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Monocytes as gene therapy vectors for the treatment of Alzheimer's disease

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Title:
Monocytes as gene therapy vectors for the treatment of Alzheimer's disease
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English
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Lebson, Lori Ann
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University of South Florida
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Tampa, Fla
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Transgenic mouse model
Cell therapy
Abeta
Neprilysin
Protease
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Summary:
ABSTRACT: The accumulation of amyloid-β; protein (Aβ) in Alzheimer's disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of Aβ; is therefore thought to serve as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of Aβ; and defects in the degradation of Aβ; could contribute to AD disease progression. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genetically modified monocytes to carry exogenous genes of interest into the brain or other organs for the purposes of gene therapy.To determine the time course of monocyte recruitment into the brain during the neurodegenerative damage characteristic of Alzheimer's disease, we used transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. We determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found this time to be 1.5 hours post injection. We also examined the effects of the APP+PS1 transgene on the recruitment of peripheral monocytes and showed that these cells are actively recruited to the brains in AD transgenic mouse models compared to non transgenic mice. As an approach to increase expression of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice.These monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. Monocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS. In addition, we found significant reductions in both Aβ and Congo red staining in the NEP-S injected mice only. These studies show that putting monocytes together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatment of AD.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
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Includes bibliographical references.
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by Lori Ann Lebson.
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Title from PDF of title page.
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Document formatted into pages; contains 146 pages.
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Includes vita.

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aleph - 002007297
oclc - 402473638
usfldc doi - E14-SFE0002793
usfldc handle - e14.2793
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Monocytes as Gene Therapy Vectors for the Treatment of Al zheimers Disease by Lori Ann Lebson A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Molecular P harmacology and Physiology College of Medicine University of South Florida Major Professor: Marcia N. Gordon, Ph.D. David Morgan, Ph.D. Keith Pennypacker, Ph.D. Paula Bickford, Ph.D. Amyn Rojiani, M.D., Ph.D. Date of Approval November 7, 2008 Keywords: transgenic mouse model; cell th erapy, Abeta, neprilysin, protease Copyright 2008, Lori Ann Lebson

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i TABLE OF CONTENTS LIST OF FIGURES ii ABSTRACT iv INTRODUCTION Alzheimers Disease 1 Transgenic Mouse Models of Amyloidogenesis 4 Inflammation and AD 7 Monocytes 10 Gene Therapy 16 PAPER 1: SPATIAL AND TEMPORAL KI NETICS OF MONOCYTE MIGRATION INTO THE CNS. 27 PAPER 2: INTRACRANIAL INJECTION OF NEPRILYSIN TRANSFECTED MONOCYTES REDUCES AMYLOID IN AP P TRANSGENIC MICE. 51 PAPER 3: TRAFFICKING MONOCYTES DELIVER THERAPEUTIC GENES TO THE BRAIN OF AMYLOID DEPOSITING APP+ PS1 TRANSGENIC MICE 86 CONCLUSIONS 116 ABOUT THE AUTHOR End Page

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ii LIST OF FIGURES PAPER 1 Figure 1: The half life of injected GFP+ monocytes in circulation is approximately 1.5 hours. 47 Figure 2: Migration of GFP+ monocytes to organs in the presence and absence of Alzheimer-like pathology. 48 Figure 3: GFP+ monocytes entry into organ tissues. 50 PAPER 2 Figure 1: Characterization of neprilysin constructs. 74 Figure 2: Staining and transfection effi ciencies of neprilysin constructs. 76 Figure 3: Total A immunohistoc hemistry is significantly r educed in the frontal cortex and hippocampus after intracranial injecti on of NEP-S plasmid. 78 Figure 4: Compact Congophilic amyloi d deposits are reduced by NEP-S plasmid. 80 Figure 5: There is no change in GFAP staining in the frontal cortex and hippocampus following NEP-S injection. 82 Figure 6: There is no change in microglia l staining in the frontal cortex and hippocampus following NEP-S injection. 84 PAPER 3 Figure 1: Total A immunohistochemistry is significantly reduced in NEP-S treated mice. 107 Figure 2: Congo red staining is significant ly reduced in NEP-S treated mice. 110 Figure 3: HA is detected in brai n tissue but not in plasma. 113

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iii Figure 3: GFP positive monocytes migr ate to plaques in APP+PS1 transgenic mice and retain hematopoietic marker. 114 Figure 4: Subpopulation of GFP+ cells expr ess a macrophage phenotype. 115

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iv Monocytes as Gene Therapy Vectors for the Treatment of Al zheimers Disease Lori Ann Lebson ABSTRACT The accumulation of amyloidprotein (A ) in Alzheimers disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of A is therefore thought to serv e as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of A and defects in the degradation of A could contribute to AD disease progression. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genet ically modified monocytes to carry exogenous genes of interest into the br ain or other organs for the purposes of gene therapy. To determine the time course of monocyte recruitment into the brain during the neurodegenerativ e damage characteristic of Alzheimers disease, we used transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. We determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found th is time to be 1.5 hours post injection. We also examined the effects of t he APP+PS1 transgene on t he recruitment of

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v peripheral monocytes and showed that these cells are ac tively recruited to the brains in AD transgenic mouse models compared to non transgenic mice. As an approach to increase expressi on of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. Thes e monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. M onocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS In addition, we found significant reductions in both A and Congo red staining in the NEP-S injected mice only. These studies show that putting mono cytes together with an amyloid degrading enzyme such as neprilysin offers a pow erful novel therapeutic tool for the treatment of AD.

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INTRODUCTION ALZHEIMERS DISEASE Alzheimers Disease (AD) is an age-related neurodegenerative disorder and is the most common form of senile dementia among the elderly. Clinically AD is characterized by a decline in c ognitive performance, memory deficits, confusion and behavioral changes (Selkoe 2001). Currently there is not a direct objective test for AD, therefore only post mortem assessment of the brain offers a diagnosis based on the presence of the classic histopathological features of AD including the presence of amyloid plaques, neurofibrillary tangles and neuronal atrophy (Hardy and Selkoe 2002). Although the cause of the disease remains unknown and most of the reported cases are sporadic, a small fraction of AD patients genetically inherit the disease through autosomal dominant inheritance which results in disease onset before the age of 65. Mutations that increase the A deposition are associated with th e familial form of AD termed familial AD (FAD). Currently, mutations in amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 ( PS2) are thought to have a part in the development of the diseas e and are implicated in the transport and processing of APP (Nunan and Small 2002). These mutations all result in an increased production of A42 which is more prone to fibrillo genesis. The ability of the APP gene mutation to give rise to AD neuropathology suggests that amyloid

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deposition is one of the pivotal events in the pathogenesis of AD. This supports the amyloid cascade hypothesis which proposes that the accumulation of A results in AD pathogenesis. APP STRUCTURE, FUNCTION, AND METABOLISM The proteolytic processing of the amyloid precursor protein plays a key role in the development of AD. APP is located on chromosome 21 and is a single transmembrane glycoprotei n that is translocated in to the ER via its signal peptide, then postranslationa lly modified through the se cretory pathway (Nunan and Small 2002). APP is expressed in t he heart, kidneys, lungs, spleen and intestines as well as in the brain. The cleavage of the APP can occur through two different pathways, both of which in volve the action of proteases known as secretases. Cleavage of APP by -secretase at the N-terminus results in APPs and a membrane bound C-terminal fr agment (CTF) C99 (Selkoe 2001). Further cleavage of the CTF C99 by secretases releases A 1-40 and A 1-42. An alternative pathway that involves the cleavage of APP is through secretase which releases a large -APPs fragment into the extracellular space and also generates another CTF, namely C83. C83 is further processed by secretase, releasing a fragment known as p3 (Selkoe 2001). Amyloid (A ) is a 4 kDa peptide derived fr om the proteolytic cleavage of APP (Morishima-Kawashima and Ihara 2002). There are two major forms of A species found in vivo which include A 40 and A 42. The presence of two additional hydrophobic resides on A 42 results in enhanced hydrophobicity and 7

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increased aggregation potent ial. Approximately 90% of secreted A contains the soluble residue (A 40) while only 10% of the total A is comprised of A 42 (Price, Tanzi et al. 1998). A 42 becomes fibrillar, displays a beta sheet conformation and is most often a ssociated with diffuse plaques. A40 is more consistently seen in the blood vasculature and is involved in the development of cerebral amyloid angiopathy (C AA) (Weiner and Frenkel 2006). The accumulation of A 42 in the brain of AD patients is closely associated with neurodegenerati on. The identification of a number of mutations in the APP gene has linked A to the pathological development of AD. Ultimately, these mutations have all bee n shown to increase the production of A 42 in the brain (Selkoe 2001). PRESENILIN STRUCTURE, FU NCTION, AND METABOLISM PS1 and PS2 are homologous membrane proteins. The normal physiological functions of the presenilins are unknown but it is thought that they are involved in intracellular sorting or transport as indicated by their structure. PS1 and PS2 are located in the ER and ci s-golgi compartments of the cell and are cleaved by a protease known as presenilinase, generating a 30 kDa Nterminal fragment (NTF) and a 20 kDa CTF (Turner, O'Connor et al. 2003). Presenilin forms the active site of the secretase complex a nd is involved in the production of A (Spires and Hyman 2005). More than 75 missense mutations in the PS1 gene and three in the PS2 gene have been found, and these FAD mutations cause an increased production of A 42 (Hardy and Selkoe 2002). 8

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The genes for PS1 and PS2 are located on chromosomes 14 and 1, respectively, and mutations in the presen ilin genes appear to be responsible for AD in certain families, with patients exper iencing symptoms starting in their 40s or 50s. The lack of presenilins has been found to reduce A production, while increasing the amounts of and secretase cleaved products. Furthermore, mutations in presenilins affect the secretase cleavage site, increasing the amount of A 42 produced. PS1 mutations cause the earliest and most aggressive form of AD commonly leading to onset of symptom s before the age of 50 (Jellinger and Bancher 1996). In summary, each of the three genes identified are all associated with an increased A42 production or its enhanced deposit ion in the brain. These data support the amyloid cascade hypothesis wh ich states that missense mutations in APP, PS1, or PS2 genes lead to an increas ed accumulation of A 42, resulting in activated micr oglia, the release of neurotoxic species, and oxidative stress which ultimately leads to the neuronal death seen in AD. TRANSGENIC MOUSE MODELS OF A AMYLOIDOGENESIS With the knowledge that A plays a critical role in the development of Alzheimers disease, several groups have created transgenic mice that pathologically develop A deposits similar to those found in human brains. Agedependent increases in learni ng deficits were also observed. The first transgenic mouse model to show amyloid dep osits was developed by Games and colleagues in 1995 (Games, Adams et al 1995). This model known as the 9

