, A. F., Adema, G. J., et al. (2007). Circulati ng CXCL16 is not related to circulating oxLDL in patients with rheumatoid arthritis. Biochem Biophys Res Commun, 355(2), 392-397. Veerhuis, R., Janssen, I., Hack, C. E., & Eikelenboom, P. (1996) Early complement components in Alzheimer's disease brains. Acta Neuropathol, 91 (1), 53-60. Walker, D. G., Yasuhara, O., Patston, P. A ., McGeer, E. G., & McGeer, P. L. (1995). Complement C1 inhibitor is produced by br ain tissue and is cleaved in Alzheimer disease. Brain Res, 675 (1-2), 75-82. Webster, S., Bradt, B., Rogers, J., & Cooper, N. (1997). Aggregation state-dependent activation of the classical complement pathway by the amyloid beta peptide. J Neurochem, 69(1), 388-398. Wiesmann, C., Katschke, K. J., Yin, J., Helmy, K. Y., Steffek, M., Fairbrother, W. J., et al. (2006). Structure of C3b in complex with CRIg gives insights into regulation of complement activation. Nature, 444(7116), 217-220. Wilcox-Adelman, S. A., Wilkins-Port, C. E., & McKeown-Longo, P. J. (2000). Localization of urokinase type plasminoge n activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell Adhes Commun, 7 (6), 477-490. Wu, H., Liu, X., Jaenisch, R., & Lodish, H. F. (1995). Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell, 83 (1), 59-67. Wyss-Coray, T., Yan, F., Lin, A. H., Lambris, J. D., Alexander, J. J., Quigg, R. J., et al. (2002). Prominent neurodegeneration a nd increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci U S A, 99 (16), 10837-10842. Xu, W. D., Firestein, G. S ., Taetle, R., Kaushansky, K., & Zvaifler, N. J. (1989). Cytokines in chronic inflammatory arth ritis. II. Granulocyte-macrophage colonystimulating factor in rheu matoid synovial effusions. J Clin Invest, 83 (3), 876-882. Yang, L. B., Li, R., Meri, S., Rogers, J., & Shen, Y. (2000). Deficiency of complement defense protein CD59 may contribute to neurodegeneration in Alzheimer's disease. J Neurosci, 20 (20), 7505-7509. Yang, S. P., Kwon, B. O., Gho, Y. S., & Chae, C. B. (2005). Specific interaction of VEGF165 with beta-amyloid, and its prot ective effect on beta-amyloid-induced neurotoxicity. J Neurochem, 93 (1), 118-127. Yasojima, K., McGeer, E. G., & McGeer, P. L. (1999). Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease. Brain Res, 833 (2), 297-301. Yasuhara, O., Aimi, Y., McGeer, E. G., & McGeer, P. L. (1994). Expression of the complement membrane attack complex and its inhibitors in Pick disease brain. Brain Res, 652 (2), 346-349. Zanjani, H., Finch, C. E., Kemper, C., Atki nson, J., McKeel, D., Morris, J. C., et al. (2005). Complement activation in very early Alzheimer disease. Alzheimer Dis Assoc Disord, 19 (2), 55-66.

PAGE 98

88 Zhou, J., Fonseca, M. I., Pisalyaput, K., & Tenner, A. J. (2008). Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease. J Neurochem, 106(5), 2080-2092. Zlokovic, B. V., Martel, C. L., Matsubara, E ., McComb, J. G., Zheng, G., McCluskey, R. T., et al. (1996). Glycoprot ein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone an d in a complex with Alzheimer disease amyloid beta at the blood-brain a nd blood-cerebrospinal fluid barriers. Proc Natl Acad Sci U S A, 93(9), 4229-4234.

PAGE 99

89 CHAPTER 3 DEVELOPMENT OF BILATERAL BRAIN INFUSION METHODOLOGY Introduction In 2007, at the start of the experimental investigation into whether or not the hematopoietic colony stimulating factors (C SFs) would affect AD pathology in mice, several reports suggested that M-CSF might be the most promising CSF to reverse amyloidosis. For instance, it was reported th at osteopetrotic (op/op) mice, that do not produce functional M-CSF, developed amyl oid plaques in their brain, and had neurodegeneration (Kaku et al., 2003; Kawata et al., 2005). When 30 day old op/op mice were intracranially-injected with M-CSF, th ey had reduced brain plaques, even though their microglial populations only increased to tw o-thirds that of wild -type mice (Kaku et al., 2003). Furthermore, in vitro studies showed that M-CSF was induced in A -treated microglia cells (Ito et al., 2005) suggesting that the brains A -activated microglial might secrete hematopoietic growth factors in an attempt to increase phagocytic microglial populations. Additionally, microgl ia that overexpressed the receptor for M-CSF were reported to be neuroprotective (Mitrasinovic et al., 2005). However, there were no in vivo reports that had administered M-C SF into the CNS. Since a proposed endpoint of our study was to show an altera tion in cerebral amyloidosis, due to M-CSF administration, we wanted an adequate amount of M-CSF delivered. Thus we decided to use a novel bilateral brain infusion methodol ogy (Bennett et al., 2009), that we were developing in 2007, to continuously infuse M-C SF into the lateral ventricles of aged AD mice. This chapter explains the construction of the bilateral brain infusion catheters, and

PAGE 100

90 the results from the usage of these cathet ers in transgenic AD mice with infusion of alpha-1-antichymotrypsin (ACT) or M-CSF. Available Commercial Catheters The only commercially-available infusi on system for delivering drugs or investigational materials in vivo is the Alzet pump system. There are certain characteristics of this system that make it unusable for our experiment. For instance, since the average mouses skull measures be tween 8-9mm in width, there can physically be only one catheter placed into the test s ubjects brain due to th e 5.9mm diameter round catheter head (Figure 4). Additionally, the thickness of the catheter head does not allow for the scalp to be completely sutured closed, without tightness of the scalp and subsequent chronic irritation to the mouse durin g the trial period, in which they will rub it against their cage or scratch at it. There will also be chronic inflammation and propensity for infection, due to the s calp not closing appropriatel y. Moreover, the tubing that connects to the catheter head of ten lies partially outside of the skin, allowing the mouse to sometimes scratch it loose and compromise the experiment. Furthermore, the catheter head is flat and does not contour well to th e skull, requiring a larg e amount of cement to be applied to secure the catheter head to th e skull. Thus, the cure time for the cement causes the mouse to remain long times under anaesthesia, and contribute to increased surgery mortalities. These issues combine to make the commercial Alzet catheters not ideal for our experimentation, especially sin ce both ACT and M-CSF are mediators of the immune system, and their effects ma y be skewed throughout the trial.

PAGE 101

91 Figure 4. Commercial Alzet catheter The 5.9mm diameter round catheter head glues to the skull of th e test animal and has the 3.0mm stainless tubing extending through a drilled hole in the skull and into the brains parenchyma. There are 0.5mm washer-like plastic spacers that can be added underneath the catheter head to reduce the distance that the tubing penetrates the brain. The 3.4mm pedestal, used for mechanical placement of catheter, can be clipped off after the glue cures.Bilateral Brain Infusion Catheter Construction The catheters (patent pendingPCT/US08/ 73974) used for this experiment were made by first taking a 1.25 cm length of 30 gauge stainless steel tubing (Small Parts, Inc. Miramar, Fl) and carefully bending it at 2. 5 mm under a dissecting microscope (Leica, Heerbruug, Switzerland), to approximately 90 degrees, being careful not to crimp the tube. The remaining length of the tube was bent again at 5 mm to an angle of 120-160 degrees, approximating the contour of each animals skull. These metal cannulae were inserted into a 3 cm length of polyethylene (P E-10) tubing with an internal diameter of 0.28 mm and an outer diameter of 0.61 mm. One centimeter of the PE-10 was then itself inserted into a 4.5 cm lengt h of sterile polyvinyl tubing (P V-50-I.D. 0.69 mm/ O.D. 1.14 mm, Durect Corp., Cupertino, Ca), held in place using a bead of Locktite 454 adhesive (Plastics One. Roanoke, VA.), and cured overnig ht. The following day, sterile water was forced through each catheter assembly to ensu re that the lines were not obstructed. The osmotic pumps were then filled with solution, attached to the catheters and primed and implanted as described below. The proven ca theters were later custom manufactured by Braintree Scientific (Braintree, MA) with some minor changes: The adhesive was

PAGE 102

92 supplanted by heat sealing PE-10 to PE-50 tubing and, because the diameters of the PV50 and new PE-50 were not exactly the same, a sheath of silicone (SIL 047) was placed over the PE-50 with an extra 2 mm overhang to attach it to the flow modulator of the pump, forming a tight, leak-proof seal (Figure 7). Transgenic Mice PS/APP (presenilin 1/amyloid precursor protein) mice and PS/APP/ACT mice were generated by crossing heterozygous PDGF-hAPP (V71 7F) mice with PDGF-hPS1 (M146L) on both Swiss Webster and C57BL/ 6 backgrounds. In some cases, PS/APP mice were bred with mice harboring a ge ne for human ACT (hACT) under a GFAP promoter (Nilsson et al., 2001) were used to be compared to ACT-infused animals. Genotyping was performed using compara tive real-time PCR (Bio-Rad iCyclerHercules, CA). Pathogenically, these Alzh eimers mouse models are characterized by robust accumulation of amyloid plaques and the development of microgliosis between 610 months. The mice used in these studies ranged from 9-10 months of age. Materials Purified human -1-antichymotrypsin (ACT) was purchased lyophilized from Fitzgerald (Concord, MA) and purified mouse M-CSF was purchased from R&D Systems (Minneapolis, MN). Both were recons tituted in artificial CSF (aCSF) (Harvard Apparatus, Holliston, MA) to a concentration of 1mg/mL. ACT was infused directly into the hippocampal parenchyma or lateral vent ricles for 28 days and M-CSF was infused into the lateral ventricles for 14 days using the novel catheters attached to Alzet osmotic minipumps (Alzet model 1004, Durect Corp. Cupertino, CA) with an average flow rate of

PAGE 103

93 0.12 L/hour. The pumps and catheters were submerged in 0.9% sterile saline at 37oC and primed for 48 hours prior to implantation. Intracranial Infusions All procedures involving experimentation on animals were performed in accordance with the guidelines set forth by the University of South Florida Animal Care and Use Committee. Animals (9 month-old PS/APP25-35g) were anesthetized with 12% isoflurane, shaved and scrubbed with 10% Betadine solution at the site of incision, and placed into a dual arm stereotaxic frame (Kopf Instruments. Tujunga, Ca.). A small (5 cm) incision was made, exposing the skull and neck, and double bladed scissors (10 cm curved Strabismus, Fine Science Tools, Foster City, CA.) were used to form a subcutaneous pocket along the back of the an imal into which 2 osmotic minipumps were inserted with catheters attached. One of th e stereotaxic arms held an Ideal Micro-drill (Roboz Surgical Instrument Co., Gaithers burg, MD) holding a 0.32 mm diameter carbide drill bit. Using the drill bit on the stereota xic arm to find the proper coordinates (from Bregma -2.2-2.5 mm anterior-pos terior, +/2.2-2.5 mm medial -lateral), two holes were carefully drilled into the skull and a 30 ga uge needle from a Hamilton syringe (Hamilton Co., Reno, Nevada) was attached to the second arm of the stereotaxic frame. The needle was inserted to the appropriate depth ( 2.2-2.5 mm corresponding to the posterior portion of the CA1/CA2 hippocampus) and allowed to sit for 5 minutes for the surrounding tissue to adjust. Alternatively, holes were drille d corresponding to the late ral ventricles at the following coordinates: from Bregma -0.2 mm anterior-pos terior, +/1.0 mm mediallateral, and to a depth of 2.2-2.5 mm. After removing the needle, the tips of the cannulae were held directly over the holes with forceps and then gently inserted straight into place.

PAGE 104

94 Pulling back on the catheters from the base of the osmotic pumps while inserting the cannulae provided sheer which prevented th em from moving and potentially damaging tissue while they were firmly affixed. The cannulae were affixed to the skull with Locktite 454 adhesive (Plastics One. Roa noke, VA.) and secured down with a piece of nitrile, approximately 1 cm in diameter. Af ter the adhesive cured, the scalp was closed with 6-7 silk sutures. For analgesia, the an imal received an immediate subcutaneous dose of ketoprofen (10mg/kg) and up to every 6 hours, as needed, for a maximum of 48 hours post-operatively. After the 28 day period, the animals were given an overdose (~150mg/kg) of sodium pentobarb ital (i.p.) and were transcar dially perfused with 0.9% saline. The brains were carefully removed and analyzed as outlined below. At this time, the integrity of the catheters was confirmed again by forcing water through them, and the pumps were also determined to be empty. Histology and Immunohistochemistry Mouse brain tissues were fixed in 10% formaldehyde (formalin) for 72 hours and then passed through a series of sucrose soluti ons (10-30%) over anothe r 72 hours. Brains were frozen to the peltier stage (Physit emp, Clifton, NJ) of a histoslide (Leica, Heerbruug, Switzerland) and s ectioned coronally at 25 m. After incubating the sections with blocking buffer (Tris-buffered saline with 10% normal goat serum, 0.1% Triton X100, and 0.02% sodium azide) for 60 min, primar y antibodies against ACT (1:500Dako, Glostrop, Denmark) and A (6E10,1:1000Covance, Princeton, NJ) were applied and incubated at 4 o C overnight. After thorough washing, th e sections were incubated with secondary antibodies, Alexa 488 and 594 fluor ophores (1:1000, 1:4000-In vitrogen) in the dark at ambient temperature for 2 hours. This incubation was followed by a Hoechst

PAGE 105

95 (1:10,000, Sigma, St. Louis, MO) nuclear stain, and the sections were washed and sealed with Gel-Mount (Electron Microscopy Sciences, Hatfield, PA). The images were analyzed on a Zeiss Imager Z1 fluorescence microscope with a Zeiss Axiocam Mrm camera (Oberkochen, Germany) using Axiovi sion 4.7 software, and ACT sections were quantified using Image J. For animal to animal, hemisphere to hemisphere plaque comparison, 5 sections from 6 mice (30 total) we re stained using Thioflavin S to label the beta-pleated amyloid deposits from anterior to posterior hippocampus in each hemisphere of each mouse. Coronal sections of brain (25m) were mounted to slides and let to adhere overnight. The sections were then rinsed with deionized water and submerged in a 1% Thioflavin S aqueous solution (Sigma-A ldrich, St. Louis, MO) for 5 minutes. The sections were differentiated in 70% ethanol for 5 minutes, rehydrated in 30% ethanol for 5 minutes, washed with deionized wate r coverslipped, image d, and quantified. Results Animal Recovery. Intracranial delivery of mol ecules, though invasive, is a common procedure to confirm resu lts from in vitro experiments in an in vivo system and to test various compounds in transgenic anim als via targeted delivery. As an additional advantage, the novel method described here bypasses concerns related to the blood-brain barrier permeability of the administered compound. ACT, a serine protease inhibitor that is known to contribute to the formation of am yloid pathology in Alzheimer's disease, was infused into the hippocampi or lateral ve ntricles of 9-month-old PS/APP mice for a period of 28 days. ACT was infused into one hemisphere and aCSF (vehicle) into the other hemisphere, thus allowing a comparativ e analysis to be made within the same animal. In all cases, the animals recovered very quickly from the procedure and resumed

PAGE 106

96 eating and grooming within minutes of awaken ing. All animals were monitored closely during the 4 week infusion. In only a few inst ances did animals scratch at the incisions, but in no case were they able to open the wound and remove or damage any of the implanted pumps and catheters. In most cases the hair grew back within 2 weeks, and the animals became virtually indistinguishab le from non-treated animals, with the exception of bulges on the haunches from the implanted osmotic pumps (Figure 9A). The implants caused minimal discomfort to the animals, and procedural mortality was practically eliminated from these studies. When harvesting tissues following the 28 day infusion, the catheters were checked to ensure they were still connected to the pumps (Figure 9B) and to the cannulae (F igure 9C). The nitrile cap is indicated by the arrow in figure 9D. The entire assembly is shown a ttached to an Alzet osmotic minipump model 1004 (Durect Corp., Cupertino, CA.) in Figure 9E. Finally, when removing the brains only 2 small holes are visible (Figure 9F) show ing that there is no lateral movement of the cannulae during infusion. No serious damage or impairment was caused by the cannula, although tissue is disp laced where inserted. Figure 10 shows, on the left hemisphere, exactly where the cannula was placed and that surrounding tissue was intact following infusion. Figure 11 A and B show a region of the hippocampus anterior to the infusion site which also does not show signs of damage. To be noted, the gauge of the cannulae used in this experiment (30g or 0.30 mm O.D.) is the same of those used in commercially available cannulae (Alzet, Plastics One, Braintree Scientific), but without the irritation to the anim al, inflammation, occasional motor impairment, and high mortality rates associated with them.

PAGE 107

97 Bilateral Brain Infusions Due to the increased vari ability in plaque load between animals, even within a largely inbred transgenic mouse line, it is often difficult to compare treated and untreated mice with a reasonably small number of animals. This phenomenon was observed upon staining the M-CSF-infused mice for amyloidosis (Figure 5), and can be caused by phenotypic va riability and/or differential expression of transgenes. Upon analysis of 6 untreated PS/APP mice at 10 months of age, we found that plaque load can vary significantly from animal to animal (75% mean standard deviation in pixel count). This fact makes it difficult to dr aw conclusions on the effects that different molecules might have on amyloid deposition or other features of the transgenic brain when comparing animals wh ere hemispheres vary only about half as much (39%). Figure 6 and Figure 7 demonstrat es the variation in plaque load between hemispheres and within untreated animals of th is genotype. Therefore, in order to study promoters and inhibitors of AD pathology directly in the brain, it would be preferable (almost twice the efficiency) to compare th e individually-treated hemispheres of each animal rather than to compare the brains of littermates or matche d cohorts from other litters. Immunocytochemical techniques demo nstrated that our catheters functioned properly and that ACT was de livered successfully to the ventricles and parenchyma, respectively (Figures 10 and 11). If ACT is infused into a PS/APP mouse that has plaques for it to interact with, it binds to th e plaques and diffuses very little (Figure 11C & D). However, if ACT is delivered in to the hemispheres of nontransgenic mice it diffuses, but still remains in the inte nded hemisphere (Figure 11A and B).

PAGE 108

98 Interaction of ACT and Amyloid. It is well established that ACT is localized, together with the A peptide, in the amyloid plaques of Alzheimers disease (Abraham, Selkoe, & Potter, 1988). However, the mechanis m and effect of this interaction are still under investigation. Previous in vitro expe riments indicated that ACT can bind to A and become integrated into amyloid filaments as they are formed, and the same mechanism was presumed to occur in vi vo (Ma, Brewer, & Potter, 1996; Ma, Yee, Brewer, Das, & Potter, 1994; Mucke et al., 2000). Using the application method described here, we successfully delivered ACT into individual brain hemispheres of adult PS/APP mice and found that ACT binds to the perimeter of existing plaques in a similar manner as found for ACT association with am yloid plaques in PS/APP/ACT transgenic mouse (Nilsson et al., 2001) (F igure 12). While the application of exogeneous ACT did not lead to increased amyloid load during th e time frame of these experiments, it should be noted that ACT was infused for only 28 da ys. A longer exposure could result in an increased plaque load and is currently being investigated. At the same time, while ACT is known to enhance plaque formation, the perimetrical binding of ACT to pre-exisiting A plaques, as shown here, might indicate th at ACT involvement in AD pathogenesis not only occurs concurrent with pl aque formation but also afte rwards. This conclusion is consistent with the fact that ACT is highly upregulated in adult AD brains (Abraham et al., 1988) and also induces the phosphorylation of tau (Padmanabhan, Levy, Dickson, & Potter, 2006). Conclusions. Although there is evidence that AC T's involvement in AD is not restricted to its participation in plaque formation, the purpose of using ACT in this model was to demonstrate the utility of our system. The catheters and cannulae described here

PAGE 109

99 served as an outstanding t ool for targeted delivery of ACT into individual brain hemispheres and will facilitate further examination of the mechanistic involvement of ACT and other molecules in the pathology of Alzheimer's disease. As for the bilateral M-CSF infustions into the lateral ventricles, we were not able to determine whether M-CSF had any affect on amyloidosis due to the significant variati on in plaque load between animals. Nor could we be certai n that the M-CSF extravasated from the ventricles and into the brain s parenchyma. However, the data from the ACT infusions, of which the infused recombinant ACT peptide re mained localized to the hemisphere in which it was delivered, allowed us to form ulate a research plan to give bolus intrahippocampal injections of each C SF (M-CSF, G-CSF, and GM-CSF) into a respective hemisphere and artificial cerebrospinal fluid control into the contralateral hemisphere. The following chapter will discu ss the results from these experiments.