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PDAPP model was created us ing a platelet derived growth factor (PDGF) -chain promoter and a human APP mini gene containing the APP 717V F mutation in an APP cDNA with portions of intronic sequences allowing for alternative splicing. These mice began to deposit amyloid in the hippocampus, corpus collosum and cerebral cortex at approximat ely six to nine months of age. The majority of plaques were surrounded by GFAP positive reactive astrocytes, activated microglia and distorted neur ites. Plaque density appeared to increase with age in these transgenic mice as it does in humans, suggesting that progressive A deposition exceeds clearance as is proposed for AD. There were no neurofibrillary tangles observed as we ll as no significant neuronal loss. It was noted that PDAPP mice were impaired in a novel spatial memory task in an agedependent manner that correlat ed with the increases in plaque density (Chen, Chen et al. 2000). Another transgenic mous e model of AD with simi lar phenotype findings was reported by Hsiao and her colleagues (Hsiao, Chapm an et al. 1996). This model termed Tg2576 or APPSW expresses the Swedish mutation of APP, the 695 amino acid form of hum an APP containing the K670 N/M671L FAD mutation, placed under the control of the hamster prion gene promoter. At 9-11 months of age, A deposition is observed in numer ous brain regions. However neurofibrillary tangles are not observed. It is well documented t hat this mutation causes increases in A42 and A40 (Scheuner, Eckman et al. 1996). As Tg2576 mice age, classic neuritic plaques with Congo red-positive amyloid cores appear that are similar to those seen in AD (Irizarry, McNamara et al. 1997). In 10

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addition, Tg2576 mice develop agedependent behavioral deficits on several memory tests including the Y maze, T maze and Morris water maze testing despite the fact that no significant neuronal loss is observed (Hsiao, Chapman et al. 1996; Chapman, White et al. 1999). T he TgCRND8 mouse model contains an APP-695-cDNA with bot h the K670N/M671L and V717F mutations under the control of the prion gene promoter (Chishti, Yang et al. 2001). The APP transgene is expressed at a fivefold higher level than endogenous APP and A deposits are observed as early as three months of age. Mice transgenic for the human APP gen e (Hsiao, Chapman et al. 1996) and the PS1 gene (Duff, Eckman et al. 1996) were crossed to produce a doubly transgenic mouse presenting accelerated amyloid pathology (Holcomb, Gordon et al. 1998). In the double transgenic mouse model, the diffuse deposits are primarily composed of A 42, while the compact deposits and the vascular A are primarily composed of A 40. Double transgenic APP+PS1 mice deposit increased amounts of the A peptide in the cerebral cortex and hippocampus. Compared to the single transgenic Tg2576 or PS1 mutant mice, the APP+PS1 model displays increased GFAP reactive astrocytes, activated microglia and dystrophic neurites (Gordon, Holcomb et al. 2002). Neur ofibrillary tangles are also absent in this mouse model as s een in the other previous models. The APP+PS1 mouse model demonstrates memo ry impairments in the Y maze and radial arm water maze when compared to the non-transgenic mice (Holcomb, Gordon et al. 1998). 11

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A triple transgenic model of AD (3xTg-AD) has recently been developed that carries three Alzheimers diseas e relevant genetic mutations: a human Presenilin M146V knock in mutation (PS1M146V), human amyloid precursor protein Swedish mutation (APPswe), and the human tauP301L mutation (Oddo, Caccamo et al. 2003). These mice develop plaques and tangle pathology in the cerebral cortex, hippocam pus and amygdala, which pa rallels the pathology seen in AD patients. The developm ent of tau pat hology and A deposition simultaneously might allow th is mouse model to give researchers a better insight to therapeutically treating the disease. Although none of the tr ansgenic models of AD develop all of the neuropathological features of the disease, these transgenic mouse models have already provided important insights into the pathogenesis of the disease (Hock and Lamb 2001). The correlation between cognitive decline and increases in amyloid plaque deposition and location em ulate those seen in the human disease. Therefore, despite limitations, AD transgenic mice ar e a valuable model for understanding the role of amyloid de position in AD an d will aid in the development of immunotherapy for t he treatment of this disease. INFLAMMATION AND ALZHEIMERS DISEASE The process of aging and AD is intimately associated with microglial activation, increased production of cytokines, reactive oxygen species and neuronal deficits. Inflammation of the CNS may be the result of both innate and adaptive immune responses. When 5 and 30 month old non transgenic mice 12

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were compared, it was obser ved that there was an increas e in the expression of many inflammatory and stress genes in the aged mice, including complement factors, microglial activation markers, cyclophilin and heat shock proteins, demonstrating a change in the inflammatory process with age (Lee, Weindruch et al. 2000). Evidence from studies with post mo rtem brain tissues and transgenic mouse models of AD implicat e microglial activation as a key process in dictating the inflammatory responses seen in AD. Microglia, the resident macrophages of the CNS, comprise about 12% of the cells in the CNS and participate in activities such as phagocytosis, antigen presentati on, production and release of cytokines, complement components, reactive oxyg en species as well as neurotrophins (Minagar, Shapshak et al. 2002). Microglia are reactive not only in response to trauma and injury, but also play an important role in sensing small changes in the brain homeostasis which often precede t hat of pathological damage (Kreutzberg 1996). Found throughout the CNS parenc hyma, microglial activation is characteristic of many CNS diseases in cluding, stroke, multiple sclerosis, AD, and AIDS dementia (Flaris, Densmore et al. 1993; McGeer, Kawamata et al. 1993; Shrikant and Benveniste 1996; Sriram and Rodriguez 1997). Microglia associated with A plaques have an increased expression of many inflammatory markers, including IL-1, IL-6, TNF MIP-1 and MCP-1. Microglia express cell surface receptors required for interaction with different types of immune cells as well as A These receptors include MHC I and II which are needed in antigen present ation, the complement re ceptors CD11a, b, and c, 13

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and Fc receptors (Huang, Han et al. 2000). While microglial activation has traditionally been viewed as adverse, recent data suggests that microglia may exert beneficial effects in AD as well (Wyss-Coray and Mucke 2002). Microglia in cell culture have been shown to ingest and phagocytose A and the presence of microglia encompassing A deposits may imply that microglia are phagocytosing amyloid in an attempt to remove A plaques (Wyss-Coray, McConlogue et al. 2001). Recent observa tions with brain sections from both APP transgenic mice and AD patients indicate that Fc-R mediated phagocytosis by activated microglia may be implicated in the removal of antibodyopsonized A plaques (Schenk and Yednock 2002). Astroglial overproduction of TGFas well as intracerebral injections of LPS have both demonstrated a decrease in A plaque load in vitro and in vivo (DiCa rlo, Wilcock et al. 2001; Wyss-Coray, McConlogue et al. 2001). The lack of knowledge c oncerning the role of the in flammatory reaction in AD makes it imperative that we understand the role of the innate and adaptive immune response within the context of th e development of AD. Microglial cells may ultimately play a key role in prot ecting against or resolving Alzheimer-like pathology. Changes in microglia l activation states that increase their ability to clear A or adjust their response to A may lead to a powerful therapeutic treatment for AD (Schenk and Yednock 2002). 14

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MCP-1 MCP-1 belongs to the C-C family of chemokines and acts as a chemoattractant for monocytes, activated T cells, basophils and NK cells. MCP-1 binds solely to CCR2, a seven transmemb rane spanning protein that functions through G-protein coupled receptors (Izikson, Klein et al. 2002). MCP-1 is likely to be essential in this recruitment of bone marrow derived cells because the release of this chemokine is the key mechanism for the chemoattraction of monocytes. MCP-1 has been found immunohistochemically in mature, but not in immature senile plaques and in reactive microglia of brain ti ssues from patients with AD. In an adult Japanese population, MCP-1 levels in serum were significantly increased from patients that demonstrated MCI and early AD, but not in late stage AD cases. Galimberti et al (2006) hypothesized that increases in MCP-1 were highest during a transitional state between mild cognitive decline and the development of AD, and that MCP-1 might be serving a neuroprotective effect by signaling to microglial cells to remove A plaques (Galimberti, Fenoglio et al. 2006). In addition, many groups hav e demonstrated that the injection of leukocyte chemoattractant or chemokines into the mouse CNS can increase the process of leukocyte migration (Bell, Taub et al. 1996; Stamatovic, Shakui et al. 2005). MONOCYTES Monocytes are bloodderived leuko cytes that develop in the bone marrow, are released into the blood st ream, and migrate into the brain and 15

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various other organs (Gordon and Taylor 2005). This process occurs not only during development but conti nues throughout life (Wyss-Coray and Mucke 2002). Monocytes are defined as blood mononuc lear cells with beanshaped nuclei, expression of CD11b, CD11c, and CD14 in humans and CD11b and F4/80 in mice, and represent about 5-10% of peripheral blood leukocytes. The total leukocyte population in mi ce is approximately 106 cells, which equates to about 6 x 104 circulating monocytes per ml of mous e blood (Tacke and Randolph 2006). Monocytes are components of the m ononuclear phagocyte system which is comprised of liver Kupffer cells, l ung alveolar macrophages, peritoneal macrophages, brain microglia, skin dendritic cells and bone osteoclasts. Under steady state conditions in the mouse, about of the circulating monocytes leave the blood stream each day, where these cell s enter into different tissues of the body. Dying monocytes are destroyed in the spleen (Muller 2001). The life span of monocytes is still und er debate, but it is thought that the half life of monocytes in blood is relatively short: about three days in humans and one day in mice (Muller 2001). The short half life of monocytes in the blood has led to the idea that these cells may be continuously replenished in order to preserve tissue homeostasis and during times of an innat e or adaptive immune response (Tacke, Ginhoux et al. 2006). In mice respondin g to an inflammatory challenge, the number of monocytes leavi ng the circulation per day is at least double. As with other immune cells, the i mmune function and differentiation of monocytes rely on communication with othe r cells through cell surface proteins. The LFA-1 integrin (Leukocyte function-as sociated molecule 1) is composed of 16

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two subunits, CD11a and CD18 and is involved in monocyte adhesion. Mac-1 is composed of the subunits CD11b and CD18 and is indu ced during inflammation. PECAM-1 mediates the migration of monocytes and other immune cells into inflamed tissues through interaction wit h vascular endothelial cells. VLA-4 is another integrin that plays a role in adhesion through interaction with VCAM-1 or intercellular adhesion molecule 1 (ICAM-1). Once the monocyte is tightly bound, it then migrates between endothelial cell s in response to MCP-1 (Gordon and Taylor 2005; Tacke, Ginhoux et al 2006; Tacke and Randolph 2006). Peripheral blood monocytes display mo rophological heterogeneity in size, granularity, and nuclear mor phology. The initial criteria used to differentiate monocytes from other leukocyte types in clude mononuclearity, high expression of CD11b/Mac-1, high phagocytic capac ity and the ability to develop into macrophages upon stimulation with MCSF (Tacke and Randolph 2006). Monocytes can be classified into two gr oups: a shortlived inflammatory group which homes to tissue during times of damage, and a resident group that functions in the preservation of tissue homeostasis. In order to characterize peripheral blood monocytes, various groups demonstrated the differential expression of CD14 and CD16 mono cytes in human blood (Lagasse and Weissman 1996; Sunderkotter, Nikolic et al. 2004). These populations can be defined into two major subsets: the cl assical CD14high CD16monocytes which express CCR2 and the nonclassical CD 14low CD16+ monocytes which have higher expression of MHCII, CCR5 and CD32 and resemble mature tissue macrophages. Other groups have also identified that di fferential expression of 17