PAGE 110

Figure 5. Significant Variation of Amyloid Plaque Load Between Mice Infused with M-CSF. Standard fluorescent immunohistochemist ry used 6E10/Alexa 488 and Hoechst nuclear stain (blue). Bright green spots indi cate amyloid plaques. Pictures taken at 5X. Mouse numbers 148,160,171, and 211 received bilateral intracerebrove ntricular infusions of M-CSF and mice 164, 170, 176, and 177 rece ived bilateral intracerebroventricular infusions of artificial cerebrospinal fluid. 100

PAGE 111

Figure 6. Significant Variation of Amyloid Plaque Load Between Mice. Thioflavin S staining on 25 m coronal sections (montages) of 3 different PS/APP mice at 10 months of age showing plaque variability in the hippocampus and neighboring cortex. 101

PAGE 112

Figure 7. Scatter-plot of Plaque Load Between Mice. Data from 5 brain sections through 6 ten-month-old PS/APP mice comparing standard deviation (plaque variance) between local hemspheres of the same animal (28%), between sides of the same section (11%), and between animals (78%). All the da ta from the left hemisphere are compared with the right for each mouse (Hemi-Hemi, 1 data point for each mouse); the left half of a single slice is compared wth the right side (L eft-Right, 1 data point for each slice); and a permuted comparison of each mouse with every other mouse (Mouse-Mouse, 1 data point for each pair of mice). Where data were aggregated (HemiHemi and Mouse-Mouse, the mean of the aggregared counts was used to make the standard deviation comparable to the single slice data (standard deviation of two numbers). 102

PAGE 113

Figure 8. Catheter Assembly AFirst bend of 30g stainless steel tubing. BSecond contour bend. Cpolyethylene PE-10 tubing. DHeat seal. EPE-50. FSilicone sleeve (SIL047). Figure 9. Catheters Usage in vivo. A 10 month-old PS/APP mouse with 2 osmotic pumps following 28 days of infusion. While still active (A) and after transcardial perfusion showing the Alzet pumps (B) and cannulae (C) still securely attached. The arrow in figure D shows the nitrile cap used to affix the catheters to the skull and Figure E shows the entire assembly attached to an Alzet 1004 osmotic minipump. When the brain is removed, only small holes are visi ble where the cannulae were inserted (F). 103

PAGE 114

Figure 10 Double Immunohistochemistry of A and ACT. A) A montage of images taken at 10x from 25 m sections of an animal treated with ACT on the left and aCSF control on the right. Th e top section is immuno-labelled with the A antibody 6E10 and the bottom with ACT antibodies Inlays (40x) show A in green, ACT in red, and Hoechst nuclear stain in blue. 104

PAGE 115

Figure 11. Recombinant Peptides Remain Localized to Infused Hemispheres. Montage images of 25 m coronal sections of 10 month old nontransgenic (A & B) and PS/APP mice (C & D) immune-labeled with and antibody against 1 antichymotrypsin following 28 day infusion demonstrating that ACT stays on the infused hemisphere with or without the presence of plaques. 105

PAGE 116

Figure 12. Replication of Transgenic Animal Models. Comparison of ACT with amyloid plaques (40x) from a PS/APP/ACT transgenic mouse (A) and a PS/APP ACTinfused mouse (B) both at 9 months of age. Immuno-labeling of A is shown in green (6E10 antibody), ACT in red, and Hoechst nuclear stain in blue. References Abraham, C. R., Selkoe, D. J., & Potter, H. (1988). Immunochemical identification of the serine protease inhibitor alpha 1-antic hymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell, 52 (4), 487-501. Bennett, S. P., Boyd, T. D., Norden, M., Padm anabhan, J., Neame, P., Wefes, I., et al. (2009). A novel technique for simultaneous bilateral brain infusions in a mouse model of neurodegenerative disease. J Neurosci Methods, 184 (2), 320-326. Ito, S., Sawada, M., Haneda, M., Fujii, S ., Oh-Hashi, K., Kiuchi, K., et al. (2005). Amyloid-beta peptides induce cell pr oliferation and macrophage colonystimulating factor expression via the PI3-kinase/Akt pathway in cultured Ra2 microglial cells. FEBS Lett, 579 (9), 1995-2000. Kaku, M., Tsutsui, K., Motokawa, M., Kawata T., Fujita, T., Kohno, S., et al. (2003). Amyloid beta protein deposition and neur on loss in osteopetrotic (op/op) mice. Brain Res Brain Res Protoc, 12 (2), 104-108. Kawata, T., Tsutsui, K., Kohno, S., Kaku, M., Fujita, T., Tenjou, K., et al. (2005). Amyloid beta protein deposition in oste opetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor. J Int Med Res, 33 (6), 654660. Ma, J., Brewer, H. B., Jr., & Potter, H. (1996). Alzheimer A beta neurotoxicity: promotion by antichymotrypsin, ApoE4; inhibition by A beta-related peptides. Neurobiol Aging, 17 (5), 773-780. Ma, J., Yee, A., Brewer, H. B., Jr., Das, S ., & Potter, H. (1994). Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature, 372 (6501), 92-94. Mitrasinovic, O. M., Grattan, A., Robinson, C. C., Lapustea, N. B., Poon, C., Ryan, H., et al. (2005). Microglia overexpressing the macrophage colony-stimulating factor 106

PAGE 117

107 receptor are neuroprotective in a micr oglial-hippocampal organotypic coculture system. J Neurosci, 25 (17), 4442-4451. Mucke, L., Yu, G. Q., McConlogue, L., Rocken stein, E. M., Abraham, C. R., & Masliah, E. (2000). Astroglial expression of huma n alpha(1)-antichymotrypsin enhances alzheimer-like pathology in amyloid protein precursor transgenic mice. Am J Pathol, 157(6), 2003-2010. Nilsson, L. N., Bales, K. R., DiCarlo, G., Go rdon, M. N., Morgan, D., Paul, S. M., et al. (2001). Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease. J Neurosci, 21 (5), 14441451. Padmanabhan, J., Levy, M., Dickson, D. W., & Potter, H. (2006). Alpha1antichymotrypsin, an inflammatory protei n overexpressed in Alzheimer's disease brain, induces tau phosphorylation in neurons. Brain, 129(Pt 11), 3020-3034.

PAGE 118

108 CHAPTER 4 INTRAHIPPOCAMPAL ADMINISTRATION OF CSFs Introduction To investigate the consequences of in teractions between the innate immune system and AD, we studied the effects on AD pathology of the three colony-stimulating factors (M-CSF, G-CSF, and GM-CSF), whic h are up-regulated in the synovium and serum during RA pathogenesis (Kawaji, Y okomura, Kikuchi, Somoto, & Shirai, 1995; Nakamura et al., 2000; Olszewski et al., 2001; Xu, Firestein, Taetle, Kaushansky, & Zvaifler, 1989). The first objective was to induce the specific leukocytic phenotype of each CSF in the brains of aged transgenic AD mice. In the periphery, these CSFs enhance the survival and function of their respective leukocytes and drive their proliferation and differentiation from myeloid lineage precursors. GM-CSF pr imarily induces dendritic cells, macrophages, and granulocytes (neutr ophils, basophils, and eosinophils), while M-CSF and G-CSF respectively induce the m acrophage and granulocyte subsets of the innate immune system. These innate cells have the ability to diapedese from the circulatory system and to differentiate furt her into various specialized immune cells within organs (microglia, Langerhans cells, etc.). However, these specific leukocytic populations have not been de scribed in the brains parenchyma and leukocytes are broadly classified as just microglia. Since the bilateral intracerebroventricular infusions of M-CSF into similar-aged AD mice showed significant variations in amyloidosis and since we observed that infused recombinant proteins remain localized within their respective-infused brain hemispheres (Chapter 3), we determined th e effects of each CSF by bolus intrahippocampal injection into th e ipsilateral brain hemisphere and bolus

PAGE 119

109vehicle injection contralatera lly. This chapter shows the e ffects that each CSF induced into the brains of aged transgenic AD mice. Specific microscopy and image analysis procedures, and a novel device for paraffinembedded immunohistochemistry were also developed to analyze these eff ects (Boyd et al., 2010 In Press). Methodology of Transgenic Mouse Studies Involving Intrahippocampal Administration of CSFs Transgenic Mice. PS/APP mice in this study, which begin accumulating robust amyloid plaques at 6-8 months, were ge nerated by crossing he terozygous PDGF-hAPP (V717F) mice with PDGF-hPS1 (M146L) on both Swiss Webster and C57BL/6 backgrounds. All procedures involving experimentation on animals were performed in accordance with the guidelines set forth by the University of South Florida Animal Care and Use Committee. Transgene detections were performed using QPCR (Bio-Rad iCycler, Hercules, CA). Intrahippocampal Injections of CSFs All three CSFs were stereotaxicallyinjected (5 g/injection) into the (ipsilater al) hippocampus, with artificial cerebrospinal fluid vehicle injected contralaterally into four PS/APP mice each (all 10-12 months old, 25-35 g, both genders). Two holes were dri lled into the skull (from bregma -2.5 mm anterior-posterior, +/2.5 mm medial-lateral, and the 30 gauge needle inserted to a depth of 2.5 mm (Figure13)). Mice were perfused w ith 0.9% cold saline 7 days later and their brains placed in 10% neutral buffered formalin. Recombinant mouse GM-CSF (rmGM-CSF), recombinant murine G-CSF (rmG-CSF), and recombinant mouse M-CSF (rmM-CSF) (R&D Systems, Minneapolis, MN) will be referred to as GM-CSF, G-CSF, and M-CSF throughout this publication.

PAGE 120

110 Immunohistochemistry and Image Anal ysis of Intrahippocampal-injected Mice. Formalin-fixed brains were either coro nally cryosectioned at 14-m, or paraffinembedded and sectioned at 5-m, with stan dard deparaffination and antigen retrieval steps (boiled in 10mM Sodium Citrate buffer for 20 minutes) performed before immunohistochemical staining. To significantly reduce cost of reagents and antibodies with paraffin-embedded slides, a novel ma gnetic immunohistochemical staining device (Figure 13) was developed (patent pending, Tech ID# 09A015). Standard fluorescent immunohistochemical techniques used primary anti-A antibodies 6E10 (Covance, Emeryville, CA, 1:1000), and MabTechs 3740-5 (MabTech, Cincinnati, OH, 1:5000) to immunolabel amyloid deposition coupled with Alexa fluorophore-labelled secondary antibodies (Molecular Probes, Eugene, OR, 1:1000, 1:4000), and Hoechst (Sigma) nuclear staining. Immunofluorescence was dete cted and all pictures per section were taken at the same exposure on a Zeiss Imag er.Z1 microscope (Oberkochen, Germany) using Axiovision 4.7 software. Digital images were quantified using a novel ImageJ protocol (see Appendix D for slides outling the complete ImageJ ). Microscopy and Method of Image J An alysis of Amyloid Deposition. Lateral rows of photomicrographs were taken of coronal sections in a manner that ensured minimal overlap between each photomicrogra ph and corresponding anatomical areas in each hemisphere. Hoechst nuclear staining was used as a tool to scroll through sections, allowing for minimal overlap between each photomicrograph, and then the image was taken with the appropriate fl uorescence. After all photomicr ographs of the section were taken, they were montaged into a full coronal picture of th e section, with the outline of

PAGE 121

111each photomicrograph displayed (Axiovi sion 4.7 Panorama Module software). Photomicrograhs were then selected for anal ysis from each hemisphere (Figure 15). The histogram of each photomicrograph was analyz ed and the resulting means and standard deviations were entered into a spreadsheet. One photomicrogr aph of the section was then thresholded to select the amyloid plaques. This thresholded value was used in the spreadsheet to normalize the threshold values of the other photomicrographs to the same standard deviation from their respecti ve histogram mean (Figure 16a). Each photomicrograph was then analyzed for Area, Perimeter, Feret Diameter, and Integrated Density values from their predetermined thre shold values. Any threshold-selected data, which came in contact with the edges of a ny photomicrograph, were deleted via the Image J Analyze Particles dialogue box, so th at overlap of photomicrographs did not allow quantification of any amyl oid deposition more than once. Within the Image J Analyze Particles dialogue box, the Oulines mask was also selected to visually confirm that the pla ques were quantified a ccurately (Figure 16b). This Outline mask also numbered each plaque, which was used for selecting and eliminating artifact from the results. Th e results from each individual photomicrograph were copied into a 2nd separate spreadsheet, such that th e data were separated into their respective hemispheres (CSF-treated versus ar tificial cerebrospinal fluid-treated). After all photomicrographs from a coronal section ha d been analyzed and data entered into the spreadsheet, the Area and Perimeter values were totaled and averaged. The Feret Diameter and Integrated Density values were also averaged. The ove rall data from each section (sums and averages) were then entered into a 3rd spreadsheet A total of 5 sections per mouse, anterior to posterior, were analyzed and entered into this 3rd

PAGE 122

112spreadsheet. A total of 4 mice per intrahippocampal-injected CSF were analyzed. For GM-CSF-injected mice, each section quantified contained analysis of 15-25 individual 10X pictures of each hemisphere, with fewer pi ctures quantified in the anterior brain and more in posterior brain. For G-CSFand MCSF-injected mice, each section quantified contained analysis of 7-9 individual 5X pictures of each hemisphere Statistical significance was obtained from co mparing parameter values of ipsilateral CSF-injected hemispheres versus contralateral artificial cerebrospinal fluid-injected hemispheres. Significance per mouse and over all 4 mice pe r CSF was determined by paired Students t-test with p values < 0.5 considered significant Results Bolus Intrahippocampal Injections of CSFs. M-CSF intrahippocampal injections into mice resulted in swelling of the entire hemisphere as compared to the control side, and in one mouse, an apparent hyperplasia had formed at the injection site (Figure 17 a-c). Quantification of amyloid plaque loads from anterior to posterior of each mouse showed similar deposition in the M-CSFinjected hemispheres as compared to the control sides (data not shown). In contrast to M-CSF, G-CSF intrahippocampal injections did not induce swelling and showed some modest reductions of amyloid deposition (Figure 18 a-b). Since the analyses we re conducted on low magnification (5X) photomicrographs, only the area and integrated density values achieved significance (p<0.05). Reductions in amyl oidosis was subsequently corroborated by independent observations from fellow investigators following peripheral G-CSF administration (Sanchez-Ramos et al., 2009).

PAGE 123

113GM-CSF-injections, however, demonstrat ed pronounced decreases in amyloid deposition, as compared to control hemisphere s, in visual observati ons of coronal tissue sections (Figure 19 a-d). High magnification (10X) quantification of amyloid plaques anterior to posterior revealed significant reductions within individual mice and significant overall reductions for all plaque paramete rs measured (Figures 20 and 21 a-f). Conclusion. The differential effects from each injected CSF was surprising and exciting. Although M-CSF did not show any expected alteration of amyloidosis, its ability to rapidly induce a hyperplasia, as opposed to G-CSF and GM-CSF, supports similar literature and helps advance the fields knowledge with the parenchymal overexpression of M-CSF (Kirma et al., 2004). Further investigation by other researchers into the biology of M-CSF-induced hyperplasi as would be welcomed. However, based upon the very pronounced and significant reductions in amyloidosis, after only one week of ipsilateral intrahippocampal bolus injection of GM-CSF as compared to contralateral vehicle injection, we wanted to investigate whether GM-CSF would also show benefit in cognitive function. Although G-CSF also showed some reductions in amyloidosis, it was not as robust and was currentl y being researched by fellow investigators (Sanchez-Ramos et al., 2009). Therefore, we d ecided to immediately focus our efforts on the investigation of GM-CSF by subcutaneous delivery, since th at would likely be the potential route of treatment to AD patients. The following chapter will discuss the daily subcutaneous treatment of mice with recombinant m ouse GM-CSF and its effects thereof.

PAGE 124

Figure 13. Schematic of the Intrahippocampal Injection Sites Either M-CSF, G-CSF, or GM-CSF were injected into the hippocampus between the CA2 and CA3 regions of one hemisphere, and artificial cerebro spinal fluid would have been injected in the same contralateral site. 114

PAGE 125

115 Figure 14. Magnetic Immunohistochemical Device This novel device was designed to minimize the amount of reagents needed dur ing the immunohistochemical staining of paraffin-embedded slide-mounted tissue secti ons. The washer-style magnets have an inside diameter of 0.5 inches that encircles the coronal tissue section. Once the slide and magnet are placed upon the bracket, the magne tic force holds the slide in place and provides a barrier to the liquid reagents. Further detail is provided in the patent application attached in Appendix E. (a ) Illustration of pipetting 250 300L of reagent to magnetic well. ( b) Illustration of slide tray contain 3 tissue slides with pipet, that would have to have to deliver 8 10mL of reagent to cover slides, as opposed to the magnetic immunohistochemical device contai ning 500-600 L of reagent on two tissue slides.

PAGE 126

116 Figure 15. Method of Identifying Individual Photomicrographs for Analysis ( a ) Montaged coronal section of individual 10X photomicrographs that were taken in a lateral row proceedure with mininal overlap be tween each one, that was then demarcated to show which individual photomicrographs w ould be analyzed for amyloid deposition.

PAGE 127

Figure 16 Method of ImageJ Analysis of Amyloid Plaques ( a ) Depiction of the Thresholding of one photomicrograph and the subsequent usage of its threshold value that was entered into a spreadsheet to find the threshold value, at the same standard deviation from the mean, for all other photomic rographs that are to be analyzed and which are contained within the same coronal section. 117

PAGE 128

Figure 16 Method of ImageJ Analysis of Amyloid Plaques (continued) ( b ) Example of an ImageJ Oulines mask, that allows for visual confirmation of the plaques that were analzyed, as well as the underlying ImageJ spreadsheet data that will be entered into a compilation spreadsheet containing the data of the other photomicrographs of the same coronal section. 118

PAGE 129

Figure 17. Intrahippocampal Injection of M-CSF. M-CSF injected left hemisphere and artificial cerebrospinal flui d (aCSF)-injected right hemisphere ( a ) The image is a montage of ~35 5X pictures and is represen tative of the effects seen from anterior hippocampus to posterior in all 4 M-CSF-injected mice. ( b) This photo shows enlargement of the M-CSF-injected left hemisphere, as seen following saline perfusion. Note the small bump at the site of injection (arrow). ( c ) Image shows cyst or tumor-like growth formed in the needle track at the site of M-CSF injection. Cryosectioned at 14 m and stained with 6E10/Alexa 488 and Hoechst. Picture taken at 20X. 119

PAGE 130

Figure 18 Intrahippocampal Injection of G-CSF. G-CSF injected left hemispheres and artificial cerebrospinal fluid (aCSF)-injected right hemispheres ( a ) Images are montages of ~35 5X pictures each. Amyloid plaques i ndicated as white spots. Cryosectioned at 14 m and stained with 6E10/Alexa546 and Hoechst nuclear stain. Sections numbered 1 through 6 and co rrespond with anterior to posterior. 120

PAGE 131

Figure 18 Intrahippocampal Injection of G-CSF (continued). ( b) Amyloid plaques show a modest reductio n of plaque in the left G-CSF-injected hemisphere. Error bars are Standard Error of the Mean. (Are a, Integrated Density: p < 0.05). 121

PAGE 132

122

PAGE 133

Figure 19 Intrahippocampal Injection of GM-CSF. GM-CSF injected left hemispheres and artificial cerebrospinal fl uid (aCSF)-injected right hemispheres. ( a-d ) Representative sections of each mouse proxima l to injection site. Ti ssue sections stained with MabTech -A /Alexa 488. White spots indicate amyloid plaque immunolabelling. Images are montages of about 145 pi ctures taken at 10X. Figures 18 ( a c ) are from 14 m frozen sections, and 18 ( d) is from a 5 m paraffin-embedded section. 123

PAGE 134

124 Figure 20 Overall Percent Reductions in A Burden by GM-CSF Averages of all four GM-CSF-injected mice showing significan t overall plaque reductions in all four plaque parameters measured from 5 quantif ied sections per mouse (n = 4 mice). Error bars are Standard Error of the Mean. (Ar ea: p < 1.11E-07; Perimeter: p < 1.41E-06; Feret Diameter: p < 2.36E-09; Inte grated Density: p < 1.11E-07).

PAGE 135

Figure 21 Individual Parameter Reductions in A Burden by GM-CSF. GM CSF injected left hemispheres versus artificial cerebrospinal fluid (aCSF) injected right hemispheres. There were 5 montaged sections per mouse quantified. Each montaged section contained over 140 10X pictures and of these, 15 25 pictures per hemisphere were selected to quantify as described in Appendix D. Each figure shows the total or average values from the five sections pe r mouse with statistical significance per individual mouse and significance over all four mice. Error ba rs are Standard Error of the Mean: ( a b ) plaque areas ( c d ) perimeter values 125

PAGE 136

Figure 21 Individual Parameter Reductions in A Burden by GM-CSF (continued). ( e ) average feret diameters ( f ) average integrated densities. References Boyd, T. D., Bennett, S. P., Mori, T., Govern atori, N., Runfeldt, M., Norden, M., et al. (2010 In Press). GM-CSF Upregulated in Rheumatoid Arthritis Reverses Cognitive Impairment and Amyloidosis in Alzheimer Mice. J Alzheimers Dis. Kawaji, H., Yokomura, K., Kikuchi, K., Somoto, Y., & Shirai, Y. (1995). [Macrophage colony-stimulating factor in patients with rheumatoid arthritis]. Nippon Ika Daigaku Zasshi, 62 (3), 260-270. Kirma, N., Luthra, R., Jones, J., Liu, Y. G., Nair, H. B., Mandava, U., et al. (2004). Overexpression of the colony-stimulating factor (CSF-1) and/or its receptor c-fms in mammary glands of transgenic mice results in hyperplasia and tumor formation. Cancer Res, 64 (12), 4162-4170. Nakamura, H., Ueki, Y., Sakito, S., Matsumoto, K., Yano, M., Miyake, S., et al. (2000). High serum and synovial fluid granulocyt e colony stimulating factor (G-CSF) concentrations in patients with rheumatoid arthritis. Clin Exp Rheumatol, 18 (6), 713-718. Olszewski, W. L., Pazdur, J., Kubasiewicz, E ., Zaleska, M., Cooke, C. J., & Miller, N. E. (2001). Lymph draining from foot joints in rheumatoid arthritis provides insight into local cytokine and chemokine production and transport to lymph nodes. Arthritis Rheum, 44 (3), 541-549. 126

PAGE 137

127Sanchez-Ramos, J., Song, S., Sava, V., Catlow, B., Lin, X., Mori, T., et al. (2009). Granulocyte Colony Stimulating Factor (G-CSF) Decreases Brain Amyloid Burdenand Reverses Cognitive Impairment in Alzheimer's Mice. Neuroscience. Xu, W. D., Firestein, G. S ., Taetle, R., Kaushansky, K., & Zvaifler, N. J. (1989). Cytokines in chronic inflammatory arth ritis. II. Granulocyte-macrophage colonystimulating factor in rheu matoid synovial effusions. J Clin Invest, 83 (3), 876-882.

PAGE 138

128 CHAPTER 5 SUBCUTANEOUS ADMINISTRATION OF GM-CSF Introduction Based upon the positive results from th e GM-CSF intrahippocampal injections (Chapter 4), we investigated the effect of subcutaneous GM-CSF administration on cognitive function and AD pathology in aged transgenic AD mice. Prior to these experiments, there were no published reports involving in vivo behavioral tasks after administration of Colony Stimulating Factors. We determined an appropriate dosage of GM-CSF for in vivo administration, which would be co mparable to dosages prescribed for Leukine (sargramostim), the human GM-C SF analogue. This chapter describes the types of animal behavioral tasks performe d, and the alteration of animal cognitive function and pathophysiology after 20 days of daily recombinant mouse GM-CSF administration (Boyd et al., 2010 In Press). Methodology of Behavioral Transgenic Mouse Study Involving Daily Subcutaneous Treatment with GM-CSF Transgenic Mice. Mice in this study were derive d from the Florida Alzheimers Disease Research Center m ouse colony, wherein heterozygou s mice carrying the mutant APPK670N, M671L gene (APPsw) are routinel y crossed with hetero zygous PS1 (Tg line 6.2) mice to obtain APPsw/PS1, APPsw, PS1, and non-transgenic (NT) genotype offspring with a mixed C57/B6/SW/SJ L background. All procedures involving experimentation on animals were performed in accordance with the guidelines set forth by the University of South Florida Anim al Care and Use Committee. Transgene detections were performed using QPCR (Bio-Rad iCycler, Hercules, CA).

PAGE 139

129 Radial Arm Water Maze (RAWM) and Cognitive Interference Tasks. Eleven APPsw and 17 NT mice, all 12-months old, were selected and evaluated for 8 days in the RAWM task of working memory to obtain baseline cognitive performance before GM-CSF treatment (Arendash et al., 2001). Fo r RAWM testing, aluminum inserts were placed into a 100 cm circular pool to create 6 radially-distributed swim arms 30.5 cm in length and 19 cm wide emanating from a cen tral circular swim area 40 cm in diameter. The inserts rise 5 cm above the surface of th e water. A submerged escape platform (9 cm diameter) is placed with equal distance from th e back and side walls in one of the arms for each day of testing. The platform location was changed daily to a different arm, with different start arms for each of the 5 trials semi-randomly selected from the remaining 5 swim arms. On the outside of each arm lies a visual cue that is different for each arm. Mice do not prefer to be in water, and they readily seek out the submerged escape platform to get out of the water (Figure 22a). For any given trial, the mouse is placed into that trials start arm facing the center swim area and given 60 seconds to find the platform with a 30 second stay. Each time the mouse enters a non-platform containing arm it is gently pulled back into the start arm and an error is recorded (Figure 22b). An error is also recorded and the mouse is pulled back in to the start arm for that trial if the mouse fails to enter any arm within 20 seconds or if a mouse enters the platform-containing arm but does not find the platform. If the mouse does not find the platform within a 60-second trial, it is guided by the experimenter to th e platform, allowed to stay for 30 seconds, and assigned a latency of 60 seconds. An error is al so assigned to any animal that, for any one minute trial, does not find the goal arm and re fuses to make at least 3 choices on their

PAGE 140

130own during that trial (such as just swimming in a circular pattern). This number of -choices was calculated by averaging errors for all animals that do not locate the platform for Block 1 (day 1 through day 3) on trial 1. Both the number of errors (incorrect arm choices) and escape latency were recorded for each daily trial. The number of errors prior to locating wh ich one of the 6 swim arms contained the submerged escape platform was determined for 5 trials per day. The numbers of errors during trials 4 and 5 are both considered indices of working me mory and are temporally similar to the standard registration/recall te sting of specific items used clinically in evaluating AD patients. Following the 8 days of pre-treatment RAWM testing (Figure 23a), the 11 Tg mice were divided into two groups, balanced in RAWM performance. The 17 NT mice were also divided into two groups, bala nced in RAWM performance. Two weeks following pre-treatment testing, one group of Tg mice ( n = 5) and one group of NT mice ( n = 9) were started on a 10-day treatment protocol with GM-CSF (5 g/day given subcutaneously), while animals in the c ontrol Tg group (n=6) and control NT group ( n = 8) concurrently received daily vehicle (sal ine) treatment subcutaneously. On the 11th day of injections, all mice began f our days of RAWM evaluation, were given 2 days of rest, then evaluated in 4 days of Cognitive Interference task testing. This task was designed measure-for-measure from a Cognitive Interf erence task used to discriminate normal aged, MCI, and AD patients from one anothe r (Echeverria et al., 2009; Loewenstein et al., 2004). This analogous interference task for mice involves two radial arm water maze (RAWM) set-ups in two different rooms, each w ith different sets of visual cues. The task requires animals to remember a set of visual cues, so that following interference with a

PAGE 141

131different set of cues, the initial set of cu es can be recalled to successfully solve the RAWM task. A set of four be havioral measures were examined. Behavioral measures were: A1A3 (Composite three-trial recall scor e from first 3 trials performed in RAWM A), B (proactive interferen ce measure attained from a single trial in RAWM B), A4 (retroactive interference measure attained during a single trial in RAWM A), and A5(delayed-recall measure attained from a single trial in RAWM A following a 20 minute delay between A4 and A5). As a distract er between trials, anim als are placed in a Y-maze and allowed to explore for 60 seconds between successive trials of the three-trial recall task, as well as during the proactive in terference task. As with the standard RAWM task, this interference task involves the platform locati on being changed daily to a different arm for both of the RAWM set-ups utilized, and different start arms for each day of testing for both RAWM set-ups. For A1 and B trials, the animal was initially allowed one minute to find the platform on their own before they were guided to the platform. Then the actual trial was performe d in each case. As with the standard RAWM task, animals were given 60 seconds to find th e escape platform for each trial, with the number of errors recorded for each trial. An imals were tested for cognitive interference performance on four successive days, with st atistical analysis pe rformed for the two resultant 2-day blocks. For both RAWM (c ombined T4 and T5 overall) and cognitive interference testing (e ach of the four measures overall), swim speed was analyzed by dividing error numbers by latency and statisti cal significance was determined by one-way ANOVA followed by post hoc Fishers LSD (l east significant difference) test to determine significant group differences at p < 0.05.

PAGE 142

132Daily GM-CSF and saline injections were continued throughout the behavioral testing period. After completion of behavioral testing at 20 days into treatment, all mice were euthanatized, brains fixed in 10% Fo rmalin, and paraffin-embedded. Careful visual examination of all tissues upon necropsy revealed no morphological abnormalities, and the mice tolerated daily subcutaneous injectio ns well. Each analysis was done by a single examiner blinded to sample identities, and st atistical analyses were performed by a single examiner blinded to treatment group identitie s. The code was not broken until analyses were completed. Immunohistochemistry and Image An alysis of Subcutaneous GM-CSFtreated Mice. Five 5m sections (150m apart) were made of formalin-fixed, paraffinembedded sections throughout the hippocampus of each mouse and immunoreactivity was developed using the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) coupled with the diaminobenzidine reaction, according to the manufacturers protocol. Immunostaining us ed biotinylated anti-A clone 4G8 (Covance, Emeryville, CA, 1:200), synaptophysin (DAKO, Carpinte ria, CA, undiluted), and Iba1 (Wako, Richmond, VA, 1:1000) as primary antibodie s. Since the 4G8 antibody was obtained with biotin label, the secondary step of the ABC protocol was omitted. However, treatment with 70% formic acid prior to the pre-blocking step was necessary. For 4G8 immunohistochemistry, phosphate-buffered salin e (0.1 mM, pH 7.4) was used instead of primary antibody or ABC reagent as a negative control. For Iba 1 and synaptophysin immunohistochemistry, normal rabbit serum was used instead of primary antibody or ABC reagent as a negative control. Imag es were acquired using an Olympus BX60 microscope and digital images were quantif ied using SimplePCI software (Compix Inc.,

PAGE 143

133Imaging Systems, Cranberry Township, PA), according to previous methods (SanchezRamos et al., 2009). Briefly, a threshold opti cal density was obtaine d that discriminated staining from background, and each region of interest was manually edited to eliminate artifacts. Data are reported as percenta ge of immunolabeled area captured (positive pixels) relative to the full area captured (t otal pixels). To evaluate synaptophysin immunoreactivity, after the mode of all images was converted to gray scale, the average intensity of positive signals from each image was quantified in the CA1 and CA3 regions of hippocampus as a relative number fr om zero (white) to 255 (black). Statistical significance between GM-CSF-treat ed versus saline-tr eated groups was determined by two-tailed homoscedastic St udents t-test with a p value of < 0.05 considered significant. Each analysis was done by a single investigator blinded to sample identities and genotype. Results Daily Subcutaneous Injections of GM-CSF. Prior to GM-CSF treatment, APPsw (Tg) mice were first confirmed by RAWM testing to be cognitively-impaired for working memory (Figure 23a). Both the nontransgenic control mice (NT) and the Tg mice were then sub-divided into two cogniti vely-balanced groups, for either GM-CSF or saline treatment. RAWM test ing, that began on the 11th day of injections, re-confirmed that Tg control mice were substantially impa ired compared to NT control mice. This impairment was not only evident in individual blocks of testing, but also over all 4 days of testing (Figure 23b). In sharp contrast, GM-CSF-treated Tg mice performed equally well or better than NT control mice duri ng individual blocks and overall. GM-CSFtreated NT mice performed as well as or sligh tly better than NT cont rols (Figure 23b).

PAGE 144

134 Before evaluation in the Cognitive Inte rference Task, the mice rested two days, while daily injections continued. This task mimics human interference testing, which discriminates between normal aged, MCI, and AD patients (Loewenstein et al., 2004). In three of four cognitive interf erence measures assessed over 4 da ys of testing (Figure 23c), Tg control mice were clearly impaired compar ed to NT mice, and Tg mice treated with GM-CSF exhibited significantly better three-trial recall and delayed recall compared to Tg controls. Indeed, for all four cognitiv e measures, GM-CSF-treated transgenic AD mice performed similarly to NT mice. A part icularly strong effect of GM-CSF treatment in Tg mice was evident for the proactive in terference measure duri ng the first half of testing (Figure 23d), wherein GM-CSF-treated Tg mice performed substantially better than Tg controls and identically to both groups of NT mice. Includ ing this strong effect on proactive interference testing, GM-CSF tr eatment resulted in significantly better performance of Tg mice for all four measures of cognitive interferen ce testing. Proactive interference susceptibility has been repor ted to be a more sensitive marker for differentiating MCI and AD patients from aged normals than traditional measures of delayed recall and rate of forgetting (Loewenste in et al., 2004). Pare nthetically, even the GM-CSF-treated NT mice showed a trend towards improved cognition in behavioral studies, albeit not statis tically significant. Analysis of swim speed for both the RAWM and cognitive interference tasks revealed that Tg control mice were significantly faster than the other three groups in the RAWM ta sk and for two of f our measures in the Cognitive Interference task (3-trial recall a nd delayed recall). However, since error numbers were utilized for statistic al analysis of both tasks, this difference in swim speed was negated since it is important only if latency measures had been used.

PAGE 145

135Following completion of all behavioral eval uations, subsequent analysis of brains from Tg mice of this study revealed that GM-CSF treatment induced large reductions in amyloid burdens within entorhinal cortex ( 55%) and hippocampal ( 57%) compared to control Tg mice (Figure 24). The improved cognitive function and reduced cortical amyloidosis of GM-CSF-treated Tg mice were paralleled by increased microglial density as compared to saline-treated Tg mice (Figure 25), implying an augmented ability to bind and remove amyloid deposition (El Khoury et al., 2007; Hickman, Allison, & El Khoury, 2008). The GM-CSF-treated Tg mice similarl y demonstrated increased synaptophysin immunoreactivity in both CA1 and CA3 regi ons (Figure 26), indicating increased synaptic area in these hippocampal regions. Pr ior work has shown that adult neural stem cells in hippocampal dentate gyrus ( DG) express GM-CSF receptors, and GM-CSF increases neuronal differentiation of these ce lls in a dose-dependent fashion (Kruger, Laage, Pitzer, Schabitz, & Schneider, 2007) Thus, one mechanism for the observed GM-CSF-induced cognitive improvement is enhanced removal of deposited A in hippocampus, with ensuing neuronal growth/s ynaptic differentiation of DG mossy fiber innervation to CA3, resulting in increased innervation/synaptogenesis of Schaffer collaterals into CA1. Removal of deposited A from entorhinal cortex may also increase perforant pathway viability to hippocam pal projection fields in DG and CA1. Conclusions. This is the first study that ha s examined GM-CSFs effects on cognitive function in vivo and the reversal of pre-determined cognitive impairment gives hope that the human recombinant GM-CSF, Le ukine (sargramostim) may have the same effects in AD patients. GM-CSF-induced re duction of amyloidosis and enhancement of hippocampal/entorhinal cortex circuitry, crit ical for working (short-term) memory, may

PAGE 146

136underlie GM-CSFs reversal of working memo ry impairment in Alzheimers Tg mice. Furthermore, there may be other mechanisms which are induced by GM-CSF administration, both peripherally and in the brai n, that contribute to the restoration of cognitive function and revers al of AD pathology. The following chapter will discuss recent reports of GM-CSF in relation to othe r neurodegenerative disorders that may shed light on additional mechanisms for GM-CSFs pronounced effects in AD models.

PAGE 147

Figure 22. Radial Arm Water Maze Task. ( a ) RAWM non-error. Depiction of mouse that has found the submerged escape platform. ( b ) RAWM error. Depiction of mouse performing an error in the RAWM task. 137

PAGE 148

138

PAGE 149

139Figure 23. Behavioral analysis following daily subcutaneous GM-CSF injections. (a)Standard Radial Arm Water Maze Errors Prior to Treatment. The final block and overall performance of the Tg and NT mice during 8 days of consecutive daily pre-treatment testing in the RAWM maze. The data were analyzed in four 2-day blocks and overall (Blocks 1-4). ( *p < 0.02 or higher significance). (b) Standard Radial Arm Water Maze Errors. Tg control mice (n = 6) show substantial impairment on working memory tria ls T4 and T5 compared to NT control mice (n = 8) in individual blocks of testing ( upper), and over all 4 days of testing (lower). GM-CSF-treated Tg mice (n = 5) performed as well as or better than NT control mice on working memory trials T4 and T5 during in dividual blocks and ove r all. GM-CSF-treated NT mice (n = 9) performed similarly to or slightly better than NT controls (Note significantly better performan ce of NT+GMCSF group versus NT group for T4 of Block 1), although this effect was not significant overall. ( **p < 0.02 or higher significance versus all other groups; p < 0.02 or higher significan ce versus Tg+GM-CSF and NT+GM-CSF). (c) Cognitive Interference Ta sk. Overall (4 Days) Tg control mice are impaired compared to NT mice on all four cognitive measures assessed. GM-CSF-treated Tg mice exhibited significantly better 3trial recall (A1-A3) and delaye d recall (A5) compared to Tg controls and performed similarly to NT mice in all four cognitive measures. GM-CSF treatment of NT mice did not result in significantly better pe rformance compared to NT controls, although trends for a beneficial GM-CSF effect in NT mice were evident overall. (*Tg significantly diffe rent from NT+GM-CSF, **Tg si gnificantly different from all other groups). (d) Cognitive Interference Task. Proactive Interference Testing (First 2 days). GM-CSF-treated Tg mice performed significantly better than Tg controls and equally to NT and GM-CSF-treated NT mice.

PAGE 150

Figure 24. Amyloid Deposition in Subcut aneous GM-CSF-injected Mice. (a-d) Photomicrographs of coronal 5-m paraffi n-embedded sections immunolabelled with anti-A antibody (clone 4G8) in Entorrhinal cort ex (E) and hippocampus (H)). Pictures are representative of amyloid load closest to the mean of the GM-C SFor saline-treated Tg groups. Scale bar = 50 m. 140

PAGE 151

Figure 24. Amyloid Deposition in Subcutaneous GM-CSF-injected Mice (continued). (e) Percent of amyloid burden from the average of five 5m sections (150m apart) through both anatomic regions of interest (hippocampus and entorhinal cortex) per mouse of GM-CSF-treated (n = 5) versus salinetreat ed (n = 6). Entorhinal cortex (*p < 0.026), and Hippocampus (p = 0.12). 141

PAGE 152

Figure 25. Microglial Immunostaining in Subcutaneous GM-CSF-injected Mice. (a-d) Photomicrographs of coronal 5m para ffin-embedded sections immunolabelled with Iba-1 antibody in Entorrhinal cortex (E) and hippocampus (H). Pictures are representative of Iba-1 immunolabelling closest to the mean of the GM-CSFor saline controltreated groups Scale bar = 50m. 142

PAGE 153

143 Figure 25. Microglial Immunostaining in Subcutaneous GM-CSF-injected Mice (continued). (e) Percent of Iba1 burden from the average of five 5-m sections (150 m apart) through both anatomic regions of interest (H and EC) per mouse of GM-CSFtreated (n = 5) versus sali ne-treated (n = 6). H(p < 0.02), EC(p < 0.05).

PAGE 154

Figure 26. Synaptophysin Immunostaining in Subcutaneous GM-CSF-injected Mice. (a-d) Photomicrographs of coronal 5m paraffin-embedded sections immunolabelled with anti-synaptophysin an tibody. Pictures are representative of synaptophysin immunolabelling closest to the mean of the GM-CSFor saline controltreated groups. Scale bar = 50 m. 144

PAGE 155

145 Figure 26. Synaptophysin Immunostaining in Subcutaneous GM-CSF-injected Mice (continued). (e) Percent of synaptophysin immunoreactivity from the average of 5 sections per mouse of GM-CSFtreated (n = 5) versus saline control-treated (n = 6). CA1(p < 0.0013), CA3(p < 0.0023). References Arendash, G. W., King, D. L., Gordon, M. N., Mo rgan, D., Hatcher, J. M., Hope, C. E., et al. (2001). Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes. Brain Res, 891(1-2), 42-53. Boyd, T. D., Bennett, S. P., Mori, T., Governat ori, N., Runfeldt, M., Norden, M., et al. (2010 In Press). GM-CSF Upregulated in Rheumatoid Arthritis Reverses Cognitive Impairment and Amyloidosis in Alzheimer Mice. J Alzheimers Dis. Echeverria, V., Burgess, S., Gamble-George, J., Zeitlin, R., Lin, X., Cao, C., et al. (2009). Sorafenib inhibits nuclear factor kapp a B, decreases inducible nitric oxide synthase and cyclooxygenase-2 expressi on, and restores working memory in APPswe mice. Neuroscience, 162 (4), 1220-1231. El Khoury, J., Toft, M., Hickman, S. E., Mean s, T. K., Terada, K., Geula, C., et al. (2007). Ccr2 deficiency impairs mi croglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med, 13 (4), 432-438. Hickman, S. E., Allison, E. K., & El K houry, J. (2008). Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci, 28 (33), 8354-8360.

PAGE 156

146Kruger, C., Laage, R., Pitzer, C., Schab itz, W. R., & Schneider, A. (2007). The hematopoietic factor GM-CSF (granul ocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neur al stem cells in vitro. BMC Neurosci, 8, 88. Loewenstein, D. A., Acevedo, A., Luis, C ., Crum, T., Barker, W. W., & Duara, R. (2004). Semantic interference deficits a nd the detection of mild Alzheimer's disease and mild cognitive impairment without dementia. J Int Neuropsychol Soc, 10(1), 91-100. Sanchez-Ramos, J., Song, S., Sava, V., Catlow, B., Lin, X., Mori, T., et al. (2009). Granulocyte Colony Stimulating Factor (G-CSF) Decreases Brain Amyloid Burdenand Reverses Cognitive Impairment in Alzheimer's Mice. Neuroscience.

PAGE 157

147 CHAPTER 6 DISCUSSION Introduction The disappointing results from NSAID clini cal trials prompted us to consider immunological factors within RA pathogene sis as being protec tive against AD onset. Since it appeared most likely that cellula r effects and not fluid-phase factors would account for RAs protective effect, we decide d to induce leukocyte populations within Tg AD mouse models to study whether these induc ed leukocytes would have effects against cerebral amyloidosis, which these PS/APP mice begin accumulating at about 6 months of age. There are three major hematopoietic factors (M-CSF, G-CSF, GM-CSF) which contribute to most of the pathogenic le ukocytosis in RA, and under disease and non-disease conditions, they are known to contribute to specifi c peripheral innate cellular phenotypes. For instance, G-CSF induces gran ulocytic leukocytes, which are the most abundant type, usually constituting over half of all circulating leukocytes. On the other hand, M-CSF induces the differentiation of monocytic precursors towards macrophage phenotypes, although M-CSF can also contribut e to other cellular types, such as osteoclasts and Myeloid Derived Suppresso r Cell (MDSC) populati ons. Macrophages are usually characterized as either an M1 phenot ype, in which they are classically activated by microbial infection and drive a T-helper (Th) -1 response, or as an M2 phenotype, in which they are described as alternativ ely activated and considered to be immunosuppressive, driving a Th-2 reponse. Although considered immunosuppressive, M2 macrophages also contribu te to disease, such as in tumor-associated macrophages

PAGE 158

148 (TAMs) which drive angiogenesis and tumor development (Mantovani, Sozzani, Locati, Allavena, & Sica, 2002; Murdoch, Muth ana, Coffelt, & Lewis, 2008). Since peripheral leukocyte populations ar e greatly increased in RA and possess the ability to infiltrate into the brain, we wanted to differentially express each leukocyte phenotype by M-CSF, G-CSF, or GM-CSF induc tion after direct admi nistration of these CSFs into the brain, in order to determine which CSF might most affect amyloidosis. In the vasculature, all three CSFs work to drive the proliferatio n, differentiation, and survival of their respective innate leukocyt es from monocytic precu rsors (see Figure 3) and we hypothesized that they might also ha ve similar differential effects on microglial populations. Chronology of Initial Experiments In our studies, we initially began with the investigation of M-CSF administration into the brains of Tg AD mice. In the begi nning of 2007 at the time of these experiments, the literature suggested that a deficiency of M-CSF in osteopetro tic mice could cause a deficit in microglial populations, neuronal lo ss, and an accumulation of cerebral amyloid plaques (Kaku et al., 2003; Kawa ta et al., 2005). Also, at that time, there was no guiding literature to suggest dosage amounts for intra cerebral administration of M-CSF. Thus we decided to maximize the dosage through con tinuous bilateral intr acerebroventricular infusion for two weeks using our newly-developed osmotic catheter system (Chapter 3 and Appendix A). Subsequent immunohistochemical analyses of the plaque burden in the mice revealed significant unexpected variations of amyloidosis between mice of the same genotype and of similar age (F igures 5, 6). Furthermore, there was concern whether M-

PAGE 159

149 CSF was able to readily extravasate from the ventricles and into the brains parenchymya in order to induce an effect on amyloidosis. Meanwhile, we had contacted Dr. Kaku to request his expertise for M-CSF dosage amounts. Although he c ould not provide guidance for dosage, he did inform us that subsequent studies had shown that the antibody, in which he used for his 2003 study, was non-specifically immunolabeling some blood cells, and that they could not confirm amyloidosis in osteopetrotic mice. Moreove r, a coinciding report was soon published by another group, which confirmed that alt hough there was a deficiency in microglial density, there was no amyloid de position or loss of dopaminergic neurons in osteopetrotic mice (Kondo, Lemere, & Seabrook, 2007). Although these results with M-CSF were contrary to our initial thoughts, we nevertheless wanted to investigate each CSF s effects within the brains parenchyma in relation to already-formed amyloid depos ition. Our subsequent work with the development of the osmotic bilateral cathete rs had revealed that infused recombinant proteins remained localized into the resp ective brain hemisphere of which they are administered. Thus, we decided to perf orm intrahippocampal injections of each recombinant CSF protein into one hemisphere of the brain, and inject vehicle control contralaterally. Bilateral Infusion Catheters Our findings with the development of th e bilateral catheter system has several important implications, not only in our work, but broadly fo r other future studies of intracerebral delivery of material s into murine models. We we re the first to bilaterally infuse recombinant peptides and to show th at the infused recombinant proteins remain

PAGE 160

150 localized to the hemisphere in which they are infused. This concept will effectively reduce the number of animals that are needed for these types of experiments, since each animal can now be its own control and allow fo r comparisons of treatment or injury from one hemisphere of the brain versus the other. This effect will provide great cost benefit to researchers who will now have more flexibility in selecting murine cohorts, since they will no longer need age-matched controls. To further this idea of cost-effectiveness, the ability to infuse compounds, biologicals, etc. into one hemisphere and not the other, can provide for conditional transgenic models ipsilaterally versus control contralaterally, as we showed with replication of the trip le transgenic PS/APP/ACT model in one hemisphere versus doube transgenic PS/APP in the other (Figures 10 12). As is widely known, a large amount of breeding, genotyping, housing, and other costs of animal husbandry are incurred when going from double to triple transgenicity. Other significant benefits are also prov ided by the develoment of the bilateral catheters. For instance, in the commercially-available Alzet delivery system, the catheter head is not only too large in diamet er to place two of these upon a mouses skull, it is also too thick, in that closure of the scalp after pl acement is difficult, if not impossible sometimes. Furthermore, the cathet er tubing sometimes pr otrudes outside of the skin. In all of these case s, there is obvious discomfort to the animal, and the animals are known to scratch at the tubing and cathete r head, as well as rubbing them against the housing cage. As a result, it can be logically -assumed that stress hormone levels are chronically-affected during the course of these trials, and that there is chronic inflammation in the open wound, as the mice will of tentimes scratch their entire scalp off. Both of these issues may compromise an experiment, contribute to mortality, cause

PAGE 161

151 discomfort to the animal, increase research costs for the investigat or, and possibly skew results due to the intrinsi c immunological effects from this catheter pathology. Our bilateral catheter system is adhered directly in contour and upon the skul l, so that the skin can readily close with sutures, staples, or sk in adhesive and can then heal with new fur and absence of inflammation (Figure 9). The mice tolerate these cat heter extremely well, and have significant reductions in mortality rate (almost non-existent). Furthermore, these animals, once healed in a couple of weeks, can be used in water-based behavioral tasks, such as the Morris or RAWM. We hope that these improvements to the current methods of intracerebral infusions will become standard protocol for these types of experiments in all research vivariums around the country. Thus, using our gained knowledge from the catheter development, we injected each CSF unilaterally into one hippocam pus and vehicle contralaterally. Upon subsequent examination of the brains one week post-injectio n, we found different functional effects for each intrahippocampal-injected CSF. In the M-CSF injected mice, there was no effect on amyloidosis, but path ological changes were noticed, such as swelling and hyperplasia (Chapter 4). Pare nchymal overexpression of M-CSF in any organ is probably not advisable as overexpr ession of M-CSF and/or its receptor within mammary glands has similarly resulted in tumor formation and hyperplasia (Kirma et al., 2004). We also examined several microglial ma rkers in each CSF-tr eated group of mice, such as Iba-1, CD68, CD11b, CDllc, F4/80, and CD45 (results not shown). However, we did not find any differences with any of the microglial stainings between each brain hemisphere, except for differences in microglia l density which directly correlated with the amyloid plaque burden, as the microglia were found to abundantly decorate the

PAGE 162

152 plaques. Interestly, the mi ce in our study do not get NFTs, but in a recent study of post-mortem human brains of various age and neuropathology (Streit, Braak, Xue, & Bechmann, 2009), microglia are shown to be dystrophic and extremely fragmented around degenerating neurons with tauopathy, such as neuropil threads, NFTs, and neuritic plaques. However, the microglia in non-diseas ed brains, and the microglia in DS and AD brains,which are colocalized with amyloid de position, are shown to be fully ramified and non-activated, while the microglia in patients wi th systemic infectious disease were found to hypertrophic and activated, even in absen ce of identifiable CNS disease. Furthermore, the dystrophic microglial phenotypes preceded tau pathology, according to Braak staging of AD neuropathology, indicating that microglia normally play an important role in neuroprotection, and that a decl ine in their populations within the brain may contribute to onset of AD. M-CSF and AD Although we did not find any difference in amyloidosis from intrahippocampal injections of M-CSF, a recent study, reported th at chronic intraperitoneal (i.p.) injection of M-CSF into Tg AD mice expressing G FP+ bone marrow prevents and reverses amyloid deposition and cognitive impairment, while inducing a large accumulation of GFP+ bone marrow-derived microglia into the brain (Boissonneault et al., 2009). The authors also confirmed previous research (Simard, Soulet, Gowing, Julien, & Rivest, 2006) that bone marrow-derived microglia efficiently phagocytose and internalize A However, differences between these data a nd ours point directly to different study lengths, route of administration, and dosage effects, with Bois sonneault et al. delivering

PAGE 163

153 peripheral chronic 1.3g M-CSF per i.p. injection, as compared to our 5g intrahippocampal bolus. Another recent study has also revealed that M-CSF has significantly diminished levels in AD patients, and that M-CSF ma y have diagnostic potential within an AD biomarker panel (Britschgi & Wyss-Coray, 2008; Ray et al., 2007). Th is same group also investigated whether M-CSF admini stration would improve AD pathology in vivo They found that i.p. injection of M-CSF into 7-8 month old APP transgenic mice (Thy1-hAPP), tested 10 months later, had improved performance in the Morris water maze (Luo J., 2008). Interestingly, there were no changes in microglial activation, or levels of soluble and insoluble A levels in the brains. Moreov er, i.p injection of M-CSF, one day prior to kainite administration, pr evented neurodegeneration and microgliosis, but did not change microglial density. Although these different authors did not re late their findings to RAs inverse relationship with AD, their data does provide evidence that up-regulated M-CSF in RA pathogenesis and systemic administration of M-CSF in AD patients may both impart protection against AD onset or progression. Although the authors have proposed M-CSF as a curative therapy against AD (Boissonneau lt & Rivest, 2009), MCSFs activation of mature osteoclasts, macrophages, and other innate leukocytes, its induced thrombocytopenia (Hubel, Dale, & Liles, 2002; Nemunaitis et al., 1991; Nemunaitis et al., 1993; Zeigler, Rosenfeld, Nemunaitis, Besa, & Shadduck, 1993), and its autocrine signaling in some tumors (Pat sialou et al., 2009) may limit it s usefulness in the clinical setting.