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markers are present in mouse m onocyte populations. These studies demonstrated that CCR2+CD62L+Cx3CR1+ or Ly6C+ or Gr-1 high mouse monocytes correspond to CD14low CD1 6+ classic human monocytes which seem to be migrate to non inflamaed si tes. CCR2+Cx3CR1low or Ly6C-/low or Gr-1 low correspond to CD14highCD16human monocytes which seem to be recruited to inflamed peripheral si tes through recognition of CCL2/MCP-1 (Geissmann, Jung et al. 2003; Tacke, Ginh oux et al. 2006; Tacke and Randolph 2006). The identification of various subsets of monocytes has allowed researchers to address the in vivo rele vance of human monocytes by studying mouse monocytes. Inflammation, trauma and immune stimuli all cause an increased recruitment of monocytes to peripheral tissues, aiding in host defense and tissue repair (Taylor, Martinez-Pomares et al. 2005). The commonalities between mouse and human monocyte subsets confirm a conserved system between species (Gordon and Taylor 2005). Increas ed technical skill in isolating monocyte subsets, as well a better understanding of monocyte antigen composition will hopefully aid in a better understanding of the various physiological roles the monocytic lineage plays. MONOCYTE RECRUITMENT IN APP TRANSGENIC MICE The importance of the peripheral i mmune system in the development of AD pathology remains debatable. Howeve r, these pathological changes are consistently seen to be connected with an inflammatory response involving 18

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microglia and astrocytes (Akiyama, Barger et al. 2000). In vitro studies have shown the potential for A to stimulate the production of cytokines, chemokines and inflammatory mediators from human mi croglia. These signals may be able to recruit peripheral immune cells into t he brains of AD patients (McGeer and McGeer 2001; Rogers and Lue 2001). Howeve r, the lack of a single definitive marker to label monocytes makes it difficult to distinguish invading cells from resident brain microglia (Guillemin and Brew 2004). It is known that microglia cells ar e found associated with senile plaques in humans (Haga, Akai et al. 1989; Itagaki, McGeer et al. 1989) as well is in transgenic mice that develop amyloid (Perlmutter, Scott et al. 1992; Sheng, Mrak et al. 1997; Wegiel, Wang et al. 2001; Wegiel, Imaki et al. 2003). One approach used to track bone marrow derived cells (BMDCs) within the CNS uses bone marrow chimeras, whose endogenous hematopoietic systems have been destroyed by irradiation, then replenished with geneti cally tagged bone marrow obtained from transgenic donors. By studyi ng these transplantation models it has been estimated about 20-40% of adult micr oglia undergo continuous turnover from BMDCs in adulthood (K ennedy and Abkowitz 1997; Hess, Abe et al. 2004). These observations have provided the ba sis for work aimed at using autologous bone marrow to deliver therapeutic gene s to damaged regions of the CNS (Makar, Trisler et al. 2004). Previous st udies have demonstrated engraftment of marrow derived cells within the CNS, primarily as microglia l cells (Priller, Flugel et al. 2001; Vallieres and Sa wchenko 2003; Simard, Soulet et al. 2006). Malm and colleagues showed that overexpres sion of the mutant human APP+PS1 19

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genes in the AD mouse model resulted in an increased migration of bone marrow derived monocytes into the brain during the progression of AD pathology. They demonstrated a 17fold higher increas e in the number of bone marrow derived cells associated with A deposits in mice transplanted before the development of AD pathology compared to aged AD mice transplanted after A deposition (Malm, Koistinaho et al. 2005). Simards group studied the migration of bone marrow transplanted cells in different aged irradiated APP23 mice and found that bone marrow cells could cross the BBB and that approximately 20% of the cells were associated with congophilic plaques. Si mards group also injected different isoforms of A and found that A 40 and 42, but not A 31 and 57 isoforms, were capable of inducing the infiltrati on of blood-derived monocytes as well as activation of inflammatory genes such as TLR-2, IL-1 and MCP-1 (Simard, Soulet et al. 2006). Wegiel and colleagues (Wegiel, Imaki et al. 2003) found that most of the A plaques in APPSwe mice were associated with blood vessels and that monocytes are found at the interf ace between the blood vessels and the A plaques, supporting the hypothesis that the recruitment of bone marrow derived monocytes into the brain occurs during the development of A deposits. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea t hat these cells could be used to deliver therapeutic genes to the brain (Kokovay a nd Cunningham 2005). Bone marrow monocytes could serve as an alternative source to stem cells, because they are safe, readily available and ethically acc eptable source for cell and g ene therapy (Vallieres and Sawchenko 2003). Microglial cells are cons idered a promising therapeutic target 20

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in AD because of their potential role in A plaque evolution and even phagocytosis of A deposits. At least some of these A associated microglial cells are produced in the bone marrow and ci rculate in the blood prior to and during the development of AD (Malm, Ko istinaho et al. 2005) The signal that causes the infiltration of blood derived microg lia in this model of AD has yet to be determined. Moreover the ability of different -amyloid isoforms to recruit and activate microglial cells in vivo is still under debate. Underst anding the relative contribution of these cells to the pathological conditions seen in AD will be important when designing futu re immunotherapies that specifically target A deposits in the brain. GENE THERAPY The ability to correct a disease phen otype in vivo through the use of a gene as a pharmacological agent has been termed gene therapy (Friedmann 1989). This type of therapy is based on t he notion that the disease phenotype can be corrected either through the modifi cation of the resident mutant gene or through the introduction of new genetic info rmation into the defective cells or organs in vivo. The main lim iting step of treating CNS di sorders is the blood brain barrier which limits the influx of cells into the brain parenchyma (Priller, Flugel et al. 2001). The BBB is crucial for the brain to maintain homeostasis, but presents a major obstacle when developing gene th erapy treatments for CNS diseases. Presently, CNS gene transfer studies have re lied on direct injections of cells or vectors into the brain (Eglitis and Mezey 1997). Transplantat ion studies have 21

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been performed in different neurodegenerative diseases and have even gone to clinical trial in Parkinsons disease. Ho wever, the ability of these transplanted cells to survive is low, with only about 5-10% of the tr ansplanted embryonic neurons surviving. Also, in these early studies the damage resulting from drilling holes into the skull or the introduction of a catheter to allow for cell infusion all lead to the potential for high risk. Thus noninvasive gene therapy methods are needed. Previous studies have demonstrated engraftment of marrow derived cells within the CNS (Priller, Flugel et al. 2001; Vallieres and Sawchenko 2003; Simard and Rivest 2004). These observa tions have provided the basis for work aimed at using autologous bone marrow to deliver therapeutic genes to damaged regions of the CNS (Park, Eglitis et al. 2001; Makar, Wilt et al. 2002; Makar, Trisler et al. 2004). Bone marrow cells have been used as a delivery system to express transgenes in the CNS such as glial derived neurotrophic factor and interferon-beta (Makar, Trisler et al. 2004). However, much of our knowledge about bone marrow derived microglia in the normal and injured CNS has been generated by the usage of lethally irradiated mice in which the population dynamics of cells in the brain and in t he bone marrow may differ from that of a normal, non-irradiated mouse (Gaugler Squiban et al. 2001). It has been suggested that it may be possible to use genetically modified monocytes to carry exogenous genes of interest into the brai n or other organs for the purposes of gene therapy. The evaluation of this novel approach to CNS gene therapy will require considerable preclin ical analysis, but a critical first step lies in the 22

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development of efficient methods for ex vivo gene transfer into primary mouse monocytes. Experiments using retroviral and lentiviral vectors have already established the ability of gene engineered hematopoietic stem cells to investigate the nature and fate of monocytes under physiological and pathophysiological conditions in the brain (Mezey and Chandro ss 2000; Priller, Flugel et al. 2001; Djukic, Mildner et al. 2006) With growing interest in the use of monocytes as a novel cellular vehicle for gene therapy in the CNS, it is of in terest and importance to develop optimized in vitr o transfection techniques for genetic modification of primary monocytes ex vivo. NEPRILYSIN One contributing and potentially causal factor in the accumulation of A in late onset AD is the decrease in pr oteases that normally clear soluble A In general A levels are determined by the balance between A production and removal. Thus, even small changes in the rate of proteolytic degradation of A peptides could contribute to AD. Several A degrading proteases have been identified by their ability to cleave A including Neprilysin (NEP), Insulin Degrading Enzyme (IDE), Endothelin Converting Enzyme (ECE), Matrix Metalloproteinase-9 (MMP-9) and Matrix Metalloproteinase-2 (MMP-2). Each protease appears to have di stinct cleavage sites and generates variable A fragments. However, only NEP, IDE and ECE have been reported to affect A levels in the brains of experimental ani mal models. Overexpression of IDE or NEP in AD -related animal models has shown a decrease in levels of A 1-42, 23

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confirming the importance of th ese enzymes in regulating A turnover in vivo (Leissring, Farris et al. 2003; Leissring, Lu et al. 2003). Neprilysin (NEP) is a type II memb rane metalloendopeptidase comprised of approximately 750 residues. NEP is al so referred to as enkephalinase, common acute lymphoblastic leukem ia antigen (CALLA) and neutral endopeptidase and can catalyze the hydrolysis of specific signaling peptides in order to inactivate and degr ade their signal (Turner, Fi sk et al. 2004). Peptides that NEP targets include bradykinin, glucagon, and neuropeptides such as neurotensin and enkephalin (Johnson, Stevenson et al. 1999; Sakurada, Sakurada et al. 2002). NEP is made of 3 parts: an N-terminal domain of 321 residues known as domain 1, a C-termi nal domain of 286 residues known as domain 2 and 4 linker fragm ents connecting domains 1 and 2. Domain 1 contains the active site with a HExxH motif, a motif that is a common feature of many zinc peptidases (Turner, Fisk et al. 2004). NEP is strongly inhibited by phosphoramidon (a protease inhibitor m ade by streptomyces), thiorphan, and EDTA but can be reactivated by zinc (Roques 1993; Iwata, Mizukami et al. 2004). NEP is activated by cytokines, gl ucocorticoids, and protein kinase C and is found in many different types of cells in the body such as the kidney, intestine, lungs, CNS, neutrophils, fibroblasts, and epi thelilal cells (Roques, Noble et al. 1993; Wang, Dickson et al. 2006). Lentiviral overexpression of NEP in the brain has been shown to decrease A deposits in APP transgenic mice with significant plaque loads (Marr, Rockenstein et al. 2003). Iwata et al (I wata, Mizukami et al. 2004) showed that 24