PAGE 164

154 G-CSF and AD While our study of intrahippocampal ad ministration of G-CSF showed only a modest reduction in amyloid burden after one week post-injecti on (Figure 18), other studies have shown greater effects agai nst AD pathology from chronic peripheral administration (Sanchez-Ramos et al., 2009; Tsai, Tsai, & Shen, 2007). In the recent study by Sanchez-Ramos et al. (2009), the authors used PS/APP mice which had been lethally-irradiated and transpla nted with GFP+ bone marrow at 2 months of age. At 6 to 8 months of age, the GFP+ mice showed a significant increase in bone marrow-derived microglia which corresponded with about 40% amyloid reductions. A second cohort of aged predetermined cognitively-impaired mice, which were treated chronically for only 2.5 weeks with G-CSF, demonstrated restorat ion of cognitive function to about half of the difference in errors between the Tg and age-matched control group. This partial reversal of cognitive deficit correlated with decreased amyloid burden, increased microglial density, increased neuronal synaptic area, and increased neurogenic markers. The prior study by Tsai et al. (2007) had also subcutaneously administered G-CSF into Tg AD mouse models and observed some resc ue of cognitive function, which correlated with increased neurogenic staining and with in creased expression of the neurotransmitter, acetylcholine. As with M-CSF, these results were not associated with RAs protective role against AD, but the increased ameliora tive effects of both M-CSF and G-CSF from peripheral adminstration versus intracerebral administration is consistent with the observation that RA, a peripheral disease, can prevent onset of AD, a CNS disease.

PAGE 165

155 GM-CSF and AD Although the G-CSF intrahippocampal findi ngs were encouraging, our GM-CSF intrahippocampal injections into an AD mouse model demonstrated a much more pronounced reduction of amyloidosis (Figures 19 21). These data led us to further examine GM-CSF on the behavior of aged cognitively-impaired AD mice. Daily subcutaneous administration of GM-CSF for 20 days resulted in nearly complete reversal of cognitive impairment, resulting in function similar to that of wild-type mice, with a corresponding average of about 50% reduction of amyloidosis in Entorrhinal cortex and Hippocampus. In contrast, M-CSF and G-C SF were shown to onl y partially reverse cognitive impairment in aged AD mice, although the preventativ e chronic study of MCSF administration into young AD mice show ed equal cognitive function over time compared to wild-type controls. One explanation for this increased benefit with GM-CSF could be due to GM-CSFs overlapping functi ons of both M-CSF and G-CSF, resulting in a combinatory effect of increased macrophage and granulocyte populations derived from bone marrow-derived monocytic precursors in the periphery, as well as induction or activation of other innate le ukocyte subsets in both the pe riphery and brain. Indeed, GMCSF has been shown to cross the blood brai n barrier (McLay, Kimura, Banks, & Kastin, 1997), and a recent study showed that GM-CSF injected into the brains of normal mice activates microglia (Reddy et al., 2009). As mentioned in Ch apter 2 (Malm et al., 2005), only after LPS injection into the hippocampi of the 25-month-old aged mice with established amyloid burden, did reductions in amyloidosis and recruitment of BMDCs occur, as opposed to the 21-month-old non-treated and normally aged Tg mice. A following study also showed that LPS activation of microglia was able to reduce

PAGE 166

156 amyloidosis, possibly through a mechanism of LPS-induced up-regulation of CR3 (CD11b) (Herber et al., 2007). Thus GM-CSF may function to have a role in both the activation of resident microglia, and also in the recruitment of BM DCs to the brain. There may also be further explanations for the very robust cognitive benefits of GM-CSF, including significant re ductions in amyloidosis (Fig ure 24), increased neuronal plasticity (Figure 26), augm entation of peripheral erythropoietic amyloid-clearance mechanisms by increasing CR1 availability (Fisher, 2003; Helmy et al., 2006; Rogers et al., 2006), increased cerebral an giogenesis (U. C. Schneider et al., 2007), neuroprotection from apoptosis (Schabitz et al., 2008) and incr eased neurogenesis (Kruger, Laage, Pitzer, Schabitz, & Schneider, 2007). These effects may also help explain some of the benefits against AD that were desribed in the M-C SF and G-CSF studies. All three CSFs in the periphery function in the mobilization, pro liferation, and differentiation of innate leukocyte populations, but G-C SF and GM-CSF are known to ha ve additional roles in the CNS. Following is a more detailed description of these putative effect s for all three CSFs. Angiogenic Functions of CSFs in the AD Brain Introduction. All three CSFs, when given periphe rally, result in the reduction of amyloid plaques within the brain. This may be due to the increased leukocyte populations that contain receptors, such as CR3 or CR4, which may aid in the phagocytosis of complement-bound amyloid, or by the secretion of proteases from the bone marrow-derived microglia, which may then proteolytically degrade the plaques. The exact mechanisms are not completely known, just that the plaques ar e decreased and that there is an influx of bone marrow-derived mi croglial cells which decorate the plaques. Since A itself has been shown to be anti-angi ogenic (Paris et al., 2010; Patel et al.,

PAGE 167

157 2010), the removal of amyloid plaque would t hus remove this inhibition of angiogenesis and allow for neovascularization, which would in turn allow for increased cerebral blood flow and aid in the availability of more er ythrocytes and leukocyt es to the brain for further plaque clearance and delivery of oxygen and glucose. Much research within the field of tumor biology has shown the ability of innate leukocytes to play a central role in angiogenesis. Thus it would be logical to presume that neovascularization occurs simultaneously with amyloid removal, given that the same myeloi d-derived leukocytes participate in both roles. Angiogenic Mediators. There are several studies that show that various mediators of angiogenesis are found in the AD brain, implying a need and attempt of the brain for neovascularization, although this e ffort is intrinically stymied in AD, and aberrant vascular protrusions emanate from the ends of truncated and occluded vessels around plaques (Figure 1). For instance, angi opoietin-2 and VEGF are expressed in AD microvessels, but are not in control mi crovessels (Thirumangalakudi, Samany, Owoso, Wiskar, & Grammas, 2006). However, the tissu e inhibitors of matrix metalloproteinases (TIMP)-1,2 are also up-regulated in the AD microvessels and in amyloid plaques (Lorenzl et al., 2003; Peress, Perillo, & Zuck er, 1995), while matrix metalloproteinase (MMP)-9 activity, which the TIMPs regulate, is undetectable (Thirumangalakudi et al., 2006). MMPs play a crucial role in promoting angiogenesis by degrading the extracellular matrix (Bergers et al., 2000; Coussens, Tinkle, Hanahan, & Werb, 2000). Studies in PS/APP mice show that MMP-9 and other A -degrading enzymes, such as insulysin and neprilysin, as well as A -binding receptors, such as scavenger receptor A (SRA), CD36, and receptor for advanced-glycation endproducts (RAGE), are all

PAGE 168

158 decreased with age (H ickman, Allison, & El Khoury, 2008). Thus the increase of innate leukocytes, which contain these receptors and proteases, could help remove the plaque and normalize angiogenic mechanisms, that otherwise have both pro-angiogenic and anti-angiogenic factors being simultaneous ly produced in AD microvasculature. Macrophages. Macrophages are very versatile, multifunctional cells that can adapt their functions to the environment in wh ich they reside. As me ntioned earlier, they are usually classified at either M1 or M2 phenotypes. The LPS-induced clearance of amyloid (Herber et al., 2007; Malm et al., 2005 ) could well have been from M1 types of microglia, which would also induce angi ogenic Cox-2 expre ssion within them (Hoozemans et al., 2001). In tumors associ ated macrophages (TAMs), these macrophages obtain an M2-like phenotype that also pr omotes angiogenesis through the release of pro-angiogenic factors, such as VEGF, ba sic fibroblast growth factor (bFGF), TNF IL-1 IL-8, COX-2, plasminogen activator, urokina se (uPA), platelet derived growth factor (PDGF ), and MMPs-7,-9, and -12 (Reviewed in (Dirkx, Oude Egbrink, Wagstaff, & Griffioen, 2006)). TAMs are chemot axically-attracted to areas of hypoxia, where they up-regulate HIF-relate d genes, such as VEGF and NF B, which in turn skews them into their M2 phenotype (Hagemann et al., 2008). Furthermore, recent studies have shown that under sustained stimulation of a ngiogenic growth factors, that monocytes are able to transdifferentiate into endothelial cells (Fernandez Pujol et al., 2000; Kuwana et al., 2006). Another specific subpopulation of circul ating monocytes, which have much more potent pro-angiogenic activity, are identified through their expression of the angiopoietin receptor, TIE2 (Venneri et al., 2007). Thes e TIE2-expressing monocytes (TEMs) have

PAGE 169

159 been found to reside in both perivascular and hypoxic, avascular areas of tumors (De Palma et al., 2005; Venneri et al., 2007), and although TEMs have not been identified in AD, its ligand, angiopoietin-2 is specifically expressed in the AD (Thirumangalakudi et al., 2006) truncated, and occluded microvasculature, thus impl ying that there may indeed be potential involvement of TEMs in the dys regulated vasculogenic activity surrounding amyloid plaques. Myeloid-Derived Suppressor Cells. Another group of pro-angiogenic leukocytes are the myeloid-derived suppre ssor cells (MDSCs), a hetero geneous group of immature precursor cells for neutrophils, monocytes, a nd dendritic cells (DCs). MDSC populations are upregulated by COX-2, prostaglandins, st em-cell factor (SCF) (Pan et al., 2008; Serafini et al., 2004; Sinha, Clements Fulton, & Ostrand-Rosenberg, 2007), VEGF (Gabrilovich et al., 1998), IL-6, M-CSF (Bunt et al., 2007), and GM-C SF (Serafini et al., 2004). While MDSCs are precursors cells eith er resembling an immature neutrophil phenotype (characterized as CD11b+Gr1hi) or an immature monocyte phenotype (characterized as CD11b+Gr1low) (Movahedi et al., 2008; Sawanobori et al., 2008), they are potently immunosuppressive as opposed to mature neutrophils and macrophages. Thus MDSCs are of great interest in can cer research, since th ey suppress immune activation against tumors, and because th ey contribute to angiogenesis via high expression of MMPs (Yang et al., 2004; Ya ng et al., 2008). Therefore in M-CSF and GM-CSF treated AD mice, it could be hypothe sized that the induced MDSCs could release MMPs into the vasc ulature around the plaques and which may counteract the overexpressed TIMPs, thus allowing unobstruc ted VEGF and angiopoietin2 vasculogenic

PAGE 170

160 effects. However, there are no studies yet th at have examined MDSC activity in Tg AD mice, following peripheral administ ration of GM-CSF or M-CSF. Neutrophils. Like their precursor MDSCs, neutr ophils are also pro-angiogenic by their high expression of MMP-9 (Coussens et al., 2000), and studies show that their number correlates with the microvessel de nsity within tumors (Benelli et al., 2002; Mentzel et al., 2001; Van Coilli e et al., 2001). Furthermore, neutrophils do not express TIMPs, allowing them to rapidly promote a ngiogenesis by uninhibited release of MMP-9 (Ardi, Kupriyanova, Deryugina, & Quig ley, 2007). Factors, such as TNF and GM-CSF cause the neutrophils to de-granulate and re lease intracellular stores of pro-angiogenic chemokines, CXCL1 and CXCL8, and MMP-9 (Cassatella, 1999). Ne utrophils, although the most abundant phagocytic leukocyte, are also the shortest lived, with a half-life of within 24 hours from leaving the bone marrow before undergoing apoptosis (Cohen, 1991). In DS patients, neutrophil survival is shortened by up to 30% compared to non-DS individuals, implying the DS patients may have even more anti-angiogenic mechanisms than those described in Chapter 1. Intere stingly, GM-CSF and IL-5 could prevent apoptosis in the neutrophils of control individuals, but not in neutrophils from DS patients (Yasui, Shinozaki, Nakazaw a, Agematsu, & Komiyama, 1999). The administration of G-CSF and GM-CSF into AD mice would have pro-angiogenic effects through induced expression of neutrophil popul ations by both G-CSF and GM-CSF, but GM-CSF administration may also sustain the neutrophil populations against apoptosis longer, providing for increased angi ogenic and phagocytic acitivity. Dendritic Cells. Dendritic cells, the major antig en-presenting cells of the body which are critical in the regulat ion of the adaptive immune re sponse, also play a major

PAGE 171

161 role in angiogenesis, as immature DCs (iDCs) release TNF and CXCL8, which triggers other cells, like neutrophils, to secrete thei r various angiogenic growth factors (Caux et al., 1994). iDCs release othe r factors, such as osteopontin, which has increased concentrations in the cerebrospinal flui d of AD and triggers the release of IL-1 (Comi et al., 2010; Wung et al., 2007), a highly angiogenic cytokine (Naldini et al., 2006). Cytokines, such as VEGF (Dikov et al., 2001; Laxmanan et al., 2005), hepatocyte growth factor (HGH) (Okunishi et al., 2005), TGF (Alard, Clark, & Kosiewicz, 2004), prostaglandin E (Pockaj et al., 2004), and osteopontin (Konno et al., 2006) suppress DC maturation and contribute to the maintenan ce of this pro-angioge nic iDC phenotype Conclusion. Given the angiogenic potential of all of the innate leukocytes described above, and the increased cere bral blood flow that would result from neovascularization, it would be expected for th ese hematopoietic growth factors, M-CSF, G-CSF, and GM-CSF, to have therepeutic eff ects in ischemic diseases. While M-CSF has not been shown to be a potential therapeuti c for treatment of stroke, both G-CSF and GM-CSF have undergone investigation in various models of ischemia. In an experimental model of cerebral focal ischemia, G-CSF in jections up to 3 days post-infarct, was demonstrated to enhance angiogenesis, with increased vascular surface area, vascular branching, de novo endothelial cell prolif eration, and eNOS/angiopoi etin-2 expression, as compared to PBS control injected rats (L ee et al., 2005). Although, there have not been any further experimental cerebral ischemic studies performed with G-CSF which have reported on neovascularization, G-CSF has been shown in a small clinical trial of 5 patients who received G-CSF after acute myocar dial infarction (AMI), as compared to 5 control patients, that six days of daily GCSF treatment increased the vascular growth

PAGE 172

162 factors, VEGF, bFGF, and MCP-1, while im proving ejection fraction and myocardial perfusion and function (Kuethe et al., 2006). GM-CSF, on the other hand, has been s hown to induce neovascularization in several models of experimental stroke. In a rat model of vertebral plus left carotid artery occlusion, s.c. administration of GM-CSF over a course of 3 weeks significantly increased arteriogenesis and functional he modynamic measurements, via up-regulated macrophage populations in the posterior cerebr al artery, as compar ed to saline-treated animals (Buschmann, Busch, Mies, & Hossm ann, 2003). Two similar follow-up studies by this same group showed similar induction of arteriogenesis by GM-CSF injections following variations of experimental hypoperfusion in rat brain (Hossmann & Buschmann, 2005; Schneeloch, Mies, Busc h, Buschmann, & Hossmann, 2004). Other groups have also subsequently shown that GM-CSF treatment completely restores cerebral blood flow following bilateral caroti d artery occlusion (BCO) (U. C. Schneider et al., 2007) and restores le ptomeningeal collaterals fo llowing common carotid artery (CCA) occlusion (Todo et al., 2008). Although G-CSF and GM-CSF have shown some utility in stroke models for the restoration of cerebral blood flow and neovascul arization, they have yet to be described with the same effects in Tg AD mice. A very fine analyses of the microvessels would be needed to examine whether chronic admini stration of these CSFs over time would prevent the buildup of amyloi d deposits on the parenchymal side of the vessels before they progress to the large deposits and severly damaged cerebrovasculature, as seen in Figure 1, as well as whether the microvasculat ure can be repaired by the CSFs after the removal of amyloid deposition in aged mice. Further studies, using VCC models and with

PAGE 173

163 chronic versus acute delivery of the CSFs, w ill be needed to closely examine these effects of innate leukocytes. Neuroprotective Functions of CSFs in the AD Brain GM-CSF. Many of the experiments with stroke models have also found the CSFs to be neuroprotective after induced ischemia. A rat model of middle cerebral artery occlusion (MCAO) of 1 hour duration, in whic h an intracarotid inje ction of GM-CSF or saline upon reperfusion was administered and in which the study animals were assessed 2 days later, showed improved neurological function in the GM-CSF-injected rats, and their subsequent immunohistochemistry re vealed a reduction in infarct volume, an increase of activated microglia or macrophages, and a decrease of a poptotic cells in the penumbra surrounding the infarct, as compared to saline injected rats (Nakagawa, Suga, Kawase, & Toda, 2006). A following study examined the neuroprotective pathways that GM-CSF induces within two stroke models of MCAO and MCA. In this study, the authors identified the alpha-chain of th e GM-CSF receptor (GM-CSFR), and showed GM-CSFR to be up-regulated after ischemia, with it broadly expressed throughout the brain, and with it localized on primary corti cal neurons, and oligodendrocytes, as well as on human neuroblastoma cells. The authors also found that GM-CSF counteracts apoptotosis via the induction of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway, and by induction of the anti-a poptotic proteins Bcl-2 and Bc l-xL, as well as increasing intraparenchymal and leptomen ingeal arterioles (Schabit z et al., 2008). Another MCAO study, combining a glutamate-induced excitotoxicity neuronal injury cell culture model, showed similar alterations of apoptotic related genes and found GM-CSF to be

PAGE 174

164 neuroprotective, with decreas ed infarct volume and improved locomotor function in GM-CSF-treated rats versus controls (Kong et al., 2009). G-CSF. Although G-CSF has only one study ex amining neovascularization in experimental ischemic models (Lee et al., 2005), it has been exte nsively studied since 2003 for its neuroprotective roles after cerebral ischemia. G-CSF has also been shown to protect against glutamate-induced neurotox icity (Han, Blank, Schwab, & Kollmar, 2008; Schabitz et al., 2003), to ha ve induced expression following ischemia (Hasselblatt, Jeibmann, Riesmeier, Maintz, & Schabitz, 2007; Kleinschnitz, Schroeter, Jander, & Stoll, 2004), to reduce infarct volume and improve function after transi ent focal ischemia (Gibson, Bath, & Murphy, 2005, 2010; A. Schneider et al., 2006) and intracebral hemorrhage (Park et al., 2005), and to induce the expression of antiapoptotic proteins and pathways (Komine-Kobayashi et al., 2006; Solaroglu, Cahill, Tsubokawa, Beskonakli, & Zhang, 2009; Solaroglu, Tsuboka wa, Cahill, & Zhang, 2006; Yata et al., 2007). M-CSF. While M-CSF has not been reported to be used therapeutically in stroke models for neovascularization or neuroprot ection, its receptor, MCSFR (also known as c-fms) has been reported to be neuropro tective when overexpressed in a microglialhippocampal oganotypic cocultu re system, in which microglia overexpressing M-CSFR rescued neurons from excitoxicity (Mitrasinov ic et al., 2005). A similar follow-up study using the same coculture system, found that M-CSF treatment prevented NMDA-induced apoptosis (Vincent, Robinson, Simsek, & Murphy, 2002). Cultured microglia overexpressing M-CSF exhibited enhanced phagocytosis of A (Mitrasinovic & Murphy, 2002) and postmortem analyses of AD patients have increased expression of M-CSFR on

PAGE 175

165 microglia (Akiyama et al., 1994), although M-CSF levels itself are decreased in AD patients (Ray et al., 2007). However, as mentioned above, its side effects limit its usefulness in the clinical setting. Neurogenic Functions of CSFs in the AD Brain Both GM-CSF and G-CSF have been reporte d to have neurogenic capabilities. The human analogues to both have been FDA-approved since early 1990s for the mobilization and collection of peripheral blood stem cells (PBSCs) for transplantation (Reviewed in (Hubel et al., 2002)). As was discussed in Chapter 2, bone marrow-derived cells have the ability to ente r the brain and become neuronal or other types of brain cells. However, GM-CSF and G-CSF can both cro ss the BBB, as well as be secreted by neurons and oligodendrocytes. Not only do th ese neuronal cell types contain receptors for each CSF (Schabitz et al., 2003; Schabitz et al., 2008), neural stem cells (NSCs) have also been shown to contain receptors for bot h (Kruger et al., 2007; A. Schneider et al., 2005), and binding of either CSF to their resp ective receptor on NSCs induces time and dosage dependent proliferation and subsequent differentiation. A recent report examining both bone marrow-derived neuronal progenitors versus brain-derived NSCs found that the brain-derived NSCs have a higher cap acity for proliferat ion (Song, Song, Zhang, Cuevas, & Sanchez-Ramos, 2007), although both neuronal progenitors types would be expected to be involved from peripheral admi nstration of the CSFs. In fact the recent study by fellow investigators show parenchym al incorporation of bone marrow-derived neurons after G-CSF peripheral administration (Sanchez-Ramos et al., 2009). Although our studies were not conducted in a manner to examine this eff ect, it is also expected that bone marrow-derived neuronal cells were pr esent within our study, given that GM-CSF

PAGE 176

166 induction encompasses both bone marrow-deri ved macrophage and granulocyte subsets of leukocytes, which have independently been shown to become ne uronal and glial cells following their respective pe ripheral CSF induction Research Theory Summary To restate our theory of what is needed to inhibit and reverse AD pathology, there must first be simultaneous strategies to re move the cerebral amyloidosis and to provide neuroprotection to the micro-penumbras that surround the plaques and occluded vessels. We have shown a remarkable reduction in plaque burden from GM-CSF after just one week following intrahippocampal administra tion, and then similarly by 20 days of subcutaneous delivery. Although, G-CSF had m odest effects intrahippocampally, and MCSF did not, other researchers have shown significant reversal of amyloidosis from peripheral administration. Additionally, many st udies that have also shown each CSF to have neuroprotective properties, which actio ns would simultaneously be needed in the penumbras around the amyloid plaques as the plaques are being cleared. Next after removal of amyloid, we theori zed that there must be strategies to immediately induce neovascula rization in the infarcted areas where the plaques were located and to also induce neuritogenesis an d/or neurogenesis to re build the neural and vascular networks in order to restore cognitive function. While we did not have appropriate models to examine neovasculariz ation, many other mode ls and studies have shown both GM-CSF and G-CSF to be highly pr o-angiogenic. Other studies have also shown both of these CSFs to also be neuroge nic to NSCs, as well as to be peripheral blood progenitor cell mobilizers. Furthermore, it has been found that neurogenesis and neovascularization occur simultaneously with the same cues guiding each together. Thus

PAGE 177

167 with both G-CSF and GM-CSF showing increa sed synaptic area, with G-CSF showing evidence of induced neurogenesis, and with all three CSFs demons trating reversal of cognitive deficits, all aspects of our theory are explained by intrinsic mechanisms of RAs leukocytic pathogenesis. Furthermore, with GM-CSFs pronounced effects over such a short period of time, we are encour aged that the recombinant human GM-CSF will show similar effects in AD patients. Clinical Implications Our results mirror those reported for GCSF (Sanchez-Ramos et al., 2009), and supports the current practice of interchangeable prescription of either recombinant human GM-CSF (Leukine) or G-CSF (Granocyte, Ne upogen, and Neulasta) into patients with depressed bone marrow function. G-CSF pr imarily treats neutropenia while GM-CSF treats all leukopenia, and both have long records of safety data from two decades of FDA-approved usage. Rare adverse events are usually mild febrile incidents that quickly subside upon cessation of administration. G-CSF is currently in clinical trial for stroke and was recently approved for an AD Phase II clinical trial. However, GM-CSF/Leukine is more effective in the AD mouse mode l and while neutrophils are short-lived leukocytes, the fact that GM-CSF induces the up -regulation of all inna te cells means that it could potentially impart prolonged protective effects against AD. Furthermore, Leukine is the only FDA-approved drug to prevent fata l infections in acute myelogenous leukemia (AML) patients, and thus would also be e xpected to provide an tibiotic-free prophylaxic immunity against onset of infection in the recipient AD patients, who at their age and immunodeficient state are highly sus ceptible to microbial opportunism.

PAGE 178

168 Conclusion The failure of NSAID clinical trials in AD and the multiple studies which show that a defective innate immune system propagates AD pathology, encouraged us to develop and test our hypothesis that intrinsic pathogenic properties of RA are protective against AD. The recent 2009 ICAD news by Dr. Breitner of the ADAPT consortium, which reported a 0.33 hazard ratio for dementia within cognitively-normal individuals taking naproxen for over two years, is still more than that of RA patients, in which less than 5% ever develop concomitant AD, further strengthening our hypothesis that other intrinsic factors within RA pathogenesis must underlie the prot ective effect of RA against AD onset. Indeed the beneficial effects of all three RA-upregulat ed CSFs, especially GM-CSF, in mouse models of AD point to a potential new approach to AD therapy and indicate that age-related depressed he matopoiesis may be etiological for AD pathogenesis. References Akiyama, H., Nishimura, T., Kondo, H., Ikeda, K., Hayashi, Y., & McGeer, P. L. (1994). Expression of the receptor for macrophage colony stimulating factor by brain microglia and its upregulation in brains of patients with Alzheimer's disease and amyotrophic lateral sclerosis. Brain Res, 639 (1), 171-174. Alard, P., Clark, S. L., & Kosiewicz, M. M. (2004). Mechanisms of tolerance induced by TGF beta-treated APC: CD4 regulatory T cells prevent the induction of the immune response possibly through a mechanism involving TGF beta. Eur J Immunol, 34(4), 1021-1030. Ardi, V. C., Kupriyanova, T. A., Deryugina, E. I., & Quigley, J. P. (2007). Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis. Proc Natl Acad Sci U S A, 104 (51), 20262-20267. Benelli, R., Morini, M., Carrozzino, F., Ferrari, N., Minghelli, S., Santi, L., et al. (2002). Neutrophils as a key cellular target for angiostatin: imp lications for regulation of angiogenesis and inflammation. Faseb J, 16(2), 267-269. Bergers, G., Brekken, R., McMahon, G., Vu, T. H., Itoh, T., Tamaki, K., et al. (2000). Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol, 2 (10), 737-744.

PAGE 179

169 Boissonneault, V., Filali, M., Lessard, M., Relton, J., W ong, G., & Rivest, S. (2009). Powerful beneficial eff ects of macrophage colony-stimulating factor on betaamyloid deposition and cognitive impairment in Alzheimer's disease. Brain, 132(Pt 4), 1078-1092. Boissonneault, V., & Rivest, S. (2009). [The hematopoietic cytokine M-CSF as a cure for Alzheimer's disease]. Med Sci (Paris), 25 (8-9), 666-668. Britschgi, M., & Wyss-Coray, T. (2008). Blood Protein Signature for the Early Diagnosis of Alzheimer Disease. Arch Neurol Bunt, S. K., Yang, L., Sinha, P., Clements, V. K., Leips, J., & Ostrand-Rosenberg, S. (2007). Reduced inflammation in the tumor microenvironment delays the accumulation of myeloid-derived suppre ssor cells and limits tumor progression. Cancer Res, 67(20), 10019-10026. Buschmann, I. R., Busch, H. J., Mies, G ., & Hossmann, K. A. (2003). Therapeutic induction of arteriogenesis in hypoperfused rat brain via granulocyte-macrophage colony-stimulating factor. Circulation, 108(5), 610-615. Cassatella, M. A. (1999). Neutrophil-derive d proteins: selling cytokines by the pound. Adv Immunol, 73 369-509. Caux, C., Massacrier, C., Vanbervliet, B., Duboi s, B., Van Kooten, C., Durand, I., et al. (1994). Activation of human dendriti c cells through CD40 cross-linking. J Exp Med, 180(4), 1263-1272. Cohen, J. J. (1991). Programmed ce ll death in the immune system. Adv Immunol, 50, 5585. Comi, C., Carecchio, M., Chiocchetti, A., Nicola, S., Galimberti, D., Fenoglio, C., et al. (2010). Osteopontin is increased in the cerebrospinal fluid of patients with Alzheimer's disease and its levels correlate with cognitive decline. J Alzheimers Dis, 19(4), 1143-1148. Coussens, L. M., Tinkle, C. L., Hanahan, D., & Werb, Z. (2000). MMP-9 supplied by bone marrow-derived cells contribu tes to skin carcinogenesis. Cell, 103(3), 481490. De Palma, M., Venneri, M. A., Galli, R., Sergi Sergi, L., Politi, L. S., Sampaolesi, M., et al. (2005). Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell, 8 (3), 211-226. Dikov, M. M., Oyama, T., Cheng, P., Takahashi, T., Takahashi, K., Sepetavec, T., et al. (2001). Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells. Cancer Res, 61 (5), 2015-2021. Dirkx, A. E., Oude Egbrink, M. G., Wags taff, J., & Griffioen, A. W. (2006). Monocyte/macrophage infiltration in tu mors: modulators of angiogenesis. J Leukoc Biol, 80 (6), 1183-1196. Fernandez Pujol, B., Lucibello, F. C., Gehling, U. M., Lindemann, K., Weidner, N., Zuzarte, M. L., et al. (2000). Endothe lial-like cells derived from human CD14 positive monocytes. Differentiation, 65 (5), 287-300. Fisher, J. W. (2003). Erythropoie tin: physiology and pharmacology update. Exp Biol Med (Maywood), 228 (1), 1-14. Gabrilovich, D., Ishida, T., Oyama, T., Ran, S., Kravtsov, V., Nadaf, S., et al. (1998). Vascular endothelial growth factor inhibits the de velopment of dendritic cells and

PAGE 180

170 dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood, 92 (11), 4150-4166. Gibson, C. L., Bath, P. M., & Murphy, S. P. (2005). G-CSF reduces infarct volume and improves functional outcome after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab, 25 (4), 431-439. Gibson, C. L., Bath, P. M., & Murphy, S. P. (2010). G-CSF administration is neuroprotective following transient cerebral ischemia even in the absence of a functional NOS-2 gene. J Cereb Blood Flow Metab, 30 (4), 739-743. Hagemann, T., Lawrence, T., McNeish, I., Charles, K. A., Kulbe, H., Thompson, R. G., et al. (2008). "Re-educating" tumor-a ssociated macrophages by targeting NFkappaB. J Exp Med, 205 (6), 1261-1268. Han, J. L., Blank, T., Schwab, S., & Kollmar, R. (2008). Inhibited glutamate release by granulocyte-colony stimulating factor after experimental stroke. Neurosci Lett, 432(3), 167-169. Hasselblatt, M., Jeibmann, A., Riesmeier, B ., Maintz, D., & Schabitz, W. R. (2007). Granulocyte-colony stimulating factor (G -CSF) and G-CSF receptor expression in human ischemic stroke. Acta Neuropathol, 113 (1), 45-51. Helmy, K. Y., Katschke, K. J., Jr., Gorgani, N. N., Kljavin, N. M., Elliott, J. M., Diehl, L., et al. (2006). CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell, 124(5), 915-927. Herber, D. L., Mercer, M., Roth, L. M., Sy mmonds, K., Maloney, J., Wilson, N., et al. (2007). Microglial activation is required for Abeta clearance after intracranial injection of lipopolysaccharid e in APP transgenic mice. J Neuroimmune Pharmacol, 2(2), 222-231. Hickman, S. E., Allison, E. K., & El K houry, J. (2008). Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci, 28 (33), 8354-8360. Hoozemans, J. J., Rozemuller, A. J., Janssen, I., De Groot, C. J., Veerhuis, R., & Eikelenboom, P. (2001). Cyclooxygenase expr ession in microglia and neurons in Alzheimer's disease and control brain. Acta Neuropathol, 101 (1), 2-8. Hossmann, K. A., & Buschmann, I. R. (2005). Granulocyte-macrophage colonystimulating factor as an arteriogenic fact or in the treatment of ischaemic stroke. Expert Opin Biol Ther, 5 (12), 1547-1556. Hubel, K., Dale, D. C., & Liles, W. C. (2002) Therapeutic use of cytokines to modulate phagocyte function for the treatment of in fectious diseases: current status of granulocyte colony-stimulating fact or, granulocyte-macrophage colonystimulating factor, macrophage colony-s timulating factor, and interferon-gamma. J Infect Dis, 185 (10), 1490-1501. Kaku, M., Tsutsui, K., Motokawa, M., Kawa ta, T., Fujita, T., Kohno, S., et al. (2003). Amyloid beta protein deposition and neur on loss in osteopetrotic (op/op) mice. Brain Res Brain Res Protoc, 12 (2), 104-108. Kawata, T., Tsutsui, K., Kohno, S., Kaku, M., Fujita, T., Tenjou, K., et al. (2005). Amyloid beta protein deposition in oste opetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor. J Int Med Res, 33 (6), 654660.

PAGE 181

171 Kirma, N., Luthra, R., Jones, J., Liu, Y. G., Nair, H. B., Mandava, U., et al. (2004). Overexpression of the colony-stimulating f actor (CSF-1) and/or its receptor c-fms in mammary glands of transgenic mice results in hyperplasia and tumor formation. Cancer Res, 64 (12), 4162-4170. Kleinschnitz, C., Schroeter, M., Jander, S., & Stoll, G. (2004). Induction of granulocyte colony-stimulating factor mRNA by focal cerebral ischemia and cortical spreading depression. Brain Res Mol Brain Res, 131 (1-2), 73-78. Komine-Kobayashi, M., Zhang, N., Liu, M., Tanaka, R., Hara, H., Osaka, A., et al. (2006). Neuroprotective e ffect of recombinant human granulocyte colonystimulating factor in transient focal ischemia of mice. J Cereb Blood Flow Metab, 26(3), 402-413. Kondo, Y., Lemere, C. A., & Seabrook, T. J. (2007). Osteopetrotic (op/op) mice have reduced microglia, no Abeta deposition, and no changes in dopaminergic neurons. J Neuroinflammation, 4 31. Kong, T., Choi, J. K., Park, H., Choi, B. H ., Snyder, B. J., Bukhari, S., et al. (2009). Reduction in programmed cell death and im provement in functional outcome of transient focal cerebral ischemia after administration of granulocyte-macrophage colony-stimulating factor in ra ts. Laboratory investigation. J Neurosurg, 111 (1), 155-163. Konno, S., Eckman, J. A., Plunkett, B., Li, X., Berman, J. S., Sc hroeder, J., et al. (2006). Interleukin-10 and Th2 cytokines differen tially regulate osteopontin expression in human monocytes and dendritic cells. J Interferon Cytokine Res, 26 (8), 562-567. Kruger, C., Laage, R., Pitzer, C., Schab itz, W. R., & Schneider, A. (2007). The hematopoietic factor GM-CSF (granul ocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neur al stem cells in vitro. BMC Neurosci, 8, 88. Kuethe, F., Krack, A., Fritzenwanger, M., He rzau, M., Opfermann, T., Pachmann, K., et al. (2006). Treatment with granulocyte-col ony stimulating factor in patients with acute myocardial infarction. Evidence for a stimulation of neovascularization and improvement of myocardial perfusion. Pharmazie, 61(11), 957-961. Kuwana, M., Okazaki, Y., Kodama, H., Satoh, T., Kawakami, Y., & Ikeda, Y. (2006). Endothelial differentiation potential of human monocyte-derived multipotential cells. Stem Cells, 24 (12), 2733-2743. Laxmanan, S., Robertson, S. W., Wang, E., Lau, J. S., Briscoe, D. M., & Mukhopadhyay, D. (2005). Vascular endothelial growth f actor impairs the functional ability of dendritic cells through Id pathways. Biochem Biophys Res Commun, 334 (1), 193198. Lee, S. T., Chu, K., Jung, K. H., Ko, S. Y ., Kim, E. H., Sinn, D. I., et al. (2005). Granulocyte colony-stimulating factor enhances angiogenesis after focal cerebral ischemia. Brain Res, 1058 (1-2), 120-128. Lorenzl, S., Albers, D. S., LeWitt, P. A., Chirichigno, J. W., Hilgenberg, S. L., Cudkowicz, M. E., et al. (2003). Tissue inhibitors of matrix metalloproteinases are elevated in cerebrospinal flui d of neurodegenerative diseases. J Neurol Sci, 207(1-2), 71-76. Luo J., P. F. C., Britschgi M.,Narasimhan R.,Zhang H.,Wong G.,Relton J.,Wyss-Coray T. (2008, November 19, 2008). Macrophage colony-stim ulating factor is

PAGE 182

172 neuroprotective and ameliorates memory de ficits in an Alzheime r's disease mouse model. Paper presented at the 2008 Neuroscience Meeting, Washington, DC. Malm, T. M., Koistinaho, M., Parepalo, M., Vatanen, T., Ooka, A., Karlsson, S., et al. (2005). Bone-marrow-derived cells contri bute to the recruitm ent of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol Dis, 18 (1), 134-142. Mantovani, A., Sozzani, S., Locati, M., A llavena, P., & Sica, A. (2002). Macrophage polarization: tumor-associated macrophage s as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol, 23(11), 549-555. McLay, R. N., Kimura, M., Banks, W. A., & Kastin, A. J. (1997). Granulocytemacrophage colony-stimulating factor cr osses the blood--brain and blood--spinal cord barriers. Brain, 120 ( Pt 11) 2083-2091. Mentzel, T., Brown, L. F., Dvorak, H. F., Kuhn en, C., Stiller, K. J., Katenkamp, D., et al. (2001). The association between tum our progression and vascularity in myxofibrosarcoma and myxoi d/round cell liposarcoma. Virchows Arch, 438(1), 13-22. Mitrasinovic, O. M., Grattan, A., Robinson, C. C., Lapustea, N. B., Poon, C., Ryan, H., et al. (2005). Microglia overexpressing th e macrophage colony-stimulating factor receptor are neuroprotective in a micr oglial-hippocampal organotypic coculture system. J Neurosci, 25 (17), 4442-4451. Mitrasinovic, O. M., & Murphy, G. M., Jr. (2002). Accelerated phagocytosis of amyloidbeta by mouse and human microglia overexpressing the macrophage colonystimulating factor receptor. J Biol Chem, 277 (33), 29889-29896. Movahedi, K., Guilliams, M., Van den Bossche, J., Van den Bergh, R., Gysemans, C., Beschin, A., et al. (2008). Identificati on of discrete tumor-induced myeloidderived suppressor cell subpopulations with distinct T cell-suppressive activity. Blood, 111(8), 4233-4244. Murdoch, C., Muthana, M., Coffelt, S. B., & Lewis, C. E. (2008). The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer, 8 (8), 618-631. Nakagawa, T., Suga, S., Kawase, T., & Toda, M. (2006). Intracarotid injection of granulocyte-macrophage colony-stimulating factor induces neuroprotection in a rat transient middle cerebral artery occlusion model. Brain Res, 1089(1), 179-185. Naldini, A., Leali, D., Pucci, A., Morena, E ., Carraro, F., Nico, B., et al. (2006). Cutting edge: IL-1beta mediates the proangioge nic activity of osteopontin-activated human monocytes. J Immunol, 177 (7), 4267-4270. Nemunaitis, J., Meyers, J. D., Buckner, C. D., Shannon-Dorcy, K., Mori, M., Shulman, H., et al. (1991). Phase I trial of recombinant human macrophage colonystimulating factor in patients with invasive f ungal infections. Blood, 78 (4), 907913. Nemunaitis, J., Shannon-Dorcy, K., Appelbaum, F. R., Meyers, J., Owens, A., Day, R., et al. (1993). Long-term follow-up of patient s with invasive fungal disease who received adjunctive ther apy with recombinant hu man macrophage colonystimulating factor. Blood, 82 (5), 1422-1427. Okunishi, K., Dohi, M., Nakagome, K., Tana ka, R., Mizuno, S., Matsumoto, K., et al. (2005). A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function. J Immunol, 175 (7), 4745-4753.

PAGE 183

173 Pan, P. Y., Wang, G. X., Yin, B., Ozao, J., K u, T., Divino, C. M., et al. (2008). Reversion of immune tolerance in advanced mali gnancy: modulation of myeloid-derived suppressor cell development by blocka de of stem-cell factor function. Blood, 111(1), 219-228. Paris, D., Patel, N., Ganey, N. J., Laporte, V., Quadros, A., & Mullan, M. J. (2010). AntiTumoral Activity of a Short Decapeptide Fragment of the Alzheimer's Abeta Peptide. Int J Pept Res Ther, 16 (1), 23-30. Park, H. K., Chu, K., Lee, S. T., Jung, K. H., Kim, E. H., Lee, K. B., et al. (2005). Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage. Brain Res, 1041 (2), 125-131. Patel, N. S., Mathura, V. S., Bachmeier, C., Beaulieu-Abdelahad, D., Laporte, V., Weeks, O., et al. (2010). Alzheimer's beta-amylo id peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2. J Neurochem, 112(1), 66-76. Patsialou, A., Wyckoff, J., Wang, Y., Goswami, S., Stanley, E. R., & Condeelis, J. S. (2009). Invasion of human breast cancer ce lls in vivo requires both paracrine and autocrine loops involving the col ony-stimulating factor-1 receptor. Cancer Res, 69(24), 9498-9506. Peress, N., Perillo, E., & Zucker, S. (1995). Localization of tissue inhibitor of matrix metalloproteinases in Alzheimer's disease and normal brain. J Neuropathol Exp Neurol, 54(1), 16-22. Pockaj, B. A., Basu, G. D., Pathangey, L. B., Gr ay, R. J., Hernandez, J. L., Gendler, S. J., et al. (2004). Reduced T-cell and dend ritic cell function is related to cyclooxygenase-2 overexpression and prostaglan din E2 secretion in patients with breast cancer. Ann Surg Oncol, 11 (3), 328-339. Ray, S., Britschgi, M., Herbert, C., Takeda-U chimura, Y., Boxer, A., Blennow, K., et al. (2007). Classification and pr ediction of clinical Alzheimer's diagnosis based on plasma signaling proteins. Nat Med, 13 (11), 1359-1362. Reddy, P. H., Manczak, M., Zhao, W., Naka mura, K., Bebbington, C., Yarranton, G., et al. (2009). Granulocyte-macrophage colony-stimulating factor antibody suppresses microglial activity: implicati ons for anti-inflammatory effects in Alzheimer's disease and multiple sclerosis. J Neurochem, 111 (6), 1514-1528. Rogers, J., Li, R., Mastroeni, D., Grover, A., Leonard, B., Ahern, G., et al. (2006). Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes. Neurobiol Aging, 27(12), 1733-1739. Sanchez-Ramos, J., Song, S., Sava, V., Catlow, B., Lin, X., Mori, T., et al. (2009). Granulocyte Colony Stimulating Factor (G-CSF) Decreases Brain Amyloid Burdenand Reverses Cognitive Impairment in Alzheimer's Mice. Neuroscience. Sawanobori, Y., Ueha, S., Kurachi, M., Shimao ka, T., Talmadge, J. E., Abe, J., et al. (2008). Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice. Blood, 111(12), 5457-5466. Schabitz, W. R., Kollmar, R., Schwaninger, M ., Juettler, E., Bardut zky, J., Scholzke, M. N., et al. (2003). Neuroprote ctive effect of granulocyte colony-stimulating factor after focal cerebral ischemia. Stroke, 34(3), 745-751.