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degradation of exogenous ly administered A 42 was decreased in NEP-deficient mice when compared to nontransgenic controls. Additionally the endogenous levels of A 40 and A42 were significantly elevat ed in the brains of NEPknockout mice, validating that NEP is a physiologically relevant A degrading enzyme. Marr et al (Marr, Rockenstein et al. 2003) injected a lentiviral vector expressing human NEP unilaterally into the hippocampus of APP transgenic mice. One month later, amyloid burden wa s found to be significantly reduced on the injected hemisphere and remaining pl aques were notably smaller than those found on the contralateral side(Turner, Fi sk et al. 2004). The effect of NEP gene transfer via AAV vectors was examined in A PP transgenic mice by Iwata et al, as well as in our lab (Iwata, Mizukami et al. 2004; Carty, Wilcock et al. 2006), both demonstrating that NEP gene transfer c an reduce amyloid deposition in aged transgenic mice. These results show that ev en fairly small upregulation of NEP activity may be sufficient to reduce A accumulation in the brain. Neprilysin expression in the brain has been shown to decrease during the aging process and in the early stages of AD progression (Mueller-Steiner, Zhou et al. 2006). Recent in vitro and in vivo st udies demonstrate that the up regulation of A degrading enzymes can significantly reduce the development of the A peptide (Eckman and Eckman 2005). Gene t herapy may be a key therapeutic treatment allowing for an increased expression of A degrading proteases in the brain. Identification of a method to selectively upregulate brain neprilysin activity may provide a new therapeutic potential (S aito, Iwata et al. 2005). The ability of monocytes to enter into the brain in response to inflammatory signals and 25

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associate with amyloid plaques doubled with the fact that they are easy to obtain and can be genetically altered makes mono cytes an ideal cell choice (Priller, Flugel et al. 2001) Putting monocytes together with an amyloid degrading enzyme such as neprilysin offers a pow erful novel therapeutic tool for the treatment of Alzheimers disease (Stalder, Ermini et al. 2005). 26

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PAPER 1: SPATIAL AND TEMPORAL KINETICS OF MONOCYTE MIGRATION INTO THE CNS Lebson La, Morgan Da, Gordon MNa aAlzheimer's Research Laboratory, University of South Florid a, School of Basic Biomedical Sciences, Depar tment of Molecular Pharmacology and Physiology, 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. ACKNOWLEDGEMENTS: ACKNOWLEDGEM ENTS: This work was supported by a grant from the Alzheimers Association IIRG 07-58446 (MNG). LL is the Benjamin Scholar in Alzheimer Research. 27

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ABSTRACT The recruitment of peripheral blood m onocytes into the brain during the development of amyloid pathology is poor ly understood. Previous work by other groups as well as our own has show n that bone marrow derived monocytes have the ability to migrate to the brain and associate with amyloid deposits in the AD transgenic mouse model. Circulating m onocytes have only been studied to a limited extent in the mouse, due in part to the limited availabili ty of cells and the lack of definitive marker to identify this cell type. Consequently, the life span and migration abilities of these cells remain uncertain. To determine the time course of monocyte recruitment into the brain during the neurodegenerative damage characteristic of Alzheimers disease, we used transplanted GFP labeled bone marrow monocytes to characterize the ki netics that peripheral monocytes display once injected into the circulation. We fi rst determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found this time to be 1.5 hours post injection. We also determined where these cells migrated following their exit from the blood stream. Our data indicate that there is a recruitment of these mono cytes to the spleen, lungs, liver and brain. Through stereological and flow cytometry analyses, we also show that APP+PS1 mice have significantly more recrui tment of monocytes to the brain in areas of plaque deposition compared to cont rol nontransgenic mice. 28

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INTRODUCTION Increasing evidence suggests that t he accumulation of amyloid plaques, composed primarily of the amyloid beta protein (A ), is a common histopathological featur e in Alzheimers disease (AD). A may play a critical role in disease progression (Hardy and Selk oe 2002). Although microglial cells respond and demonstrate morphological changes when exposed to A (Bard et al., 2000; DiCarlo et al., 2001; Wyss-Coray et al., 2001; Wilcock et al., 2003). the role the peripheral immune system in t he disease process remains debatable. However, these pathological changes are consistently seen to be connected with an inflammatory response involving micr oglia and astrocytes (Akiyama et al., 2000; Ueda et al., 2004; Nagaoka et al., 2000). In vitro studies have shown the potential for A to stimulate the production of cytokines, chemokines and inflammatory mediators from human mi croglia. These signals may be able to recruit peripheral immune cells into t he brains of AD patients (McGeer and McGeer 2001; Rogers and Leu 2001). It is we ll known that microglia cells are found associated with senile plaques in humans (H aga et al., 1989; Itagaki et al., 1989) as well is in transgenic mice that dev elop amyloid (Perlmutter et al., 1992; Sheng et al., 1997; Weigel et al., 2001; Weigel et al., 2003). At least some of these A associated microglial cells are pro duced in the BM and circulate in the blood prior to and during the development of AD (Malm et al., 2005). The signal that causes the infiltration of blood derived microglia in this model of AD has yet to be determined. Moreover the ability of different -amyloid isoforms to recruit and activate microglial cells in vivo is still under debate. Understanding the 29

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relative contribution of these cells to the pathological conditions seen in AD will be important when designing future immunot herapies that specifically target A deposits in the brain. Furthermore, monocyte derived and resident CNS microglia are practically identical on the basis of current immunophenotypic markers. The lack of a single defining marker to distinguish the invading monocytes/macrophages from resident and activated microglia has made the study of the peripheral systems involvement in the CNS challenging. Their isolation therefore presents a challenge and accounts for the current scarc ity of kinetic data on pure monocytes populations in AD. Previous studies on the turnover of monocytes in the circulation used autoradiography to detect cells labeled at various times using an infusion of [3H]thymidine, which labels t he DNA of cells in S-phase. (Van Furth et al 1970) However since we are now able to track the migration of GFP labeled cells, it is possible to identify the locati on from which these cells are originating. The migratory route monocytes take onc e leaving the bone marrow is currently unclear, but previous work has suggest ed that hematopoietic cells may migrate into the brain parenchyma to give rise to microglia (Massengale et al 2005, Priller et al., 2001, Vallieres et al., 2003). O ne drawback of all of these studies, however, was the use of lethal whole b ody irradiation prior to bone marrow transplantation, which could have induced n euroinflammation, altered the state of the blood brain barrier, and artefactually promoted peripheral immune cell entry. In the present study, we used tr ansplanted GFP labeled bone marrow monocytes to discriminate per ipheral monocytes from in trinsic brain microglia. 30

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We used these identifiable donor cells to c haracterize the kinetics that peripheral monocytes display once injected into the circulation. We first determined the half life of bone marrow derived monocytes in t he peripheral circulation. Our data indicate that there is a recruitment of these monocytes into the spleen, lungs, liver and brain from circulation. Through st ereological and flow analyses, we also show that APP+PS1 mice have more recrui tment of monocytes to the brain in areas of plaque deposition compared to control non transgenic mice. MATERIALS AND METHODS Animals 16 month old double transgenic APP+PS1 mi ce that are a cross between the mAPP transgenic line Tg2576 (Hsiao, Chapman et al. 1996) and the mPS1 transgenic line 5.1 (Duff, Eckman et al, 1996) and nontransgenic littermates were used. The GFP transgenic mouse model used for bone marrow donors were C57BL/6-Tg(UBC-GFP)30Scha/J [Jackso n Laboratory, Bar Harbor, ME; Stock #004353]. These transgenic mice expr ess the enhanced green fluorescent protein (GFP) under the direction of the human ubiquitin C promoter. All mice were bred and maintained in our animal facility according to institutional guidelines. Adoptive Transfer of Monocytes Transgenic GFP mice were overdosed with pentobarbital. The femurs and tibias were removed aseptically and bone marrow was flushed from the bone using a 25G 5/8 gage needle attached to a syringe. Cells were co llected by centrifugation 31

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for 5 minutes at 300xg (4C). CD11b+ cells were separated using immunomagnetic cell depletion using MACS technology (Miltenyi Biotec, Auburn CA). 5 X 106 freshly isolated CD11b+, GFP+ cells were then resuspended in 100 l saline and injected into the left heart ventricle. Blood 1 mL of blood was drawn from each mouse via cardiac puncture. 10 ml of RBC lysis buffer was immediately added to the blood and incubated for 5 minutes. To stop the reaction, 30 ml of 1xPBS was added, and the ce lls were centrifuged at 300xg at 4C for 10 minutes. Cells were resuspended in PBS and taken for flow cytometry analysis. GFP+ and total cell nu mbers in the blood were analyzed at 1 minute, 30 minutes, 2 hours, 4 hours, 6 hours, and 24 hours following cell injection. Tissue Collection and Histochemical Procedures On the day of sacrifice, mice were overdosed with pentobarbital (100mg/kg). The brain, spleen, liver and lung were remo ved, bisected sagittally and the left half was immersed in freshly prepared 4% paraformaldehyde in 100 mM PO4 buffer (pH 7.4). The organs were postfixed in paraformaldehyde for 24 hours. The brain, liver and spleen tissue were cryoprotected in a series of sucrose solutions, frozen, sectioned in the horiz ontal or transverse plane at 25 m using a sliding microtome and stored at 4C in Dulb eccos phosphate buffered saline for immunocytochemistry and histology. The lung tissue was paraffin embedded 32

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before being sectioned using a rotary microtome. One-hal f of each organ was digested as described (Cardona et al 2006). Single-cell suspensions were analyzed by flow cytometry. Immunohistochemistry was performed on free floating sections as described in detail previously (Gordon et al, 1997). A series of 8 sections spaced approximately 600 m apart were incubated with pr imary antibody overnight at 4C, then incubated in the biotinylat ed secondary antibody (2h) followed by streptavidin-peroxidas e. Peroxidase reactions consist of 1.4 mM diaminobenzidine with 0.03% hydrogen perox ide in PBS for exactly 5 minutes. Single and multiple immunofl uorescent labeling: after incubation with the primary antibody, the free floating sections we re then incubated for 2 hours with the appropriate fluorophore coupl ed secondary antibody. As secondary antibodies, goat IgG coupled to AlexaFluor 594 (1:1500), AlexaFluor 488 (1:1500) (Molecular Probes, Eugene, OR) were used. Sections were rinsed in Dulbeccos PBS and coverslipped with VECTASHIELD Mounting Medium with DAPI. Image and Flow Cytometry Analysis GFP fluorescence, immunohistochemis try and Congo red staining were quantified with Image Pro Plus (Media Cybe rnetics) image software. All values obtained from a single mouse were averaged together to represent a single value for that animal. Statistical analysis was performed using ANOVA followed by Fischers LSD post hoc means comparis on test (Statview software from SAS). For flow cytometry cells were analyz ed on a FACSCalibur cytometer or LSR 33

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cytometer (Becton Dickins on, Mountain View, CA) using CellQuest software (Becton Dickinson). Stereology N = (Number of cells counted) x (1/ ssf) x ( 1/asf) x (1/tsf) ssf = the sampling fraction (i.e. a 1/6 seri es or 1/17). The fraction of the total samples used asf = area sampling fraction ( this is going to be 1 since you counted everything). Tsf = thickness sampling fraction (you will have to measur e with a z axis encoder) M.J. West and H.J.G. Gundersen, Unbi ased stereological estimation of the number of neurons in the human hippocampus. J. Comp. Neurol. 296 (1990) RESULTS Injected monocytes have a short half life in circulation: In order to track the migration of injected monocytes thr ough the blood stream following a single injection of 5 million monocytes, GFP mice were used as cell donors. These cells were injected into the left ventricle and t he mice were analyzed at 6 time points following the injection. Using flow cytom etry, we were able to identify the GFP monocytes in the blood and determine thei r relative numbers at each time point (Figure 1). Injected monocytes spent very little time in the blood stream following injection, with a half life of 1.5 hours. By 24 hours, we were not able to find any GFP labeled monocytes in the circulation. 34

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Monocytes can be found in the brain, liver, lung and spleen up to a week after cell injection: To determine the number of GFP bone marrow derived cells in spleen, liver, lung and the brains of APP+PS1 transgenic and nontransgenic mice, cells were visualized using a fl orescent microscope and stereological analysis (Figure 2A) or by flow cytomet ry (Figure 2B). Stereological analysis detected a significant difference in recr uitment in the liver, lung and spleens between the nontransgenic and the APP+ PS1 transgenic mice. While not significant, these trends were also conf irmed using flow cytometry analysis (Figure 2B). This significant increase in the organs of the nontransgenic mice is most likely due to the fact that the majo rity of the GFP+ monocytes in the APP+ PS1 were located in the brain. In the nontransgenic control mice, only a few cells were seen in the brain and the few that were present were associated with the blood vessels. The APP+PS1 mice however recruited a significant number of GFP positive cells to the brain following a single injection. These cells were found mainly in the cortex and hippocampal loca tions, those regions with high amyloid load. Stereological analysis revealed a 90 % increase for the transgenic mice compared to the nontransgenic 24 hours after the injection. These results were confirmed with flow cytometry, which confi rmed a significant increase of 97 % in GFP+ cell number in the brains of APP+PS1 mice. Morphologically the GFP+ monocytes look ed identical in the spleen, liver and lung when compared between the two groups of mice as seen in Figure (3). Fluorescent analysis also revealed an association of the GFP+ cells with brain 35

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amyloid deposits. The majority of these cells were ameboid in shape; however a small percentage of the cells displayed short processes. It is important to note that while some amyloid plaques we re decorated with GFP+ cells, other neighboring plaques completely lacked any association with GFP+ cells. The size of the plaque did not seem to be a fact or for the association with GFP positive cells. The residence time of these cells followi ng their migration from the blood to the various tissues also demonstrated a trans ient time course. The most number of cells were observed at the shortest time point (1 day). Al though the rate of decline seemed to vary somewhat in differe nt organs, by day 7, almost all of the GFP+ monocytes were absent from each of the organs examined. DISCUSSION Although previous studies have demonstrated infiltration of GFP+ peripheral microglia follo wing irradiation and bone marro w reconstitution, the percent contribution of circ ulating monocytes to the mi croglial reaction in brains has not been investigated. In this present study, we characterized the half life and migration of bone marro w mouse monocytes in noni rradiated mice. To track these monocytes, we utilized bone marrow monocytes from mice that express an enhanced green fluorescent protein (GFP ) under the direction of the human ubiquitin C promoter into APP+PS1 and nontransgenic mice. We demonstrated that monocytes have a short half life in blood circulation: 1.5 hours following a 36

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single injection. Ultimately these cells l eave the circulation and enter a variety of tissues including the lung, liver spleen and the brain. Despite considerable advances in understanding imm une cell migration into the CNS, the invasion of different immune cells is only understood in part. Under non-inflammatory conditions, it has been reported that there are approximately 3000 macrophages recruited to the brain of a normal mouse each day (Larson et al 1992). While we did not see a significant recruitment of GFP+ monocytes in the brains of nontransgenic mice, we di d demonstrate that an overexpression of AD linked mutant human APP and PS1 genes in mice triggers a significant increase in migration of bone ma rrow derived monocytic cells into the brain. These cells were able to access all regions of the brain and had a transient lifespan, about 7 days, once in the CNS parenchyma. Al though the percentage of GFP+ monocytes was small compared to the endogenous microglia, the majority of monocytes were located near and around amyloid plaques. These results emphasize that there is an interaction occurring between the peripheral immune system and cells in the CNS. It has been proposed that chemokines presented by the vascular endothelium regulate influx of hematopoietic cells and direct their fate from bone marrow to blood and tiss ues. However, with the presence of inflammation, changes in the cellular composition of these sites increases the emigration of bone marrow cells into the circulation (Rot A. and von Andrian, 2004; Tani et al., 1994; Pleiner et al., 1998). It has been hypothes ized that many of the signals that request cells to the brain are mediated by the release of pro and anti-inflammatory cytokines. Cytokines such as IL-1 TNF, and TGFare 37

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released from astrocytes and microglia during brain inflammation and can intensify the expression of adhesion mole cules and chemokines resulting in an increased extravasation of leukocytes (Kim and de Vellis, 2005; Campbell et al., 1998; Merrill and Murphy, 1997). MCP-1 is likely to play a role in the recruitment of bone marrow derived cells, because t he release of this chemokine by endothelial cells is the key mechanism for the chemoattraction of cells of monocytic lineage. This chemokine is rapi dly synthesized by endothelial cells in response to immune stimuli, which trigger s monocyte recruitment and rolling prior to their transmigration. MCP-1 is also re ported to be increased in the brains of amyloid-depositing transgenic mice (Simard et al 2006; Janelsins et al 2005). An up regulation of ICAM-1 could also contribu te to recruitment of immune cells from the circulation. It is likely that monocytes express receptors for ICAM-1 on their surface and endogenous brain microglia might signal to peripheral cells through an up regulation of this adhesion mole cule (Woiciechowsky et al., 2002; Merrill and Beneveniste 1996). Another important factor in the recr uitment of monocytes is to determine which population is recruited to the brain. In our study we isolated monocytes from bone marrow using a magnetic cell se paration kit that used CD11b as the marker to isolate the cells. Many groups have recently identified two subsets of CD11b monocytes: a resident and an in flammatory subset. The so called inflammatory monocytes express the cell surface protein Ly6c(gr1+), the chemokine receptor CCR2, and the adhesion molecule L-selectin. They are selectively recruited to inflamed ti ssues and lymph nodes and also have the 38

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capability to differentiate into inflammato ry dendritic cells. The second subset of monocytes has been designated resident in mice because they are located in both resting and inflamed tissues. This subset is defined by smaller size, high expression of both the chem okine receptor CX3CR1 and LFA-1 integrin, and by the lack of expression of Ly6c, CCR2 and L-selectin (Geissman et al., 2003; Sunderkotter et al., 2004; Palframan et al., 2001). Resident and inflammatory monocytes thus appear to be defined by di stinct sets of adhesion molecules and chemokine receptors, which suggests different modes of tissue trafficking. However there are still important questions that need to be addressed when profiling these monocytes. For exampl e, do these monocytes leave the bone marrow prepared to enter the tissues through the presence of a specific receptor? Or is it the pres ence of an inflammatory stimulus that causes a switch in these cells from a resident to inflamma tory state? Also it will be important to determine if there are distinct receptors that direct monocytes to specific organs following emigration from the bone marro w. Answers to these fundamental questions will be important to further under stand the recruitment of each subset in the presence of AD pathology in tr ansgenic mice and to further specify the exact population that migrat es to the brain. While it is well known that microglia cells are found associated with senile plaques in humans(Haga et al., 1989; It agaki et al., 1989) as well as in transgenic mice that develop amyloid (P erlmutter et al., 1992; Sheng et al., 1997); Weigel et al 2001; Weigel et al 2003) many groups have argued the origin of the cells. However, recently studi es have demonstrated engraftment of marrow 39

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derived cells within the CNS (Hess et al., 2004; McMahon et al., 2002; Simard and Rivest, 2004; Yagi et al., 2004). Cons istent with the data presented in this study, Malm and colleagues sh owed that overexpression of the mutant human APP+PS1 genes in the AD mouse model resu lted in an increased migration of bone marrow derived monocytes into the br ain early in the progression of AD pathology. They demonstrated a 17fold higher increase in the number of bone marrow derived cells associated with A deposits in mice transplanted before the development of AD pathology compared to aged AD mice transplanted after A deposition (Malm et al., 2005). Simards group studied the migration of BM transplanted cells in different aged irr adiated APP23 mice and found that bone marrow cells could cross the BBB and that approximately 20% of the cells were associated with congophilic plaques. Simards group also injected different isoforms of A and found that A 40 and 42, but not A 31 and 57 isoforms, were capable of inducing the inf iltration of blood-derived monocytes as well as activation of inflammatory genes such as TLR-2, IL-1 and MCP-1 (Simard et al., 2006). Wegiel and colleagues (Weigel et al., 2003) found that most of the A plaques in APPSwe mice were associated with bl ood vessels and that monocytes were found at the interface bet ween the blood vessels and the A plaques, supporting the hypothesis that the recruitment of BM derived monocytes into the brain occurs during the development of A deposits. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea t hat these cells could be used to deliver therapeutic genes to the brain (Kokovay and C unningham, 2005). BM monocytes could serve as an 40

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alternative source to stem cells, becaus e they are safe, readily available and ethically acceptable source for cell and gene therapy (Vallier es and Sawchenko 2003). Microglial cells are considered a promising therapeutic target in AD because of their potential role in A plaque evolution and even phagocytosis of A deposits. At least some of these A associated microglial cells are produced in the BM and circulate in the blood prior to and during the development of AD (Malm et al., 2005). The signal that caus es the infiltration of blood derived microglia in this model of AD has yet to be determined. Moreover the ability of different -amyloid isoforms to recruit and activa te microglial cells in vivo is still under debate. Understanding the relative contribution of these cells to the pathological conditions seen in AD wil l be important when designing future immunotherapies that s pecifically target A deposits in the brain. In summary, our data provide evidence that peripheral bone marrow monocytes are actively recruited to the brains in AD trans genic mouse models. Monocytes were localized near and ar ound plaque pathology only in the APP+PS1 transgenic mouse and not in the non transgenic mouse model. Defining the contribution of monocytes to the neuroinf lammatory response in AD and other neurodegernative diseases may re veal new therapeutic targets or gene delivery approaches. Monocytes represent a novel therapeutic vehicle for delivering drug molecules or proteases to improve recovery and repair brain damage for the treatment of AD. 41