PAGE 184

174 Schabitz, W. R., Kruger, C., Pitzer, C., Weber, D., Laage, R., Gassler, N., et al. (2008). A neuroprotective function for the hematopoi etic protein granulocyte-macrophage colony stimulating factor (GM-CSF). J Cereb Blood Flow Metab, 28 (1), 29-43. Schneeloch, E., Mies, G., Busch, H. J., Buschmann, I. R., & Hossmann, K. A. (2004). Granulocyte-macrophage colony-stimula ting factor-induced arteriogenesis reduces energy failure in hemodynamic stroke. Proc Natl Acad Sci U S A, 101(34), 12730-12735. Schneider, A., Kruger, C., Steigleder, T., Weber, D., Pitzer, C., Laage, R., et al. (2005). The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J Clin Invest, 115 (8), 2083-2098. Schneider, A., Wysocki, R., Pitzer, C., Kruger, C., Laage, R., Schwab, S., et al. (2006). An extended window of opportunity for GCSF treatment in cerebral ischemia. BMC Biol, 4 36. Schneider, U. C., Schilling, L., Schroeck, H ., Nebe, C. T., Vajkoczy, P., & Woitzik, J. (2007). Granulocyte-macrophage colony-stimulating factor-induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion. Stroke, 38(4), 1320-1328. Serafini, P., Carbley, R., Noonan, K. A., Ta n, G., Bronte, V., & Borrello, I. (2004). Highdose granulocyte-macrophage colony-s timulating factor-p roducing vaccines impair the immune response through the re cruitment of myeloid suppressor cells. Cancer Res, 64(17), 6337-6343. Simard, A. R., Soulet, D., Gowing, G., Julie n, J. P., & Rivest, S. (2006). Bone marrowderived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron, 49 (4), 489-502. Sinha, P., Clements, V. K., Fulton, A. M., & Ostrand-Rosenberg, S. (2007). Prostaglandin E2 promotes tumor progression by inducing myeloid-derived suppressor cells. Cancer Res, 67 (9), 4507-4513. Solaroglu, I., Cahill, J., Tsubokawa, T., Beskonakli, E., & Zhang, J. H. (2009). Granulocyte colony-stimulating factor protects the brain ag ainst experimental stroke via inhibition of apoptosis and inflammation. Neurol Res, 31 (2), 167-172. Solaroglu, I., Tsubokawa, T., Cahill, J., & Zha ng, J. H. (2006). Anti-apoptotic effect of granulocyte-colony stimulating factor afte r focal cerebral isch emia in the rat. Neuroscience, 143 (4), 965-974. Song, S., Song, S., Zhang, H., Cuevas, J., & Sanchez-Ramos, J. (2007). Comparison of neuron-like cells derived from bone marro w stem cells to those differentiated from adult brain neural stem cells. Stem Cells Dev, 16(5), 747-756. Streit, W. J., Braak, H., Xue, Q. S., & Bech mann, I. (2009). Dystrophi c (senescent) rather than activated microglial cells are asso ciated with tau pathology and likely precede neurodegeneration in Alzheimer's disease. Acta Neuropathol, 118 (4), 475-485. Thirumangalakudi, L., Samany, P. G., Owoso, A., Wiskar, B., & Grammas, P. (2006). Angiogenic proteins are expr essed by brain blood vessels in Alzheimer's disease. J Alzheimers Dis, 10(1), 111-118. Todo, K., Kitagawa, K., Sasaki, T., Omura-Mats uoka, E., Terasaki, Y., Oyama, N., et al. (2008). Granulocyte-macrophage colony-stimulating factor enhances

PAGE 185

175 leptomeningeal collateral growth indu ced by common carotid artery occlusion. Stroke, 39(6), 1875-1882. Tsai, K. J., Tsai, Y. C., & Shen, C. K. (2007). G-CSF rescues the memory impairment of animal models of Alzheimer's disease. J Exp Med, 204 (6), 1273-1280. Van Coillie, E., Van Aelst, I., Wuyts, A., Vercauteren, R., Devos, R., De Wolf-Peeters, C., et al. (2001). Tumor angiogenesis induced by granulocyte chemotactic protein-2 as a counter current principle. Am J Pathol, 159 (4), 1405-1414. Venneri, M. A., De Palma, M., Ponzoni, M., Pu cci, F., Scielzo, C., Zonari, E., et al. (2007). Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer. Blood, 109(12), 5276-5285. Vincent, V. A., Robinson, C. C., Simsek, D., & Murphy, G. M. (2002). Macrophage colony stimulating factor prevents NMDA-induced neuronal death in hippocampal organotypic cultures. J Neurochem, 82 (6), 1388-1397. Wung, J. K., Perry, G., Kowalski, A., Harris, P. L., Bishop, G. M., Trivedi, M. A., et al. (2007). Increased expression of the rem odelingand tumorigenic-associated factor osteopontin in pyramidal neur ons of the Alzheimer's disease brain. Curr Alzheimer Res, 4(1), 67-72. Yang, L., DeBusk, L. M., Fukuda, K., Fingleton, B., Green-Jarvis, B., Shyr, Y., et al. (2004). Expansion of myel oid immune suppressor Gr+CD11b+ cells in tumorbearing host directly promotes tumor angiogenesis. Cancer Cell, 6 (4), 409-421. Yang, L., Huang, J., Ren, X., Gorska, A. E ., Chytil, A., Aakre, M., et al. (2008). Abrogation of TGF beta signaling in mammary carcinomas recruits Gr1+CD11b+ myeloid cells that promote metastasis. Cancer Cell, 13 (1), 23-35. Yasui, K., Shinozaki, K., Nakazawa, T., Agematsu, K., & Komiyama, A. (1999). Presenility of granulocytes in Down syndrome individuals. Am J Med Genet, 84(5), 406-412. Yata, K., Matchett, G. A., Tsubokawa, T., Tang, J., Kanamaru, K., & Zhang, J. H. (2007). Granulocyte-colony stimulating fact or inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats. Brain Res, 1145 227-238. Zeigler, Z. R., Rosenfeld, C. S., Nemunaitis, J. J., Besa, E. C., & Shadduck, R. K. (1993). Increased macrophage colony-stimulating factor levels in immune thrombocytopenic purpura. Blood, 81 (5), 1251-1254.

PAGE 186

176 APPENDIX A: A NOVEL TECHNIQUE FOR SIMULTANEOUS BILATERAL INFUSIONS IN A MOUSE MODEL OF NEURODEGENERATIVE DISEASE The manuscript entitled, A novel technique for simultaneous bilateral infusions in a mouse model of neurodegenerative dis ease, by Steven P. Bennett et al., was accepted for publication on August 27, 2009 in th e Journal of Neuroscience Methods. The device developed and described within th is manuscript provided the scientific and technological framework upon which the findi ngs of GM-CSF against AD were made. Following is a copy of this manuscript as it can be found in the Journal of Neuroscience Methods and as it can be accessed through a search of the online PubMed database.

PAGE 187

177

PAGE 188

178

PAGE 189

179

PAGE 190

180

PAGE 191

181

PAGE 192

182

PAGE 193

183

PAGE 194

184 APPENDIX B: GM-CSF UP-REGULATED IN RHEUMATOID ARTHRITIS REVERSES COGNITIVE IMPAIRMENT AND AMYLOIDOSIS IN ALZHEIMERS MICE Following is the acceptance email from th e Journal of Alzheimers Disease of the latest revision of the manuscript enti tled, GM-CSF Up-regulated in Rheumatoid Arthritis Reverses Cognitive Impairment and Amyloidosis in Alzheimers Mice. Curently, we are expecting back proofs of th e manuscript as it will be presented in the journal, and thus the final version is not available for this dissertation.

PAGE 195

185

PAGE 196

186 GM-CSF up-regulated in Rheumatoid Arthritis reverses cognitive impairment and amyloidosis in Alzheimer mice Tim D. Boyd, PhDc 1,2,3, Steven P. Bennett, PhD1,2,3, Takashi Mori, DVM,PhD,7 Nickolas Governatori, BS5, Melissa Runfeldt,BS5, Michelle Norden1, Jaya Padmanabhan, PhD1,2, Peter Neame, PhD1,2, Inge Wefes, PhD2,3, Juan SanchezRamos, PhD, MD1,4, Gary W. Arendash, PhD5,6 Huntington Potter, PhD1,2,3,6 *Both authors contributed equally to this work 1-USF Health Byrd Alzheimers Center and Research Institute, 4001 Fletcher Ave., Tampa, Florida 33613, USA 2Department of Molecular Medicine, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, Florida 33612, USA 3-Eric Pfeiffer Suncoast Gerontology Center, University of South Florida, 4001 Fletcher Ave., Tampa, Florida 33613, USA 4Department of Neurology, College of Medi cine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, Florida 33612, USA 5Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 East Fowler Ave, SCA110 Tampa, Florida 33620, USA 6-Florida Alzheimers Disease Research Center University of South Florida, 4001 Fletcher Ave., Tampa, Florida 33613, USA 7-Departments of Medical Science and Pat hology, Saitama Medical Center and Saitama Medical University, 1981 Kamoda, Kawagoe, Saitama, Japan Corresponding Author :

PAGE 197

187 Huntington Potter, PhD. Johnnie B. Byrd Sr. Alzheimers Center and Research Institute at the University of South Florida 4001 E. Fletcher Ave. Tampa, Florida 33613 E-mail: hpotter@health.usf.edu phone: 813-396-0660 fax: 813-971-0373 Potential conflict of interest: Nothing to report Running title: Boyd et al: Arthritis CSF reverses AD in mice

PAGE 198

188 ABSTRACT Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimers disease (AD). While it has been commonly assumed that RA patients usage of nonsteroidal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of AD, NSAID clinical trials have proven unsuccessful in AD patients. To determine whether intrinsic factors within RA pathogenesis itself may underlie RAs protective effect, we investigated the activity of colony-stimulating factors, up-regulated in RA, on the pathology and behavior of transgenic AD mice. 5 g bolus injections of macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factors (M-CSF, G-CSF, or GM-CSF) were administered unilaterally into the hippocampus of aged cognitivelyimpaired AD mice and the resulting amyloid load reductions determined one week later, using the artificial cerebrospinal fluid-injected contralateral sides as controls. G-CSF and more significantly, GM-CSF reduced amyloidosis throughout the treated brain hemisphere one week following bolus administration to AD mice. 20 daily subcutaneous injections of 5g of GM-CSF (the most am yloid-reducing CSF in the bolus experiment) were administered to balanced cohorts of AD mice after assessment in a battery of cognitive tests. Reductions in amyloid load and improvements in cognitive function were assessed. Subcutaneous GM-CSF administratio n significantly reduced brain amyloidosis and completely reversed the cognitive impairment, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, recombinant hum an GM-CSF, in elderly leukopenic patients, suggest that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

PAGE 199

189 Keywords: Granulocyte-Macrophage Colony-Stimulating Factor, Rheumatoid arthritis, Alzheimers disease, amyloid, Radial Arm Water Maze, Cognitive Interference Task, transgenic mice, intrahippocampal, subcutaneous

PAGE 200

190 Introduction Although numerous studies have reported Rheumatoi d arthritis (RA) to reduce the risk of Alzheimers disease (AD), the mechanisms for RAs protective effect are still unknown [1]. It was proposed and commonly assumed that RA patients usage of non-steroidal anti-inflammatory drugs (NSAIDs) may help prevent the onset and progression of AD [1]. However, the largest NSAID clinical trials have not demonstrated efficacy in reducing the incidence of dementia, and recently Naproxen was reported to be detrimental, with increased risk of cardiovascular and cerebrovascular events [2]. These results suggested to us that intrinsic, probably immunological factors within RA pathogenesis itself may underlie RAs protective effect against AD. We surmised that up-regulated local cellular populations in RA would have the highest potential to enter into the brain and inhibit the development of AD pathology and/or neuronal dysfunction. Alzheimers disease is an age-related, progressive neurodegenerative disorder that presents as increasing decline in cognitive and executive function. Alzheimer dementia is associated with cerebrovascula r dysfunction [3], extracellular accumulation of amyloid (A ) peptides in the brain parenchyma and vasculature walls [4, 5] (predominantly A1-42 and A 1-40), and intraneuronal accumulation of neurofibrillary tangles consisting of hyperphosphorylated Tau proteins[6]. Associated neuroinflammation may contribute to AD pathogenesis [7], as the inflammatory proteins apolipoprotein E (apoE) and 1-Antichymotrypsin (ACT) catalyze the polymerization of A peptides into amyloid filaments in vivo and in vitro [8-11], and ACT has been shown to induce the phosphorylation of Tau [12]. Conversely, it has also been shown that amyloid plaques form rapidly and then become decorated by microglia [13, 14], both resident and bone marrow-derived, suggesting an ability and intention to remove amyloid [15-17]. Thus it is unclear whether neuroinflammation is deleterious or beneficial

PAGE 201

191 in the AD brain, and indeed the role of microglia in AD is complex and may involve different states of activation with different activities. Rheumatoid arthritis is an autoimmune disease in which inflamed synovial tissue and highly vascularized pannus form, irreparably damaging the cartilage and bone. In this inflammatory pannus, leukocyte populations are greatly expanded, perhaps as an endogenous, but ineffective attempt to remove the inflammatory insult. As a result, many proinflammatory factors are produced that work together in feed-forward mechanisms, further increasing leukocytosis, cytokine/chemokine release, osteoclastogenesis, angiogenesis, and autoantibody production (rheumatoid factors and anti-citrullinated protein antibodies) [18, 19]. Additionally, the adaptive immune system presents a Th17 phenotype within CD4+ lymphocytes, with ultimate production of interleukin 17 (IL-17) which is then responsible for inducing much of the pro-inflammatory effects [20, 21]. Further enhancements of leukocyte populations come from increased expression of structurallyunrelated colony-stimulating factors: M-CSF (macrophage), G-CSF (granulocyte), and GM-CSF (granulocyte-macrophage) [22-25]. Although up-regulated leukocytes in response to RA could potentially enter into the brain and inhibit development of AD pathology and/or neuronal dysfunction, lymphocytic infiltrates into AD patient brains have not been reported. The lack of infiltration suggests that RA-induced proliferation and activation of the innate immune system by the CSFs described above might be responsible for preventing AD pathology in RA patients. Evidence supporting the innate immune systems role in AD pathogenesis show that complement proteins are up-regulated in AD brain, and that inhibition of C3 convertase significantly in creases amyloid pathology in AD mice [26]. Bone marrow-derived microglia also play a critical role in restricting amyloid deposition, and indeed, microglia activation and many associated receptors and enzymes, such as

PAGE 202

192 CD36, scavenger receptor A, and receptor for advanced glycation end products, neprilysin, insulysin, and matrix metalloproteinases, decline with age as risk of AD pathology increases [17, 27, 28]. To investigate the interplay of the innate immune system and AD, we studied the effects on AD pathology of the three colony-stimulating factors (M-CSF, G-CSF, and GM-CSF), which are up-regulated during RA pathogenesis [22-25]. These CSFs enhance the survival and function of their respective leukocytes and drive their proliferation and differentiation from myeloid lineage precursors. GM-CSF induces dendritic cells, macrophages, and granulocytes (neutrophils, basophils, and eosinophils), while M-CSF and G-CSF respectively i nduce the macrophage and granulocyte subsets of the innate immune system. These innate cells have the ability to diapedese from the circulatory system and to differentiate further into various specialized immune cells within organs (microglia, Langerhans cells, et c.). GM-CSF and G-CSF are also known to be involved in erythropoiesis, and GM-CSF and erythropoietin act synergistically in the maturation and proliferation of the burst-forming and colony-forming erythroid units to the normoblast stage of erythropoiesis [29, 30]. Circulating A binds to complement opsonin C3b in an antibody-independent fashion, and C3-opsonized particles bind to the complement receptor, CR1, on erythrocytes and to CR1g on liver-resident kupffer macrophages [31, 32]. Thus GM-CSF could function in both the peripheral clearance of A and in bone marrow-derived microglial activity, since it is involved in the proliferation, differentiation, and maintenance of most innate leukocytes. Here, we report on experiments that investigated the effect of CSF administration on amyloid plaque deposition, microglial activation, synaptic function, and associated cognitive decline in a mouse model of AD. Our results, particularly with GM-CSF, provide a compelling explanation for RAs in verse relationship with AD. Moreover, the

PAGE 203

193 reduction of amyloidosis and enhancement of cognition by GM-CSF warrant clinical investigation of Leukine for the treatment of Mild Cognitive Impairment (MCI) and AD patients, especially with Leukines long-standing safety history in leukopenic patients.

PAGE 204

194 Materials and Methods All procedures involving experimentation on ani mals were performed in accordance with the guidelines set forth by the University of South Florida Animal Care and Use Committee. Transgene detections were performed using QPCR (Bio-Rad iCycler, Hercules, CA). Transgenic Mouse Studies Involving Intracerebral Administration of CSFs PS/APP mice in this study, which begin accumulating robust amyloid plaques at 6-8 months, were generated by crossing heterozygous PDGF-hAPP (V717F) mice with PDGF-hPS1 (M146L) on both Swiss Webster and C57BL/6 backgrounds. Bilateral Intracerebroventricular infusion of M-CSF M-CSF was bilaterally infused directly into the lateral ventricles (5 g/day) for 14 days using a novel intracranial catheter infusion system (patent pending PCT/US08/73974) [33]. This completely subcutaneously-contained system allows bilateral intracerebral infusion of test substances ipsilaterally and vehicle contralaterally, and overcomes the problem of amyloidosis variance between animals (Supplementary Fig. 1), effectively making each animal its own control. Briefly, animals (PS/APP, all 8.8 -9.6 months, numbered sequentially according to date of birth, 25-35 g, both genders) were anesthetized with 1-2% isoflurane, shaved and scrubbed with 10% Betadine solution at the site of incision, and placed into a stereotaxic frame (Kopf Instruments, Tujunga, Ca.). A small (3 cm) incision was made, exposing the skull, and curved Strabysmus surgical scissors were used to form a subcutaneous pocket along the animals back into which 2 osmotic minipumps (Alzet model 1004, average flow rate of 0.12 L/hour, Durect Corp., Cupertino, CA) were inserted. Two holes were drilled into the skull (from Bregma -0.1 mm anterior-posterior, +/0.9 mm medial-lateral), and 30 gauge catheters were inserted at a depth of 3.0 mm, corresponding to the lateral ventricles. Leading from the Alzet

PAGE 205

195 pump was a proprietary catheter system with the delivery tips fashioned to the contours of the skull rather than the commercially-available pedestal cannula. The cannulae are affixed to the skull using Locktite 454 adhesive (Plastics One, Roanoke, VA) and secured with 1 cm diameter nitrile, followed by silk sutures to close the scalp. After 2 weeks of M-CSF infusions, mice were perfused, brain tissues were fixed in 10% neutral buffered forma lin, and cryosectioned at 14 m. Intrahippocampal injections of CSFs All three CSFs were stereotaxically-injected (5 g/injection) into the (ipsilateral) hippocampus, with artificial cerebrospinal fluid vehicle injected contralaterally into four PS/APP mice each (all 10-12 months old, 25-35 g, both genders). Two holes were drilled into the skull (from bregma -2.5 mm anterior-posterior, +/2.5 mm medial-lateral, and the 30 gauge needle inserted to a depth of 2.5 mm). Mice were perfused with 0.9% cold saline 7 days later and their brains placed in 10% neutral buffered formalin. Recombinant mouse GM-CSF (rmGM-CSF) recombinant murine G-CSF (rmG-CSF), and recombinant mouse M-CSF (rmM-CSF) (R&D Systems, Minneapolis, MN) will be referred to as GM-CSF, G-CSF, and M-CSF throughout this publication. Immunohistochemistry and Image Analysis of Intrahippocampal-injected Mice Formalin-fixed brains were either coronally cryosectioned at 14-m, or paraffinembedded and sectioned at 5-m, with standard deparaffination and antigen retrieval steps (boiled in 10mM Sodium Citrate buffer for 20 minutes) performed before immunohistochemical staining. To significant ly reduce cost of reagents and antibodies with paraffin-embedded slides, a novel magnet ic immunohistochemical staining device was developed (patent pending, Tech ID# 09A015). Standard fluorescent immunohistochemical techniques used primary anti-A antibodies 6E10 (Covance, Emeryville, CA, 1:1000), and MabTechs 3740-5 (MabTech, Cincinnati, OH, 1:5000) to

PAGE 206

196 immunolabel amyloid deposition coupled with Alexa fluorophore-labelled secondary antibodies (Molecular Probes, Eugene, OR, 1:1000, 1:4000), and Hoechst (Sigma) nuclear staining. Immunofluorescence was detected and all pictures per section were taken at the same exposure on a Zeiss Imager .Z1 microscope (Oberkochen, Germany) using Axiovision 4.7 software. Digital images were quantified using ImageJ (method described in Supplementary Fig. 6 online). Briefly, each analyzed picture per coronal section was thresholded equally to the same standard deviation from the histogram mean, and analyzed for area, perimeter, feret diameter, and integrated density parameters of each plaque. Area and Perimeter data were calculated from the total and average number of plaque values in each hem isphere per section, and Feret Diameter and Integrated Density were calculated from the average values of the plaques measured in each hemisphere per section. For GM-CSF-injected mice, each section quantified contained analysis of 15-25 individual 10X pictures of each hemisphere, with fewer pictures quantified in the anterior brain and more in posterior brain. For G-CSFand M-CSF-injected mice, each section quantified contained analysis of 7-9 individual 5X pictures of each hemisphere Statistical significance was obtained from comparing parameter values of ipsilateral CSF-injected hemispheres versus contralateral artificial cerebrospinal fluid-injected hemispheres. Significance was determined by paired Students t-test with p values <0.5 considered significant. Behavioral Transgenic Mouse Study Involving GM-CSF Treatment Mice in this study were derived from the Florida Alzheimers Disease Research Center mouse colony, wherein heterozygous mice carrying the mutant APPK670N, M671L gene (APPsw) are routinely crossed with heterozygous PS1 (Tg line 6.2) mice to obtain APPsw/PS1, APPsw, PS1, and non-transgenic (NT) genotype offspring with a mixed C57/B6/SW/SJL background. Eleven APPsw and 17 NT mice, all 12-months old, were

PAGE 207

197 selected and evaluated for 8 days in the RAWM task of working memory (as previously described [34] (Supplementary Fig. 7). Briefly, an aluminum insert was placed into a 100 cm circular pool to create 6 radially distributed swim arms emanating from a central circular swim area. The number of errors prior to locating which one of the 6 swim arms contained a submerged escape platform (9 cm diameter) was determined for 5 trials per day. The platform location was changed daily to a different arm, with different start arms for each of the 5 trials semi-randomly selected from the remaining 5 swim arms. The numbers of errors during trials 4 and 5 are both considered indices of working memory and are temporally similar to the standard r egistration/recall testing of specific items used clinically in evaluating AD patients. Following 8 days of pre-treatment RAWM testing, the 11 Tg mice were divided into two groups, balanced in RAWM performance. The 17 NT mice were also divided into two groups, balanced in RAWM performance. Two weeks following pre-treatment testing, one group of Tg mice ( n = 5) and one group of NT mice ( n = 9) were started on a 10-day treatment protocol with GM-CSF (5 g/day given subcutaneously), while animals in the control Tg group (n=6) and control NT group (n = 8) concurrently received daily vehi cle (saline) treatment subcutaneously. On the 11th day of injections, all mice began four days of RAWM evaluation, were given 2 days of rest, then evaluated in 4 days of Cognitive Interference task testing as previously described [35, 36]. We desi gned this task measure-for-measure from a Cognitive Interference task used to discriminate normal aged, MCI, and AD patients from one another [34]. Our analogous interference task for mice involves two radial arm water maze (RAWM) set-ups in two different rooms, each with different sets of visual cues. The task requires animals to remember a set of visual cues, so that following interference with a different set of cues, the initial set of cues can be recalled to successfully solve the RAWM task. A set of four behavioral measures were examined.