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REFERENCES Akiyama, H., S. Barger, et al. (2000). "Inflammation and Alzheimer's disease." Neurobiol Aging 21(3): 383-421. Bard F., Cannon C., Barbour R., Burke R.L. Games D., Grajeda H., Guido T., Hu K., Huang J., Johnson-Wood K., Khan K., Kholodenko D., Lee M., Lieberberg I., Motter R., Nguyen M., Soriano F., Vasquez N., Weiss K., Welch B., Seubert P., Schenk E., Yednock T. (2000). Peripherally administered antibodies against amyloid beta peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimers disease. Nat. Med. 6 : 916-919. Campbell J.J., Hedrick J., Zlotnik A., Si ani M.A., Thompson D.A., Butcher E.C. (1998). Chemokines and the arrest of lymphocyte rolling under flow conditions. Science 279 : 381-384. DiCarlo G., Wilcock D. Henderson D., Gordon M., Morgan D. (2001). Intrahippocampal LPS injections reduce A load in APP+PS1 transgenic mice. Neurobiol. Aging 22: 1007-1012. Djukic M., Mildner A., Schmidt H., Czesni k D., Bruck W., Priller J., Nau R., Prinz M. (2006). Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology followin g meningitis in mice. Brain. 129 : 2394-2403. Geissmann F., Jung S., Littman D.R. (2003) Blood monocytes Consist of Two Principal Subsets with Distinc t Migratory Properties. Immunity 19:71-82. Gordon S. and Taylor P.R. (2005) Monocyte and macrophage heterogeneity. Nat Rev Immunol 5 :953-64. Guillemin, G. J. and B. J. Brew (2004) "Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification." J Leukoc Biol 75(3): 388-97. Kin S.U., de Vellis J. (2005). Microglia in health and disease. J Neurosci Res 81:302-313. Haga, S., Akai K., Ishii T. (1989). "Dem onstration of microglial cells in and around senile (neuritic) plaques in the Alz heimer brain. An immunohistochemical study using a novel monoclonal antibody." Acta Neuropathol (Berl) 77(6): 569-75. 42

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Hardy J. and Selkoe D.J. (2002). The amyloid hypothesis of Alzheimers disease: progress and problem s on the road to therapeutics. Science 297 : 353-356. Henderson R.B., Hobbs J.A., Mathies M., Hogg N. (2003). Rapid recruitment of inflammatory monocytes is independent of neutroph il migration. Blood 102 :328. Hess DC, Abe T., Hill W.D., Studdard A.M. Carothers J., Masuya M., Flemming P.A., Drake C.J., Ogawa M. (2004). Hematopoietic origin of microglial and perivascular cells in brain. Exp. Neurol 186 : 134-144. Itagaki, S., P. L. McGeer, et al. (1989). "Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease." J Neuroimmunol 24(3): 173-82. Jung S., Aliberti J., Graemmel P., Suns hine M.J., Kreutzberg G.W., Sher A., Littman D.R. (2000). Analysis of Frac talkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluor escent Protein Reporter Gene Insertion. Molecular and Cellular Biology. 20: 4106-4114. Kato H., Kogure K., Liu X.H., Araki T., Itoyama Y. (1996). Progressive expression of immunomolecules on ac tivated microglia and invading leukocytes following focal cerebral ischemia in the rat. Brain Res 734 : 203-212. Kokovay, E. and L. A. Cunningham (2005). "Bone marrow-derived microglia contribute to the neuroinflammatory response and express iNOS in the MPTP mouse model of Parkinson's disease." Neurobiol Dis 19(3): 471-8 Lagasse E., Weissman I.L. (1996). Flow cytometric identification of murine neutrophils and monocytes. J. Immunol. Methods 197 :139. Massengale M., Wagers A.J., Vogel H., Weissman I.L. (2005).Hematopoietic cells maintain hematopoietic fates upon entering the brain. J Exp Med 201 :1579-1589. Makar, T. K., D. Trisler, et al. (2004) "Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marro w cells as vehicles in mice." Neurosci Lett 356 (3): 215-9. Malm, T. M., M. Koistinaho, et al. (2005). "Bone-marrow-derived cells contribute to the recruitment of microglial ce lls in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice." Neurobiol Dis 18(1): 134-42. 43

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McGeer, E. G. and P. L. McGeer (2001). "Innate immunity in Alzheimer's disease: a model for local inflammatory reactions." Mol Interv 1 (1): 22-9. McMahon E.J., Suzuki K., Matsushi ma G.K. (2002). Peripheral macrophage recruitment in cuprizone-induced CNS demylination despite an intact blood brain barrier. J. Immunol 130 : 32-45. Merrill J.E., Murphy S.P. (1997). Inflammatory events at the blood brain barrier: regulation of adhesion mole cules, cytokines, and chemokines by reactive oxygen species. Brain Behav Immun 11:245-263. Merrill J.E., Benveniste E.N. (1996). Cytok ines in inflammatory brain lesions: helpful and harmful. Trends Neurosci 19:331-338. Nagaoka H., Gonzalez-Aseguinolaza G., Tsuji M., Nussenzwe ig M.C. (2000). Immunization and infection change the number of recombination activating gene (RAG)-expressing B ce lls in the periphery by altering immature lymphocyte production. J. Exp. Med 191 :2113-2120. Nimmerjahn A., Kirchoff F., Helmchen F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308 :1314-1318. Palframan R.T., Jung S., Cheng G., Weni nger W., Luo Y., Dorf M., Littman D.R., Rollins B.J., Zweerink H., Rot A., v on Andrian U.H. (2001). Inflammatory chemokine transport and pr esentation in HIV: a remote control mechanism for monocytes recruitment to ly mph nodes in inflamed tissues. J Exp. Med 9 :1361-73. Perlmutter, L. S., S. A. Scott, et al (1992). "MHC class II-pos itive microglia in human brain: association with Alzheimer lesions." J Neurosci Res 33(4): 549-58. Pleines U.E., Stover J. F., Kossmann T., Trentz O., Morganiti K.M. (1998). Soluble ICAM-1 in CSF coincides with the extent of cerebral damage in patients with severe traumatic brain injury. J Neurotrauma 15:399-409. Priller J. Flugel A., Wehner T., Boenter et M., Haas C.A., Prinz M., FernandezKlett F., Prass K., Bechmann I., de Boer B.A. (2001). Targeting gene modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat Med 7 :13561361. Rot A., von Andrian U.H. (2004). C hemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu. Rev. Immunol. 22:891-928. 44

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Rogers J., Lue L.F. (2001). Microglial chem otaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimers disease. Neurochem Int. Schoning B., Elepfandt P., Daberkow N ., Rupprecht S., Stockhammer F., Stoltenburg G., Volk H.D., Woiciec howsky C. (2002). Differences in immune cell invasion into the cerebrospinal fluid and brain parenchyma during cerebral infusion of interleukin-1 . Neurol Sci. 23:211-218. Sheng, J. G., R. E. Mrak, et al. (1997). "Neuritic plaque evolution in Alzheimer's disease is accompanied by transition of activated microglia from primed to enlarged to phagocytic forms." Acta Neuropathol (Berl) 94(1): 1-5. Siamon G., Taylor P.R. (2005). Monocyte and Macrophage Heterogeneity. Nat. Rev. Immunol 5 :953-64. Simard A.R., Rivest S. (2004). Bone marrow stem ce lls have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J. 18: 998-1000. Sunderkotter C., Nikolic T., Dillon M.J., R ooijen N., Stehling M., Drevets D.A., Leenen P. (2004). Subpopulations of M ouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response. J Immunol. 7 :4410-4417. Tacke F., Randolph G.J. (2006). Migrato ry fate and differentiation of blood monocytes. Immunobiology. 211 :609-618. Tacke, F. and G. J. Randolph (2006). "Migra tory fate and differentiation of blood monocyte subsets." I mmunobiology 211 (6-8): 609-18. Tani M., Ransohoff R.M. (1994). Do c hemokines mediate inflammatory cell invasion of the central ner vous system parenchyma?. Brain Pathol. 4 :125-143. Ueda Y., Yang K., Foster S.J., Kondo M ., Kelsoe G. Inflammation controls B lymphopoiesis by regulating chemokine CXCL12 expression. J. Exp. Med 199 :47-58. Vallieres L., Sawchenko P.E. (2006). Bone marrow derived cells that populate the adult mouse brain preserve their hematopoietic identity. J. Neurosci. 26:11753-11762. Van Furth R., Diesselhoff-Den Dulk M. (1970). The kinetics of promonocytes and monocytes in the bone marrow. J. Exper. Med 813-828. 45

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Van-de S.A., Van-der S. P. (1996). Intercellula r adhesion molecule-1. J Mol Med 74:13-33. Wegiel, J., H. Imaki, et al. (2003). "Origin and turnov er of microglial cells in fibrillar plaques of APPsw transgenic mice." Acta Neuropathol (Berl) 105 (4): 393-402. Wegiel, J., K. C. Wang, et al. (2001). "The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APP(SW) mice." Neurobiol Aging 22(1): 49-61. Wilcock D.M., DiCarlo G., Henderson D., Jackson J., Clarke K., Ugen K.E., Gordon M.N., Morgan D. (2003). Intracranially adminis tered anti-Abeta antibodies reduce beta-amyloid deposition be mechanisms both independent of and associated with microglial activation. J. Neurosci 23: 3745-3751. Wyss-Coray T., Lin C., Yan F., Yu G.Q. Rohde M., McConlogue L., Masliah E., Mucke L. (2001). TGF-beta 1 promotes microglial amyloid beta clearance and reduces plaque burden in transgenic mice. Nat. Med 7 : 612-618. Wyss-Coray T., Mucke L. (2002). Infla mmation in neurodegenerative disease-a double edged sword. Neuron 35 :419-32. Yagi T., McMahon E.J., Takiki ta S., Mohri I., Matsushima G.K., Suzuki K. (2004). Fate of donor hematopoietic cells in demyelinating mutant mouse, twitcher, following transplantati on of GFP+ bone marrow cells. Neurobiol. Dis 16: 98-109. 46

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Figure 1: THE HALF LIFE OF INJECTED GFP+ MONOCYTES IN CIRCULATION IS APPROXIMATELY 1.5 HOURS. A single injection of 5 x 106 GFP bone marrow monocytes was made into the left ventricle of the heart of nontransgenic mice At the indicated time points, blood was collected and the number of GFP+ monocytes in t he blood was measured by flow cytometry (n=4 at each time point). Peripheral Blood 0 6 12 18 24 0 1 2 3 4 Time (hr)# GFP+ Monocyte in 1 mL blood x 106 47

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Figure 2: MIGRATION OF GFP+ MONOCYTES TO ORGANS IN THE PRESENCE AND ABSENCE OF ALZHEIMER-like PATHOLOGY Recruitment of GFP+ bone marrow monocyt es to tissues in nontransgenic and APP+PS1 transgenic mice were compar ed. A single injection of 5 x 106 GFP+ bone marrow monocytes was made into the left ventricle of the heart, and blood was collected 1, 3 and 7 days later. The number of monocytes present in each organ was quantified using (A) stereology and (B) flow cytometry and is plotted as the number of GFP monocytes / mg of tissue for each organ. indicates significant differences between APP+PS 1 and non transgenic mice (P <0.05 by t-test). (n=4 at each time point). (A). Stereology 1250APP+PS1 1 3 7 1 3 7 1 3 7 1 3 7 0 250 500 750 1000Non Transgenic LiverSpleen Lung Brain Days# GFP monocytes/ mg of tissue 48

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(B). Flow Cytometry 1 3 7 1 3 7 1 3 7 1 3 7 0 100 200 300 400APP+PS1 Non Transgenic LiverSpleenLung Brain Days# GFP monocytes/ 50,000 cells 49

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Figure 3: GFP+ MONOCYTE ENTRY INTO TISSUE Representative micrographs of GFP staining in the tissues indicated above each panel. Scale bar = 25 m. 50

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PAPER 2 INTRACRANIAL INJECTION OF NEPRILYSIN-TRANSFECTED MONOCYTES REDUCES AMYLOID IN APP TRANSGENIC MICE. Lebson L1, Nash K1, Kamath S, Morgan D1, Gordon MN1 1Alzheimer's Research Laboratory, Universi ty of South Florida, Department of Pharmacology, 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. ACKNOWLEDGEMENTS: This work was s upported by grants to MNG from the Alzheimers Association (IIRG-07-58446) and the National Institute on Aging (AG15490). LL is the Benjamin Sc holar in Alzheimer Research. 51

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ABSTRACT The accumulation of amyloidprotein (A ) in Alzheimers disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of A is therefore thought to serv e as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of A and defects in the degradation of A could contribute to AD disease progression. Additionally, studies implicating the hom ing of monocytes to regions of CNS damage have led to the idea that it may be possible to us e genetically modified monocytes to carry exogenous genes of interest into the brai n or other organs for the purposes of gene therapy. The evaluation of this ne w technique in CN S gene therapy will warrant extensive preclinical investigation, but a cruc ial first step requires the characterization and optimizat ion of efficient ex vivo methods of gene transfer into primary mouse monocytes. As an approach to up regul ate levels of these A -degrading proteases in a transgenic mouse model of am yloid deposition, we developed an ex vivo gene therapy method utilizing bone marrow monocyt es from mice transgenic for green fluorescent protein (GFP) mice. Thes e monocytes were transfected with a construct directing expression of either a secreted neprilysin pr otein (NEP-S) or a mutant form (NEP-M), which lacks any enzym e activity, into the brains of APP tg mice. We found a significant reduction in both A and Congo Red staining in the NEP-S injected mice only. These results support the hy pothesis that NEP plays a role in A clearance and that pairing monocyt es together with an amyloid52

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degrading enzyme such as NEP offers a pow erful novel therapeut ic tool for the treatment of Alzheimers disease. INTRODUCTION Alzheimers disease (AD) is the mo st common cause of senile dementia and is characterized by the presence of amyloid plaques, neurof ibrillary tangles, neuron loss and gliosis. Many different studies have alluded to A as a culprit in AD disease progression because the 4042 amino acid peptide is the major component of amyloid deposits seen in postm ortem AD pathology (Selkoe, 1999, Gandy. S, 2005). Also, the genetic changes observed in the inherited forms of AD have shown an increase in the assembly of A or a mutation in the amyloid precursor protein from which A is derived (Mills et al., 1999, Tanzi, and Bertram, 2005). Decreasing the production or increasing the degradation of A is therefore thought to serve as a potential therapeutic intervention in AD. However, while the mechanisms for the production of A have been studied at length, little is known about the normal degradation processes of A in the brain. Evidence from many laboratories suggests that decreasing A levels in brain tissue is a logical approach to prevent or slow the development of AD (Q iu et al., 1997, Saido et al., 1998, Mentlein et al., 1998). Recent in vitro and in vi vo studies have suggested that certain proteases may be involved in the catabolism of A and that defects in the degradation of A might play a role in AD disease pr ogression. Proteases that may be involved in A degradation include insulin degrading enzyme (IDE), endothelin converting 53

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enzyme (ECE), matrix metalloproteinase (MMP-9), and neprilysin (NEP) (Iwata et al., 2000, Iwata et al, 2001, Eckman et al., 2001, McDermott et al 1997, Qiu et al 1998, Carvalho et al, 1997). Nepril ysin (NEP) is a type II membrane metalloendopeptidase comprised of appr oximately 750 residues, and can catalyze the hydrolysis of specific signal ing peptides in order to inactivate and degrade their signal (Iwata et al., 200 1). NEP is also referred to as enkephalinase, common acute lymphoblastic leukemia antigen (CALLA) and neutral endopeptidase. Pepti des that NEP targets incl ude bradykinin, glucagon, neurotensin and enkephalin (Johnson et al ., 1999; Sakurada et al., 2002). NEP is a presynaptic membrane associated ectoenzme with an extracellu lar active site, and it is involved in A degradation at presynaptic si tes (Iwata et al., 2004; Fukami et al., 2002; Turner et al, 2004). Down regulation of NEP in the hippocampus and cerebral cortex with aging (Iwata et al ., 2002; Caccamo et al., 2005) and during the early stages of AD development (Caccamo et al., 2005; Yasojima et al., 2001) suggests a link between NEP and AD etiology and pathogenesis. In fact, over expression of NEP in trans genic mice has revealed a decrease in the levels of A as well as an increase in the lifespan (Marr et al., 2003, Leissring et al 2003). Previous studies have demonstrated engraftment of marrowderived cells within the CNS (Priller et al. 2001; Vallieres and Sawc henko, 2003; Simard and Rivest, 2004). Although the signal that caus es the infiltration of marrowderived cells into the CNS remains to be determined, A is one candidate because the marrow-derived cells become localized arou nd amyloid plaques. In addition, the 54

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ultimate fate of the marrow-derived cells is controversial as we ll. One hypothesis is that they differentiate into micr oglia and play a potential role in A plaque evolution. These observations have prov ided the basis for work aimed at using autologous bone marrow-derived cells to deliver therapeutic genes to damaged regions of the CNS (Park et al., 2001; Makar et al., 2002). Bone marrow cells have been used as a delivery system to express transgenes in the CNS such as GDNF and INF(Makar et al., 2004). These studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genetically modified monocytes to carry exogenous genes of intere st into the brain or other organs for the purposes of gene therapy (Kokovay and Cunningham, 2005). Bone marrowderived monocytes could serve as an alter native to stem cells for cell and gene therapy, because they are a safe, readily available and ethically acceptable source (Vallieres and Sawchenko, 2003). As an approach to increase expressi on of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. Thes e monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. Mono cytes were located around the amyloid plaques and we found significant reductions in both A and Congo red staining in the NEP-S injected mice only. Combin ing monocyte gene deliv ery together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatm ent of Alzheimers disease. 55

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MATERIALS AND METHODS Neprilysin Constructs Endogenous neprilysin is a membranebound ectoprotease. Because A accumulates extracellularly in AD, we hypothesized that an extracellular localization of protease would be most effective in degrading A Consequently, the membrane binding domain was delet ed, and a sequence triggering secretion was added to the construct. As a control, a construct containing neprilysin with a single-point mutation at the catalytic si te precluding proteolysis was also generated. An HA tag was appended to each construct. Bone Marrow Collection Transgenic GFP mice obtained from Jackson Laboratories (C57BL/6-Tg(UBCGFP)30Scha/J [Stock #004353]) were over dosed with pentobarbital. The femurs and tibias were removed aseptically on a cold plate, and bone marrow was flushed from the bone using a 25G 5/8 gauge needle attached to a syringe. Single cell suspensions were prepared by repetitive pipetting and the cell suspension was then passed through a 70 m nylon mesh to remove particulate matter. Cells were then collected by c entrifugation for 5 minutes at 300xg (4C). Then cells were then incubat ed with RBC lysis buffer (Milt enyi Biotech, CA) for 5 minutes at room temperat ure. Equal volumes of PBS were added and the cells were collected by centrifugation. Cell numbers were counted and estimated using a hemocytometer after resuspending in RPMI 1640 medium with L-glutamine 56

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(Mediatech Inc., VA) supplemented with 10 % heat inactivated fetal bovine serum (FBS), streptomycin (100 g/ml), and gentamicin (100 g/ml). Magnetic Cell Separation of CD11b+ Cells After the removal of bone ma rrow from mice, CD11b+ cells were separated using Miltenyi Biotecs LS columns and MidiMa cs magnet following the manufacturers instructions. In summary, 107 freshly harvested bone marrow cells were resuspended in 90 l of medium (PBS + 0.5% BSA), mixed with 10 l of CD11b antibody conjugated to magnetic microbeads (Miltenyi Biotec, Auburn, CA), and incubated for 15 minutes at 4C. These beads can isolate cells while binding only a fraction of the antigenic sites. The cell suspension was then applied to the column in a magnetic fiel d and washed with 3 mL of buffer three times. The column was then separated from the magnet, and CD11b+ cells were eluted in 500 l of medium and st ored at 4C. Five X 106 freshly isolated CD11b+ (GFP) cells were then resuspended in 200 l of RPMI 1640 medium and subjected to transfection. Monocyte Transfections Monocytes were transfected using t he Amaxa Mouse Macrophage Nucleofector Kit. In brief, cells were centrifuged fo r ten minutes at 1000g, resuspended in 100 l of Nucleofector Media containing 10 g plasmid, and transferred to a cuvette. Program Y-001 was used. After transfect ion, cells were transferred to a cell culture dish and incubated at 37for 1 hour before use. 57

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Transgenic mice We used APP Tg2576 derived mice that we re bred at the Univ ersity of South Florida as previously described (Holco mb et al., 1998) (Gordon et al 2002). Tg2576 mice 16 months of age at the start of the expe riment were assigned to one of two groups. The first group receiv ed the Nep-S transfected monocytes via intracranial injections and the second group received the Nep-M transfected monocyte injections. Stereotaxic Intracr anial Injections Surgery was performed on animals using a stereotaxic apparatus, injections using the convection enhanced delivery method (CED) were of approximately 200,000 monocytes in 2 l PBS were dispensed into hippocampus and frontal cortex at a flow rate of 2.5ul/min over a period of 0. 8 min. using a blunt ended 27 gauge needle attached to a 10 l syringe (Hamilton Co., Reno, NV). The 27 gauge needle was attached to polyethylene s ilicone, with an outer diameter of 165.0 m and an inner diameter of 102.0 m, (Polymicro Technologies LLC, Phoenix, AZ) tubing inserted into the openi ng of the needle and cemented in place with super glue. The silicone tubing was then cut allowing approximately 1mm of tubing to extend fr om the end of the needle. Tissue Collection and Histochemical Procedures Mice were overdosed with pentobar bital (100mg/kg). The aorta was clamped and the heart was perfused with saline. The brain was removed, 58