PAGE 208

198 Behavioral measures were: A1A3 (Composite th ree-trial recall score from first 3 trials performed in RAWM A), B (proactive interference measure attained from a single trial in RAWM B), A4 (retroactive interference measure attained during a single trial in RAWM A), and A5(delayed-recall measure a ttained from a single trial in RAWM A following a 20 minute delay between A4 and A5). As a distracter between trials, animals are placed in a Y-maze and allowed to explore for 60 seconds between successive trials of the three-trial recall task, as well as during the proactive interference task. As with the standard RAWM task, this interference task involves the platform location being changed daily to a different arm for both of the RAWM set-ups utilized, and different start arms for each day of testing for both RAWM set-ups. For A1 and B trials, the animal was initially allowed one minute to find the platform on their own before they were guided to the platform. Then the actual trial was performed in each case. As with the standard RAWM task, animals were given 60 seconds to find the escape platform for each trial, with the number of errors recorded for each trial. Animals were tested for cognitive interference performance on four successive days, with statistical analysis performed for the two resultant 2-day blocks. For both RAWM (combined T4 and T5 overall) and cognitive interference testin g (each of the four measures overall), swim speed was analyzed by dividing error numbers by latency and statistical significance was determined by one-way ANO VA followed by post hoc Fishers LSD (least significant difference) test to determine significant group differences at p < 0.05. Daily GM-CSF and saline injections were continued throughout the behavioral testing period. After completion of behavioral testing at 20 days into treatment, all mice were euthanatized, brains fixed as described above, and paraffin-embedded. Careful visual examination of all tissues upon necropsy revealed no morphological abnormalities, and the mice tolerated daily subcutaneous injections well. Each analysis

PAGE 209

199 was done by a single examiner blinded to sample identities, and statistical analyses were performed by a single examiner blinded to treatment group identities. The code was not broken until analyses were completed. Immunohistochemistry and Image Analysis of Subcutaneous GM-CSF-treated Mice Five 5m sections (150m apart) were made of formalin-fixed, paraffin-embedded sections throughout the hippocampus of each mouse and immunoreactivity was developed using the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) coupled with the diaminobenzidine reaction, according to the manufacturers protocol. Immunostaining used biotinylated anti-A clone 4G8 (Covance, Emeryville, CA, 1:200), synaptophysin (DAKO, Carpinteria, CA, undiluted), and Iba1 (Wako, Richmond, VA, 1:1000) as primary antibodies. Since the 4G8 antibody was obtained with biotin label, the secondary step of the ABC protocol was omitted. However, treatment with 70% formic acid prior to the pre-blocking step was necessary. For 4G8 immunohistochemistry, phosphate-buffered saline (0.1 mM, pH 7.4) was used instead of primary antibody or ABC reagent as a negative control. For Iba 1 and synaptophysin immunohistochemistry, normal rabbit serum was used instead of primary antibody or ABC reagent as a negative control. Images were acquired using an Olympus BX60 microscope and digital images were quantified using SimplePCI software (Compix Inc., Imaging Systems, Cranberry Township, PA), according to previous methods [37]. Briefly, a threshold optical density was obtained that discriminated staining from background, and each region of interest was manually edited to eliminate artifacts. Data are reported as percentage of immunolabeled area captured (positive pixels) relative to the full area captured (total pixels). To evaluate synaptophysin immunoreactivity, after the mode of all images was converted to gr ay scale, the average intensity of positive

PAGE 210

200 signals from each image was quantified in t he CA1 and CA3 regions of hippocampus as a relative number from zero (white) to 255 (black). Statistical significance between GM-CSF-treated versus saline-treated groups was determined by two-tailed homoscedastic Students t-test with a p value of < 0.05 considered significant. Each analysis was done by a single investigator blinded to sample identities and genotype.

PAGE 211

201 RESULTS Intracerebral administration of CSFs Following bilateral intracerebroventricular infusion of M-CSF for two weeks into PS/APP mice, immunohistochemical analysis of both experimental and control mice showed considerable variances of amyloid deposition between mice of similar age (Supplementary Fig 1), significantly compromising our ability to determine M-CSFs effect in a limited mouse cohort. While improv ing our drug delivery system by developing novel bilateral brain infusion catheters [33], we found that parenchymally-infused recombinant peptides remained localized to the infused hemisphere. These findings led us to administer the CSFs as a unilateral intrahippocampal bolus with a contralateral injection of vehicle as control, thus obviating the need for large numbers of transgenic mice and age-matched littermate controls to obtain statistical significance. Each CSF was stereotaxically injected into the hippocampus of 4 mice, with artificial cerebrospinal fluid vehicle injected contralaterally. The mice were sacrificed 7 days post-injection. M-CSF intrahippocampal injections into mice resulted in swelling of the entire hemisphere as compared to the control side, and in one mouse, an apparent hyperplasia had formed at the injection site (Supplementary Fig 2). Quantification of amyloid plaque loads from anterior to posterior of each mouse showed similar deposition in the M-CSFinjected hemispheres as compared to the control sides (data not shown). In contrast to M-CSF, G-CSF intrahippocampal injections did not induce swelling and showed some modest reductions of amyloid deposition (Supplementary Fig 3). Since the analyses were conducted on low magnification (5X) photomicrographs, only the area and integrated density values achieved significanc e (p<0.05). Reductions in amyloidosis was subsequently corroborated by independent observations from fellow investigators following peripheral G-CSF administration [37].

PAGE 212

202 GM-CSF-injections, however, demonstrated pronounced decreases in amyloid deposition, as compared to control hemispheres, in visual observations of coronal tissue sections (Fig 1a; Supplementary Fig 4). High magnification (10X) quantification of amyloid plaques anterior to posterior revealed significant reductions within individual mice and significant overall reductions for all plaque parameters measured (Fig 1b and Supplementary Fig 5). Daily subcutaneous injection of GM-CSF Based upon the positive results from intrahippocampal injections, we investigated the effect of subcutaneous GM-CSF injection on AD pathology and cognitive function. Prior to GM-CSF treatment, APPsw (Tg) mice were first confirmed by RAWM testing to be cognitively-impaired for working memory (Fig 2a). Both the non-transgenic control mice (NT) and the Tg mice were then sub-divided into two cognitively-balanced groups, for either GM-CSF or saline treatment. RAWM testing post-injection re-confirmed that Tg control mice were substantially impaired compared to NT control mice. This impairment was not only evident in individual blocks of testing, but also over all 4 days of testing (Fig 2b). In sharp contrast, GM-CSF-treated Tg mice performed equally well or better than NT control mice during individual blocks and overall. GM-CSF-treated NT mice performed as well as or slightly better than NT controls (Fig 2b). Before evaluation in the Cognitive Interference Task, the mice rested two days. This task mimics human interference testing, which discriminates between normal aged, MCI, and AD patients [36]. In three of four cognitive interference measures assessed over 4 days of testing (Fig 2c), Tg control mice were clearly impaired compared to NT mice, and Tg mice treated with GM-CSF exhibited significantly better three-trial recall and delayed recall compared to Tg controls. Indeed, for all four cognitive measures, GM-

PAGE 213

203 CSF-treated transgenic AD mice performed similarly to NT mice. A particularly strong effect of GM-CSF treatment in Tg mice was evident for the proactive interference measure during the first half of testing (Fig 2d), wherein GM-CSF-treated Tg mice performed substantially better than Tg controls and identically to both groups of NT mice. Including this strong effect on proactive interference testing, GM-CSF treatment resulted in significantly better performance of Tg mice for all four measures of cognitive interference testing. Proactive interference susceptibility has been reported to be a more sensitive marker for differentiating MCI and AD patients from aged normals than traditional measures of delayed recall and rate of forgetting [36]. Parenthetically, even the GM-CSF-treated NT mice showed a trend towards improved cognition in behavioral studies, albeit not statistically significant. Analysis of swim speed for both the RAWM and cognitive interference tasks revealed that Tg control mice were significantly faster than the other three groups in the RAWM task and for two of four measures in the Cognitive Interference task (3-trial recall and delayed recall). However, since error numbers were utilized for statistical analysis of both tasks, this difference in swim speed was negated since it is important only if latency measures had been used. Following completion of all behavioral evaluations, subsequent analysis of brains from Tg mice of this study revealed that GM-CSF treatment induced large reductions in amyloid burdens within entorhinal cortex ( 55%) and hippocampal ( 57%) compared to control Tg mice (Fig 3). The improved cognitive function and reduced cortical amyloidosis of GM-CSF-treated Tg mice were paralleled by increased microglial density as compared to saline-treated Tg mice (Fig 4), implying an augmented ability to bind and remove amyloid deposition [27, 28]. The GM-CSF-treated Tg mice similarly demonstrated increased synaptophysin immunoreactivity in both CA1 and CA3 regions (Fig 5), indicating increased synaptic area in these hippocampal regions. Prior work has

PAGE 214

204 shown that adult neural stem cells in hippocampal dentate gyrus (DG) express GM-CSF receptors, and GM-CSF increases neuronal differentiation of these cells in a dosedependent fashion [38]. Thus, one mechanism for the observed GM-CSF-induced cognitive improvement is enhanced removal of deposited A in hippocampus, with ensuing neuronal growth/synaptic differentiation of DG mossy fiber innervation to CA3, resulting in increased innervation/synaptogenesis of Schaffer collaterals into CA1. Removal of deposited A from entorhinal cortex may also increase perforant pathway viability to hippocampal projection fields in DG and CA1. Thus GM-CSF-induced reduction of amyloidosis and enhancement of hippocampal/entorhinal cortex circuitry, critical for working (short-term) memory, may underlie GM-CSFs reversal of working memory impairment in Alzheimers Tg mice.

PAGE 215

205 DISCUSSION Since peripheral leukocyte populations are increased in RA and possess the ability to infiltrate into the brain, we initially investigated M-CSF, G-CSF, and GM-CSF to determine which CSF might affect amyloidosis after direct injection into the brain. In the vasculature, all three CSFs work to drive the proliferation, differentiation, and survival of their respective innate leukocytes from monocytic precursors and would be expected to have similar effects on microglial precursors. In our study, we found different functional effects for each intrahippocampalinjected CSF. In the M-CSF injected mice, there was no effect on amyloidosis, but pathological changes were noticed, such as swelling and hyperplasia (Supplementary Fig 2). Parenchymal overexpression of M-CSF in any organ is probably not advisable as overexpression of M-CSF and/or its recept or within mammary glands has similarly resulted in tumor formation and hyperplasia [39]. In a study by Boissonneault et al. (2009), the authors published that chronic intraperitoneal injection of M-CSF prevents and reverses amyloid deposition and cognitive impairment and induces a large accumulation of bone marrow-derived microglia in the brain [40]. The authors also confirmed previous research [17] that bone marrow-derived microglia efficiently phagocytose and internalize A Although the authors did not relate their findings to RAs inverse relationship with AD, their data provide evidence that up-regulated M-CSF in RA pathogenesis and systemic administration of M-CSF in AD patients may impart protection against AD onset or progression. However, M-CSFs activation of mature osteoclasts, macrophages, and other innate leukocytes, induced thrombocytopenia, and autocrine signaling in some tumors may limit its usage in the clinical setting. Peripheral administration of G-CSF has also been found to ameliorate amyloid pathology and reduce cognitive defects in AD models [37, 41], which corroborates our

PAGE 216

206 observations of modest amyloid reduction by intrahippocampal injection. In the study by Sanchez-Ramos et al. [37], the authors also show a large increase in bone marrowderived microglia with corresponding amyloid reductions, increased synaptic area, and partial reversal of cognitive impairment. Although the M-CSF and G-CSF intrahippocampal findings are encouraging, our GM-CSF intrahippocampal injections into an AD mouse model demonstrated a much more pronounced reduction of amyloidosis. These data led us to further examine GM-CSF on the behavior of aged cognitively-impaired AD mice. Subcutaneous administration of GM-CSF resulted in almost complete reversal of cognitive impairment, resulting in function similar to that of wild-type mice, with a corresponding average of about 50% reduction of amyloidosis in Entorrhinal cortex and Hippocampus. In contrast, M-CSF and G-CSF have been shown to only partially reverse cognitive impairment in aged AD mice, although the preventative chr onic study of M-CSF administration into young AD mice showed equal cognitive function over time compared to wild-type controls. This cognitive advantage of peripherally-administered GM-CSF into aged AD mice could result from a combinatory effect of increased macrophage and granulocyte populations from bone marrow-derived monocytic precursors in the periphery, as well as induction of other innate leukocyte subsets in both periphery and brain. Indeed, GM-CSF has been shown to pass the blood brain barrier[42], and a recent study showed that GMCSF injected into the brains of normal mice activates microglia[43]. Further explanations for the very robust cognitive benefits of GM-CSF in our experiments are multiple, including the aforementioned augmentation of peripheral erythropoietic amyloidclearance mechanisms [31, 32, 44], as well as increased neurogenesis,[38] increased cerebral angiogenesis [45], neuroprotection from apoptosis[46], reduction in amyloidosis (Figs 1,3, and Supplementary Fig 5), and increased neuronal plasticity (Fig 5).

PAGE 217

207 Our results mirror those reported for G-CSF [37], and supports the current practice of interchangeable prescription of either recombinant human GM-CSF (Leukine) or G-CSF (Granocyte, Neupogen, and Neulasta) into patients with depressed bone marrow function. G-CSF primarily treats neutropenia while GM-CSF treats all leukopenia, and both have long records of safety data from two decades of FDAapproved usage. Rare adverse events are usua lly mild febrile incidents that quickly subside upon cessation of administration. G-CSF is currently in clinical trial for stroke and was recently approved for an AD Phase II clinical trial. However, GM-CSF/Leukine is more effective in the AD mouse model and while neutrophils are short-lived leukocytes, the fact that GM-CSF induces the up-regulation of all innate cells means that it could potentially impart prolonged protective effects against AD. The failure of NSAID clinical trials in AD and the multiple studies that show that a defective innate immune system propagates AD pathology encouraged us to develop and test our hypothesis that intrinsic pathogenic properties of RA are protective against AD. Indeed the beneficial effects of all three RA-upregulated CSFs, especially GM-CSF, in mouse models of AD point to a potential new approach to AD therapy and indicate that age-linked depressed hematopoiesis may be etiological for AD pathogenesis.

PAGE 218

208 Acknowledgements Support for this work was provided for by the USF Health Byrd Alzheimers Center and Research Institute, the Eric Pfeiffer Chair for research on Alzheimers disease, and the Florida Alzheimers Disease Research Center (P50AG25711).

PAGE 219

209 References [1] McGeer PL, Rogers J, McGeer EG (2006) Inflammation, anti inflammatory agents and Alzheimer disease: the last 12 years. J Alzheimers Dis 9 271 276. [2] Martin BK, Szekely C, Brandt J, Piantadosi S, Breitner JC, Craft S, Evans D, Green R, Mullan M (2008) Cognitive function over time in the Alzheimer's Disease Anti inflammatory Prevention Trial (ADAPT): results of a randomized, controlled trial of naproxen and celecoxib. Arch Neurol 65 896 905. [3] Okazaki H, Reagan TJ, Campbell RJ (1979) Clinicopathologic studies of primary cerebral amyloid angiopathy. Mayo Clin Proc 54 2231. [4] Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297 353 356. [5] Glenner GG, Wong CW (1984) Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun 122 1131 1135. [6] Lee VM (1996) Regulation of tau phosphorylation in Alzheimer's disease. Ann N Y Acad Sci 777 107 113. [7] Griffin WS, Stanley LC, Ling C, White L, MacLeod V, Perrot LJ, White CL, 3rd, Araoz C (1989) Brain interleukin 1 and S100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci U S A 86, 7611 7615. [8] Wisniewski T, Castano EM, Golabek A, Vogel T, Frangione B (1994) Acceleration of Alzheimer's fibril formation by apolipoprotein E in vitro. Am J Pathol 145 1030 1035. [9] Ma J, Brewer HB, Jr., Potter H (1996) Alzheimer A beta neurotoxicity: promotion by antichymotrypsin, ApoE4; inhibition by A beta related peptides. Neurobiol Aging 17, 773 780.

PAGE 220

210 [10] Potter H, Wefes IM, Nilsson LN (2001) The inflammation induced pathological chaperones ACT and apo E are necessary catalysts of Alzheimer amyloid formation. Neurobiol Aging 22, 923 930. [11] Nilsson LN, Arendash GW, Leighty RE, Costa DA, Low MA, Garcia MF, Cracciolo JR, Rojiani A, Wu X, Bales KR, Paul SM, Potter H (2004) Cognitive impairment in PDAPP mice depends on ApoE and ACT catalyzed amyloid formation. Neurobiol Aging 25, 1153 1167. [12] Padmanabhan J, Levy M, Dickson DW, Potter H (2006) Alpha1antichymotrypsin, an inflammatory protein overexpressed in Alzheimer's disease brain, induces tau phosphorylation in neurons. Brain 129 3020 3034. [13] Koenigsknecht Talboo J, MeyerLuehmann M, Parsadanian M, Garcia Alloza M, Finn MB, Hyman BT, Bacskai BJ, Holtzman DM (2008) Rapid microglial response around amyloid pathology after systemic anti Abeta antibody administration in PDAPP mice. J Neurosci 28, 14156 14164. [14] MeyerLuehmann M, Spires Jones TL, Prada C, Garcia Alloza M, de Calignon A, Rozkalne A, Koenigsknecht Talboo J, Holtzman DM, Bacskai BJ, Hyman BT (2008) Rapid appearance and local toxicity of amyloid beta plaques in a mouse model of Alzheimer's disease. Nature 451 720 724. [15] Malm TM, Koistinaho M, Parepalo M, Vatanen T, Ooka A, Karlsson S, Koistinaho J (2005) Bone marrow derived cells contribute to the recruitment of microglial cells in response to beta amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol Dis 18, 134 142. [16] Simard AR, Rivest S (2004) Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J 18, 998 1000.

PAGE 221

211 [17] Simard AR, Soulet D, Gowing G, Julien JP, Rivest S (2006) Bone marrow derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron 49, 489 502. [18] Szekanecz Z, Koch AE (2007) Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol 19, 289 295. [19] Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, van Venrooij WJ (2000) The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43, 155 163. [20] Parsonage G, Filer A, Bik M, Hardie D, Lax S, Howlett K, Church LD, Raza K, Wong SH, Trebilcock E, Scheel Toellner D, Salmon M, Lord JM, Buckley CD (2008) Prolonged, granulocyte macrophage colony stimulating factor dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL 17 and TNFalpha. Arthritis Res Ther 10, R47. [21] Cox CA, Shi G, Yin H, Vistica BP, Wawrousek EF, Chan CC, Gery I (2008) Both Th1 and Th17 are immunopathogenic but differ in other key biological activities. J Immunol 180 7414 7422. [22] Xu WD, Firestein GS, Taetle R, Kaushansky K, Zvaifler NJ (1989) Cytokines in chronic inflammatory arthritis. II. Granulocyte macrophage colony stimulating factor in rheumatoid synovial effusions. J Clin Invest 83, 876 882. [23] Nakamura H, Ueki Y, Sakito S, Matsumoto K, Yano M, Miyake S, Tominaga T, Tominaga M, Eguchi K (2000) High serum and synovial fluid granulocyte colony stimulating factor (GCSF) concentrations in patients with rheumatoid arthritis. Clin Exp Rheumatol 18, 713 718.

PAGE 222

212 [24] Olszewski WL, Pazdur J, Kubasiewicz E, Zaleska M, Cooke CJ, Miller NE (2001) Lymph draining from foot joints in rheumatoid arthritis provides insight into local cytokine and chemokine production and transport to lymph nodes. Arthritis Rheum 44, 541 549. [25] Kawaji H, Yokomura K, Kikuchi K, Somoto Y, Shirai Y (1995) [Macrophage colony stimulating factor in patients with rheumatoid arthritis]. Nippon Ika Daigaku Zasshi 62, 260 270. [26] Wyss Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, Masliah E (2002) Prominent neurodegeneration and increased plaque formation in complement inhibited Alzheimer's mice. Proc Natl Acad Sci U S A 99, 10837 10842. [27] Hickman SE, Allison EK, El Khoury J (2008) Microglial dysfunction and defective beta amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci 28, 8354 8360. [28] El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD (2007) Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimerlike disease. Nat Med 13, 432 438. [29] Emerson SG, Sieff CA, Wang EA, Wong GG, Clark SC, Nathan DG (1985) Purification of fetal hematopoietic progenitors and demonstration of recombinant multipotential colony stimulating activity. J Clin Invest 76, 12861290. [30] Wu H, Liu X, Jaenisch R, Lodish HF (1995) Generation of committed erythroid BFU E and CFU E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83, 5967. [31] Rogers J, Li R, Mastroeni D, Grover A, Leonard B, Ahern G, Cao P, Kolody H, Vedders L, Kolb WP, Sabbagh M (2006) Peripheral clearance of amyloid beta peptide by complement C3 dependent adherence to erythrocytes. Neurobiol Aging 27, 1733 1739.

PAGE 223

213 [32] Helmy KY, Katschke KJ, Jr., Gorgani NN, Kljavin NM, Elliott JM, Diehl L, Scales SJ, Ghilardi N, van Lookeren Campagne M (2006) CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 124 915 927. [33] Bennett SP, Boyd T.D., Norden M., Padmanabhan J., Neame P., Wefes I., Potter H. (2009) A Novel Technique for Simultaneous Bilateral Brain Infusions in a Mouse Model of Neurodegenerative Disease Journal of Neuroscience Methods [34] Arendash GW, King DL, Gordon MN, Morgan D, Hatcher JM, Hope CE, Diamond DM (2001) Progressive, age related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Brain Res 891 4253. [35] Echeverria V, Burgess S, Gamble George J, Zeitlin R, Lin X, Cao C, Arendash GW (2009) Sorafenib inhibits nuclear factor kappa B, decreases inducible nitric oxide synthase and cyclooxygenase 2 expression, and restores working memory in APPswe mice. Neuroscience 162 1220 1231. [36] Loewenstein DA, Acevedo A, Luis C, Crum T, Barker WW, Duara R (2004) Semantic interference deficits and the detection of mild Alzheimer's disease and mild cognitive impairment without dementia. J Int Neuropsychol Soc 10, 91100. [37] Sanchez Ramos J, Song S, Sava V, Catlow B, Lin X, Mori T, Cao C, Arendash GW (2009) Granulocyte Colony Stimulating Factor (GCSF) Decreases Brain Amyloid Burdenand Reverses Cognitive Impairment in Alzheimer's Mice. Neuroscience [38] Kruger C, Laage R, Pitzer C, Schabitz WR, Schneider A (2007) The hematopoietic factor GM CSF (granulocyte macrophage colony stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro. BMC Neurosci 8 88. [39] Kirma N, Luthra R, Jones J, Liu YG, Nair HB, Mandava U, Tekmal RR (2004) Overexpression of the colony stimulating factor (CSF 1) and/or its receptor c fms in

PAGE 224

214 mammary glands of transgenic mice results in hyperplasia and tumor formation. Cancer Res 64, 4162 4170. [40] Boissonneault V, Filali M, Lessard M, Relton J, Wong G, Rivest S (2009) Powerful beneficial effects of macrophage colony stimulating factor on beta amyloid deposition and cognitive impairment in Alzheimer's disease. Brain 132, 1078 1092. [41] Tsai KJ, Tsai YC, Shen CK (2007) G CSF rescues the memory impairment of animal models of Alzheimer's disease. J Exp Med 204 1273 1280. [42] McLay RN, Kimura M, Banks WA, Kastin AJ (1997) Granulocyte macrophage colony stimulating factor crosses the blood brain and blood spinal cord barriers. Brain 120 ( Pt 11) 2083 2091. [43] Reddy PH, Manczak M, Zhao W, Nakamura K, Bebbington C, Yarranton G, Mao P (2009) Granulocyte macrophage colony stimulating factor antibody suppresses microglial activity: implications for anti inflammatory effects in Alzheimer's disease and multiple sclerosis. J Neurochem 111 15141528. [44] Fisher JW (2003) Erythropoietin: physiology and pharmacology update. Exp Biol Med (Maywood) 228 1 14. [45] Schneider UC, Schilling L, Schroeck H, Nebe CT, Vajkoczy P, Woitzik J (2007) Granulocyte macrophage colony stimulating factor induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion. Stroke 38 1320 1328. [46] Schabitz WR, Kruger C, Pitzer C, Weber D, Laage R, Gassler N, Aronowski J, Mier W, Kirsch F, Dittgen T, Bach A, Sommer C, Schneider A (2008) A neuroprotective function for the hematopoietic protein granulocyte macrophage colony stimulating factor (GM CSF). J Cereb Blood Flow Metab 28 2943.

PAGE 225

215

PAGE 226

216 Fig 1. Intrahippocampal injection of GM-CSF (left) and artificial cerebrospinal fluid (aCSF) (right). (a ) Representative coronal tissue cryo-sectioned at 14 m and stained with MabTech -A /Alexa 546. Image is a montage of about 145 pictures taken at 10X. White spots indicate amyloid plaque immunolabelling (see Supplementary Fig. 4 online for representative montaged sections of all 4 mice). (b) Significant overall plaque reductions seen in all 4 plaque parameters measured from 5 quantified sections per mouse (n = 4 mice). Error bars are Standard Error of the Mean. (Area: p < 1.11E-07; Perimeter: p < 1.41E-06; Feret Diameter: p < 2.36E-09; Integrated Density: p < 1.11E07).

PAGE 227

Fig 2. Behavioral analysis following daily subcutaneous GM-CSF injections. 217

PAGE 228

218 (a)Standard Radial Arm Water Maze errors prior to treatment. The final block and overall performance of the Tg and NT mice during 8 days of consecutive daily pre-treatment testing in the RAWM maze. The data was analyzed in four 2-day blocks and overall (Blocks 1-4). ( p < 0.02 or higher significance). (b) Standard Radial Arm Water Maze errors after treatment. Tg control mice (n = 6) show substantial impairment on working memory trials T4 and T5 compared to NT control mice (n = 8) in individual blocks of testing (upper), and over all 4 days of testing (lower). GM-CSF-treated Tg mice (n = 5) performed as well as or better than NT control mice on working memory trials T4 and T5 during individual blocks and over all. GM-CSFtreated NT mice (n = 9) performed similarly to or slightly better than NT controls (Note significantly better performance of NT+GMCSF group versus NT group for T4 of Block 1), although this effect was not significant overall. ( **p < 0.02 or higher significance versus all other groups; p < 0.02 or higher significance versus Tg+GM-CSF and NT+GM-CSF). (c) Cognitive Interference Task. Overall (4 Days) Tg control mice are impaired compared to NT mice on all four cognitive measures assessed. GM-CSF-treated Tg mice exhibited significantly better 3-trial recall (A1-A3) and delayed recall (A5) compared to Tg controls and performed similarly to NT mice in all four cognitive measures. GMCSF treatment of NT mice did not result in significantly better performance compared to NT controls, although trends for a beneficial GM-CSF effect in NT mice were evident overall. (*Tg significantly different from NT +GM-CSF, **Tg significantly different from all other groups). (d) Cognitive Interference Task. Proactive Interference testing (First 2 days). GM-CSF-treated Tg mice performed significantly better than Tg controls and equally to NT and GM-CSF-treated NT mice.

PAGE 229

Fig 3. Amyloid deposition in subcutaneous GM-CSF-injected mice. (a-d) Photomicrographs of coronal 5-m paraffin-embedded sections immunolabelled with anti-A antibody (clone 4G8) in Entorrhinal cortex (E) and hippocampus (H)). Pictures are representative of amyloid load closest to the mean of the GM-CSFor saline-treated Tg groups. Scale bar = 50 m. (e) Percent of amyloid burden from the average of five 219

PAGE 230

220 5-m sections (150-m apart) through both anatomic regions of interest (hippocampus and entorhinal cortex) per mouse of GM-CSF-treated (n = 5) versus salinetreated (n = 6). Entorhinal cortex (*p < 0.026), and Hippocampus (p = 0.12)

PAGE 231

221

PAGE 232

222 Fig 4. Microglial immunostaining in subcutaneous GM-CSF-injected mice. (a-d) Photomicrographs of coronal 5m paraffin-embedded sections immunolabelled with Iba-1 antibody in Entorrhinal cortex (E) and hippocampus (H). Pictures are representative of Iba-1 immunolabelling closest to the mean of the GM-CSFor saline controltreated groups. Scale bar = 50m. (e) Percent of Iba1 burden from the average of five 5-m sections (150 m apart) through both anatomic regions of interest (H and EC) per mouse of GM-CSF-treated (n = 5) versus saline-treated (n = 6). H(p < 0.02), EC(p < 0.05).

PAGE 233

223

PAGE 234

224 Fig 5. Synaptophysin immunostaining in subcutaneous GM-CSF-injected mice. (a-d) Photomicrographs of coronal 5-m paraffin-embedded sections immunolabelled with anti-synaptophysin antibody. Pictures are representative of synaptophysin immunolabelling closest to the mean of the GM-CSFor saline controltreated groups. Scale bar = 50 m. (e) Percent of synaptophysin immunoreactivity from the average of 5 sections per mouse of GM-CSF-treated (n = 5) versus saline control-treated (n = 6). CA1(p < 0.0013), CA3(p < 0.0023).

PAGE 235

Supplementary Figure 1. Significant variation of amyloid plaque load between mice. Standard fluorescent immunohistochemistry used 6E10/Alexa 488 and Hoechst nuclear stain (blue). Bright green spots indicate amyloid plaques. Pictures taken at 5X. Mouse numbers 148,160,171, and 211 received bilateral intracerebroventricular infusions of MCSF and mice 164, 170, 176, and 177 received bilateral intracerebroventricular infusions of artificial cerebrospinal fluid. 225

PAGE 236

Supplementary Figure 2. Intrahippocampal injection of M-CSF-injected left hemisphere and artificial cerebrospinal fluid (aCSF) injected right hemisphere. (A ) The image is a montage of ~35 5X pictures and is representative of the effects seen from anterior hippocampus to posterior in all 4 M-CSF-injected mice. ( B ) This photo shows enlargement of the M-CSF-injected left hemisphere, as seen following saline perfusion. Note the small bump at the site of injection (arrow). (C ) Image shows cyst or tumor-like growth formed in the needle track at the site of M-CSF injection. Cryosectioned at 14 m and stained with 6E10/Alexa 488 and Hoechst. Picture taken at 20X. 226

PAGE 237

227

PAGE 238

Supplementary Figure 3. Intrahippocampal injection of G-CSF-injected left hemispheres and artificial cerebrospinal fluid (aCSF) injected right hemispheres. (A ) Images are montages of ~35 5X pictures each. Amyloid plaques indicated as white spots. Cryosectioned at 14 m and stained with 6E10/Alexa546 and Hoechst nuclear stain. Sections numbered 1 through 6 and correspond with anterior to posterior. ( B ) Amyloid plaques show a modest reduction of plaque in the left G-CSF-injected hemisphere. Error bars are Standard Error of the Mean. (Area, Integrated Density: p < 0.05). 228

PAGE 239

229

PAGE 240

Supplementary Figure 4. Intrahippocampal injection of GM CSF injected left hemispheres and artificial cerebrospinal fluid (aCSF) injected right hemispheres. Representative sections of each mouse proximal to injection site. Tissue sections stained with MabTech -A /Alexa 488. White spots indicate amyloid plaque immunolabelling. Images are montages of about 145 pictures taken at 10X. Figures 4AC are from 14 m frozen sections, and 4D is from a 5 m paraffin-embedded section. 230

PAGE 241

231

PAGE 242

232 Supplementary Figure 5.Quantification of reduced amyloid deposition in GM CSF injected left hemispheres versus artificial cerebrospinal fluid (aCSF) injected right hemispheres. There were 5 montaged sections per mouse quantified. Each montaged section contained over 140 10X pictures and of these, 15 25 pictures per hemisphere were selected to quantify as described in Supplementary Figure 6 online. Each figure shows total or average values from the 5 sections /mouse with significance per individual mouse and significance over all 4 mice. Error bars are SEM : ( a b ) plaque areas ( c d) perimeter values ( e ) average feret diameters ( f) average integrated densities

PAGE 243

233

PAGE 244

234

PAGE 245

235

PAGE 246

236

PAGE 247

237

PAGE 248

238

PAGE 249

239 Supplementary Figure 6. Microscopy and method of Image J analysis of amyloid deposition Lateral rows of photomicrographs were taken of coronal sections in a manner that ensured minimal overlap between each phot omicrograph and corresponding anatomical areas in each hemisphere. Hoechst nuclear staining was used as a tool to scroll through sections, allowing for minimal overlap between each photomicrograph, and then the image was taken with the appropriate fluorescence. After all photomicrographs of the section were taken, they were montaged into a full coronal picture of the section, with the outline of each photomicrograph displayed (Axiovision 4.7 Panorama Module software). Photomicrograhs were then selected for analysis from each hemisphere (Figure 6A). The histogram of each photomicrograph was analyzed and the resulting means and standard deviations were entered into a spreadsheet. One photomicrograph of the section was then thresholded to select the amyloid plaques. This thresholded value was used in the spreadsheet to normalize the threshold values of the other photomicrographs to the same standard deviation from their respective histogram mean (Figure 6B). Each photomicrograph was then analyzed for Area, Perimeter, Feret Diameter, and Integrated Density values from their predetermined threshold values. Any threshold-selected data, which came in contact with the edges of any photomicrograph, were deleted via the Image J Analyze Particles dialogue box, so that overlap of photomicrographs did not allow quantification of any amyloid deposition more than once. Within the Image J Analyze Particles dialogue box, the Oulines mask was also selected to visually confirm that the plaques were quantified accurately (Figure 6C). This Outline mask also numbered each plaque, which was used for selecting and eliminating artifact from the results. The results from each individual photomicrograph were copied into a 2nd separate spreadsheet, such that the data was separated into their respective hemispheres (CSF-treated versus artificial cerebrospinal fluid-treated). After all photomicrographs from a coronal section had been analyzed and data entered into the spreadsheet, the Area and Perimeter values were totaled and averaged. The Feret Diameter and Integrated Density values were also averaged. The overall data from each section (sums and averages) were then entered into a 3rd spreadsheet A total of 5 sections per mouse, anterior to posterior, were analyzed and entered into this 3rd spreadsheet. A total of 4 mice per intrahippocampal-injected CSF were analyzed.

PAGE 250

Significance per mouse and over all 4 mice per CSF was determined by paired Students t-test with p values <0.5 considered significant Supplementary Figure 6A Coronal section taken at 10X, showing demarcation of individual photomicrographs to be analyzed for amyl oid deposition. 240

PAGE 251

Supplementary Figure 6B Thresholding of one photomicrograph and using its threshold value to find the threshold va lue for all other p hotomicrographs within the coronal section at the same standard deviation from the mean. Supplementary Figure 6C Example of the Oulines mask and underlying ImageJ spreadsheet data. 241

PAGE 252

242 Supplementary Fig 7. Behavioral Tasks Radial Arm Water Maze For the RAWM task of spatial working memory, an aluminum insert was placed into a 100c m circular pool to create 6 radiallydistributed swim arms emanating from a central circular swim area. An assortment of 2-D and 3-D visual cues surrounded the pool. The number of errors prior to locating which one of the 6 swim arms contained a submerged escape platform (9cm diameter) was deter mined for 5 trials/day. There was a 30min time delay between the 4th trial (T4; fi nal acquisition trial) and 5th trial (T5; memory retention trial). The platform lo cation was changed daily to a different arm, with different start arms for each of the 5 trials semi-randomly selected from the remaining 5 swim arms. During each trial (60 s maximum), the mouse was returned to that trials start arm upon swimming into an incorrect arm and the latency time required to locate the su bmerged platform was recorded. If the mouse did not find the platform within a 60s trial, it was guided to the platform for a 30-s stay. The numbers of errors and escape latency during trials 4 and 5 are both considered indices of working memory and are temporally similar to standard registration/recall test ing of specific items used clinically in evaluating AD patients. Cognitive Interference Task This task was designed to mimic, measure-formeasure, a cognitive interference task rece ntly utilized clinically to discriminate between normal aged, MCI, and AD patient s. The task involves two RAWM setups in two different rooms, with two sets of visual cues different from those utilized in standard RAWM te sting. The task requires animals to remember a set of visual cues (in RAWM-A), so that following interfer ence with a different set of cues (in RAWM-B), the initial set of cues can be recalled to successfully solve the RAWM task. Five behavioral measur es were examined: A1-A3 (Composite three-trial recall score from first 3 tria ls performed in RAWM-A), B (proactive interference measure attained from a singl e trial in RAWM-B), A4 (retroactive interference measure attained during a singl e trial in RAWM-A), and A5 (delayedrecall measure attained from a single tr ial in RAWM-A following a 20-min delay between A4 and A5). As wit h the standard RAWM task, this interference task involves the platform location being changed daily to a different arm for both RAWM set-ups. For A1 and B trials, the anima l is initially allowed one minute to find the platform on their own before being guided to the platform. Then the actual trial is performed in each case. As with the standard RAWM task, animals were given 60s to find the escape platform per trial, with the number of errors and escape latency recorded per trial.

PAGE 253

243

PAGE 254

244 APPENDIX C: COPYRIGHT INFORMATION In the United States, whenever an author or creator puts thei r work onto a tangible media, they automatically obtain copyright protection, for any original work of authorship. Thus the manuscr ipts that authors submit to va rious journals are regulated by copyright laws, and some journal policies require explicit permissions for any further usage of the works that they publish within th eir journals, sometimes even by the original authors themselves. Not only does this disser tation have copyright protections, but also do the original figures and text which are en closed within, and which have also been published in the Journal of Neuroscience Methods and the Journal of Alzheimers Disease. Both journals publishers have pr ovided explicit terms rega rding authorship and copyright permissions on their websites. Th e following pages, obtained directly from each publishers website, explic itly state each publishers copyright policies. These policies give permission for the original materials from both journal articles to be used within this dissertation.

PAGE 255

The Journal of Neuroscience Methods, is published by Elsevier, a part of the Reed Elsevier group based out of Amsterdam, which has the following policy stated on its website, and which can be found at the web link: http://www.elsevier.com/wps/find/a u thorshome.a u thors/copyright 245

PAGE 256

The Journal of Alzheimers Disease, is published by IOS Press, a pulishing house also based out of Amsterdam and which has the following policy stated on its website, and which can be found at the web link: http://www.iospress.nl/a u thco/copyright.html 246

PAGE 257

247 APPENDIX D: IMAGEJ ANALYSIS PROTOCOL This protocol was developed to quantify parameters of amyloid deposition in each brain hemisphere and enable comparisons to be made between each side. It was critical that the tissue sections were properly-a ligned under the microscope so that the photomicrographs could be taken in horizon tal lateral rows con tinuously across both hemispheres of the coronal sections. We examined the Axiovision version 4.7 software which was used in the acquisition of the photom icrographs to see if it had the capability to accurately quantify amyloid plaques. Howe ver, it did not provide the capability to select artifactual or non-specific immunopositivi ty, so that these selections could be eliminated from the analyses. Thus, we examined several software packages for these capabilities, and found that the freely-avail able ImageJ software, which is provided through the National Institute of Health, co uld perform these tasks. Following is the protocol that we developed using the Imag eJ software to analyze specific plaque parameters in the mouse brains, following acquisition of the photomicrographs from Axiovision version 4.7 software. The co mpilation of the photomicrographs was performed using the Axiovision version 4.7 Panorama Module.

PAGE 258

248

PAGE 259

249

PAGE 260

250

PAGE 261

251

PAGE 262

252

PAGE 263

253

PAGE 264

254

PAGE 265

255

PAGE 266

256

PAGE 267

257

PAGE 268

258

PAGE 269

259

PAGE 270

260

PAGE 271

261 APPENDIX E: MAGNETIC IMMUNOHISTOCHEMICAL STAINING DEVICE AND METHODS OF USE This device was construc ted in order to spare the usage of valuable antibody solutions and other reagents during the imm unohistochemical (IHC) staining of tissues, which had been paraffin-embedded and mounted onto glass slides. During traditional staining protocols of these types of tissue preparations, there was no adequate procedure for mounting a hydrophobic barrie r around the edges of the slide or tissue, which would allow for concentration of reagent solutions, in stead of full submersion of slides in said solutions. Upon demonstration of this device s utility in said IHC application, it was submitted for provisional patent application at the United States Patent and Trademark Office (USPTO) on June 24, 2008, and subsequently a non-provisional patent application was filed with the USPTO on June 24, 2009. Follow ing is a copy of the patent application file on Jun 24, 2009, followed by the higher resolution figures.

PAGE 272

262

PAGE 273

263

PAGE 274

264

PAGE 275

265

PAGE 276

266

PAGE 277

267

PAGE 278

268

PAGE 279

269

PAGE 280

270

PAGE 281

271

PAGE 282

272

PAGE 283

273

PAGE 284

274

PAGE 285

275

PAGE 286

276

PAGE 287

277

PAGE 288

278

PAGE 289

279

PAGE 290

280

PAGE 291

281

PAGE 292

282

PAGE 293

283

PAGE 294

284

PAGE 295

285 APPENDIX F: METHODS OF TREATI NG COGNITIVE IMPAIRMENT Given the quick and remarkable effects of GM-CSF in transgenic (Tg) mouse models of AD, especially the reversal of already-established cognitive impairment in these aged Tg mice, implies that the recombinant human GM-CSF (sargramostim/Leukine) protein may impart th e same beneficial effects wtihin AD and other cognitively-declining patie nts. Since we were the first to identify GM-CSF to have these effects, we filed to protect USFs a nd our intellectual property rights for the usage of GM-CSF in the amelioration of pathologies found in specific neurodegenerative diseases. A provisional patent entitled Methods of Treating Cognitive Impairment was filed with the United States Patent an d Trademark Office (USPTO) on August 5, 2008. On August 5, 2009, a regular patent applica tion, containing some revisions to the provisional patent applicati on, was filed with the USPTO, as well as in international filings. Following is a copy of the provisional pa tent application that was received by the USPTO on August 18, 2008.

PAGE 296

286

PAGE 297

287

PAGE 298

288

PAGE 299

289

PAGE 300

290

PAGE 301

291

PAGE 302

292

PAGE 303

293

PAGE 304

294

PAGE 305

295

PAGE 306

296

PAGE 307

297

PAGE 308

298

PAGE 309

299

PAGE 310

300

PAGE 311

301

PAGE 312

302

PAGE 313

303

PAGE 314

304

PAGE 315

305

PAGE 316

306

PAGE 317

307

PAGE 318

308

PAGE 319

309

PAGE 320

310

PAGE 321

311

PAGE 322

312

PAGE 323

ABOUT THE AUTHOR Timothy David Boyd was born in Memphis, Te nnessee, and after finishing high school in Collierville, Tennessee, he simultaneously atte nded the University of Memphis and State Technical Institute of Memphis (now a part of Southwest Tennessee Community College), where he received an Associat e of Science degree in Microcomputer Management. In 1995, he moved to Tampa, Forida, and transferred into the University of South Florida (USF). In 2000, he received dual degress, with a Bachelor of Science in Microbiology and a Bachelor of Arts in Chemistry, while concurrently working at University Community Hospital. After gra duating, he continued to take non-degreeseeking classes at USF, while working at Bausch and Lomb, Pharmaceuticals, Inc. and while volunteering at Tampa Ge neral Hospital. In Fall of 2003, he joined the Doctorate Program in the USF College of Medicine. He served as President of the Graduate and Professional Student Council from 2004 2005, an organization that advocates for policies for all graduate students campus-w ide. He began working for the Johnnie B. Byrd, Senior Alzheimers Center and Research Institute in June, 2005 and became a student with Dr. Huntington Potter, CEO of th e Institute, in June 2007. He has since filed for two patents from his work, and has assist ed in the discovery of another. All three patent subjects are described within this dissertation. He also volunteered his time for three years every Sunday to raise money fo r St. Jude Childrens Research Hospital through tips as a server at the Tampa Melti ng Pot Restaurant, where he gained several pounds from eating cheese fondue a nd melted chocolate. He is also concurrently enrolled in the Masters in Biotechnology Program, through the Department of Molecular Medicine and expects to fulfill requirements to graduate with his M.S.B. degree in December 2010, one semester after defendi ng his doctorate degree on July 2, 2010.


xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam 22 Ka 4500
controlfield tag 007 cr-bnu---uuuuu
008 s2010 flu s 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0004662
035
(OCoLC)
040
FHM
c FHM
049
FHMM
090
XX9999 (Online)
1 100
Boyd, Timothy.
0 245
The novel use of recombinant granulocyte-macrophage colony-stimulating factor (gm-csf) to reverse cerebral amyloidosis and cognitive impairment in alzheimer's disease mouse models :
b insights from the investigation of rheumatoid arthritis as a negative risk factor for alzheimer's disease
h [electronic resource] /
by Timothy Boyd.
260
[Tampa, Fla] :
University of South Florida,
2010.
500
Title from PDF of title page.
Document formatted into pages; contains X pages.
502
Dissertation (PHD)--University of South Florida, 2010.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
3 520
ABSTRACT: For many years, it has been known that Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). It has been commonly assumed that RA patients' usage of non-steroidal anti-inflammatory drugs (NSAIDs) have helped prevent the onset and progression of AD pathogenesis. Furthermore, experiments in animal models of Alzheimer's disease have looked to inhibit inflammation, and have demonstrated some efficacy against AD-like pathology in these models. Thus many NSAID clinical trials have been performed over the years, but all have proven unsuccessful in AD patients. This suggested that intrinsic factors within RA pathogenesis itself may underlie RA's protective effect. My dissertation research goal was to investigate this inverse relationship between RA and AD, in order to more precisely pinpoint critical events in AD pathogenesis toward developing therapeutic strategies against AD. It seemed improbable that any secreted factors, produced in RA pathogenesis, could maintain high enough concentrations in the circulatory system to cross the blood brain barrier and inhibit AD pathogenesis, without affecting all other organ systems. It did seem possible that the leukocyte populations induced in RA, could traverse the circulatory system, extravasate into the brain parenchyma, and impede or reverse AD pathogenesis. We thus investigated the colony-stimulating factors, which are up‑regulated in RA and which induce most of RA's leukocytosis, on the pathology and behavior of transgenic AD mice. We found that G‑CSF and more significantly, GM-CSF, reduced amyloidosis throughout the treated brain hemisphere one week following bolus intrahippocampal administration into AD mice. We then found that 20 days of subcutaneous injections of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) significantly reduced brain amyloidosis and completely reversed cognitive impairment in aged cognitively-impaired mice, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, the recombinant human GM‑CSF analogue, in elderly leukopenic patients, suggested that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD patients. It was also implied that age-related depressed hematopoiesis may be etiological for AD pathogenesis.
590
Advisor: Huntington Potter, Ph.D.
653
G-CSF
M-CSF
Neuroinflammation
Amyloid beta
Radial Arm Water Maze
Cognitive Interference task
Transgenic mice
Intrahippocampal
Subcutanteous
690
Dissertations, Academic
z USF
x Molecular Medicine
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.4662