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bisected sagittally and the left half wa s immersed in freshly prepared 4% paraformaldehyde in 100 mM PO4 buffer (pH 7.4). The brains were postfixed in paraformaldehyde for 24 hours. The tissue was cryoprotected in a series of sucrose solutions, frozen, sectioned in the horizontal plane at 25 m using a sliding microtome and stor ed at 4C in Dulbeccos phosphate buffered saline for immunocytochemistry and histology. Immunohistochemistry was performed on free floating sections as described in detail previously (Gor don et al, 1997). Antibodies used for histochemistry: A (1:10,000 rabbit polyclonal prepar ed by Paul Gottchall); Iba1(1:3000, Wako, Richmond, VA) and GFAP (1 :3000, Dako, Carpinteria, CA). Congo Red Histology: Hydrated sect ions were incubated in alkaline alcoholic saturated NaCl (2.5mM NaOH in 80% alcohol) for 20 minutes and then incubated in 0.2% Congo red in alkaline alcoholic saturated NaCl solution for 30 minutes. Sections were rinsed through th ree changes of 100% ethanol, cleared in xylene, then coverslipped with DPX. Immunoblotting Brain homogenates were loaded on SDS pol yacrylamide gel electrophoresis and transferred to a nitrocellulose membr ane for Western blot analyses. The blots were washed three times using Trisbuffered saline with Tween20 (TBST), incubated in milk solution (TBST with 3% non fat dry milk) for 1 hour, and washed three times with TBST. The blots we re incubated in the primary antibody (rat anti-HA; Roche, Indianapolis, IN) in milk solution (dilution 1:1000) overnight, 59

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washed 3 times with TBST, incubated wit h streptavadin horseradish peroxidase (1:1000, Burlingame, CA) in milk solu tion (dilution 1:1000) for two hours, and washed three times with TBST. The blots were enhanced with LumiGOLD ECL Western Blot Detection Kit (SignaGen Labs; Gaithersburg, MD) and visualized on film. Image Analysis GFP fluorescence, immunohistochemis try and Congo red staining were quantified with Image Pro Plus (Media Cybe rnetics) image software. To quantify the staining of t he sections, we randomly sampled every 8th section from the mouse brain. All values from each br ain region obtained from a single mouse were then averaged together to represent a single value for that animal. Statistical analysis was performed using ANOVA followed by Fischers LSD post hoc means comparison test (S tatview software from SAS). RESULTS NEP Constructs and Expression in Vivo: Overexpression of NEP in AD related animal models has shown a decrease in levels of A 1-42, confirming the importa nce of this enzyme in regulating A turnover in vivo (Leissring, Fa rris et al. 2003; Leissring, Lu et al. 2003). In order to study the ability of NEP to degrade extracellular A we designed a secretory form of the NEP plasmid by delet ing the membrane binding 60

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domain and inserting a signal peptide tri ggering secretion (Figure 1A). The NepM-HA construct is the mut ant form of the plasmid lacking any enzyme activity and serves as control for the presence of an exogenous protein. The constructs also contain a hemaglutinin (HA) tag to allow us to distinguish the delivered NEP gene products from endogenous N EP found in the brain. To verify expression of the plas mids, HEK cells were transfected with each of the NEP constructs as well as with empty vector. Cell lysates were collected 24 hours after transfection, and analyzed by western blotting. Both plasmids directed strong expression. Due to the deletion of the membrane binding domain, the NEP-S-HA construct runs slightly lower than the NEP-M-HA (Figure 1B). A major challenge in working with mono cytes is a difficulty in efficiently transfecting them. Transfection effi ciencies less than 10% have been accomplished using traditional transfecti on techniques, including electroporation, liposomal transfection reagents and DEAE-dextran (Mayne, Borowicz et al. 2003). However, we have developed an effective technique that is able to transfect approximately 50% of the m onocytes using a Mouse Macrophage Nucleofector II kit from Amaxa (Figure 2A). Staining the monocytes for HA expression, we show that only the tr ansfected cells are positive for HA when compared to control transfected cells (Figure 2B). NEP-S-HA Transfected Monocytes Reduce A and Congo Red After Direct Injection: 18 month old Tg2576 APP mice were used in these studies. To 61

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each of these mice, 200,000 bone marro w derived monocytes expressing NEPS-HA or NEP-M-HA were stereotaxically inje cted unilaterally into the right cortex and the right hippocampus. The left side se rved as an uninjected control. Cells were injected with a convection enhanced delivery method to increase the area of distribution of the cells within the brain (Raghaven et al., 2006). The brains of the mice were harvested 7 days after the injection and stained. To determine the extent of A clearance, horizontal brain sect ions were stained with an A antibody. Representative sections are presented in Figure 3A. The amount of staining did not appear to be altered after treatment with the monocytes transfected with the control NEP-M-HA (le ft panels) in the cerebral cortex or hippocampus. However, a reduction of A in the NEP-S-HA treated mice was observed in the cortex and the hippocampus (Figur e 3A). Percent area quantification of A immunostaining was measured using an automated image analysis program, and is shown in Fi gure 3B. Significant reductions in A immunostaining were observed in the co rtex (57% reducti on, p < 0.01) and hippocampus (53% reduction, p < 0.01) from mice injected with the NEP-S construct. To also evaluate the effectiveness of the two constructs to clear compact plaques, brain sections were stai ned with Congo red. Representative micrographs show a qualitativ e reduction of Congo Red stained plaques in the NEP-S mice in the cortex and the hippoc ampus compared to the NEP-M injected group. (Figure 4A). Comparing the rati os of the NEP-S and NEP-M groups show significant reductions at the injection site in the ipsilateral cortex ( 84% reduction, 62

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p < 0.01) and hippocampus (69% reduction, p < 0.01) from mice injected with the NEP-S construct (Figure 4B). NEP Injections Do Not Cause Microgliosis or Astrogliosis. Microglial and astrocyte activation are often a sensor for brain inflammation or injury. Transgenic mouse models of AD have demonstrated microglial accumulation around A plaques. To determine if the injection method increased presence of glia, we exam ined two common markers: IBA-1 for microglia and GFAP for astrocytes. While there were occasional detectable increases of staining around the needle track, we found no up-regulation of GFAP (Figure 5) or IBA-1 (Figure 6) in the cortex or the hippocampus comparing the injected, ipsilateral side to the unin jected side in either the Nep-S nor the Nep-M groups. Thus, the introduction of monocytes transfected with neprilysin did not promote brain microg liosis or astrocytosis. DISCUSSION Progressive accumulation of A in senile plaques is regarded as a major culprit in the onset of AD. Thus reducing levels of cerebral A represents a logical therapeutic approach towards t he disease. While most studies have focused on the mechanisms involved in am yloid accumulation, a more important therapeutic intervention might involve m anipulating the processes involved in amyloid degradation. NEP levels in the cortex and the hippocampus are 63

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significantly lower in AD brains compared to normal control brains (Iwata et al., 2002; Caccamo et al., 2005 ; Yasojima et al., 2001). This is consistent with the idea that a deficiency in NEP contributes to the accumulation of amyloid plaques. The present study established a procedure for the isol ation and transfection of primary mouse monocytes. We also used tw o forms of NEP which allowed us to clarify the role of this protease in A degradation in APP transgenic mice. Gene therapy holds immense pr omise for the amelioration of neurodegenerative diseases. AD is an excellent disease to evaluate this type of cell therapy. Amyloid exists largely out side the cells, making it accessible to externally oriented or secr eted proteins. Moreover, th e disease is spread over large portions of the cerebr al cortex and the hippocampus making it difficult to reach with viral vector injections. The blood brain barrier restricts the transport of chemicals and large molecules into the brain and has served as a considerable challenge for the development of gene t herapies. As a result, current gene therapies utilize direct intercranial inject ion of viral vectors or cells that are transfected with a gene of interest (Marr et al., 2003, Iwata et al., 2004, Hemming et al., 2007). Many cell transplantation st udies have been attempted in neurodegenerative diseases. In Parkinson disease as well as in Alzheimers disease growth factors have been transfected in cells and tr ansplanted into the brain (Freed et al, 1991, Lindvall et al 1991, Tuszynski et al., 2005). However these studies have not shown great promise as a result of survival of grafted cells as well as ethical issues involv ed with using embryonic cells. While stem 64

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cells may eventually serve an important cell vector for gene therapy, a better understanding of how these cells differentia te and proliferate is crucial before they can be used in gene ther apy studies. As a result, a search for a primary cell has been needed. Our present study show s that monocytes can be easily isolated and injected in the br ain and that after injection these cells survive up to 7 days. This is in agreement with Kennedy and Abkowitz (1998) as well as Zassler and Humpel 2006 who demonstrat ed a similar survival time of monocytes post injection in the brains of rodents. Studies showing that hem atopoietic cells can enter the brain in AD and differentiate into microglia /macrophages at sites of A plaques have resulted in multiple research groups alluding to the use of monocytes as vehicles by which genes can be delivered to the brain (Priller 2001, Biffi 2004, Asheuer 2004, Zassler and Humpel 2006). These cells ar e readily available in the bone marrow or blood, and as we have pr eviously shown, have the ability to migrate through the BBB to the brain and associate around A plaques. A monocyte gene therapy approach has a number of positive benefits compared to other cells types such as stem cells or progenitor cells. Monocytes have short lifespans in vivo allowing adverse reactions to resolve over time. The transfection of autologous monocytes should have a lo w probability of eliciting an immune reaction against the gene therapy vector. And most importantly, this approach would have the benefits of access to t he entire brain, and concentration of the therapeutic gene at the sites of brain injury. 65

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While many studies have implicated the use of monocytes as an ideal cell vector for gene therapy in AD, it is cr ucial to optimize in vitro transfection methods to genetically modify primary m onocytes ex vivo. There have only been a small number of studies focused on the separation and cultivation of monocytes (Grace et al., 1988, Agger et al., 2000, Zeng et al 2006). Typically transfection efficiencies of less than 10% have been achieved using standard transfection protocols (Burke et al 2002, Harwood et al 2002). This low level of transfection is not functional for an ideal scientific study. However in our study, we have found a method to harvest, isolate and transfect monocytes and reinject them back into an animal within hour s. To study the ability of monocytes to serve as gene therapy vectors it was essential to separate a homogeneous monocytic cell population from the tota l bone marrow cell population. While monocytes are available in the circulat ion, the percentage of monocyte cells is much greater in bone marrow than in the blood. It has also been shown that monocytes isolated from bone marrow were more receptive to viral vector mediated transduction than monocytes fr om mouse blood (Z eng et al, 2006). The lack of a specific marker on monocytes has also made these cells difficult to study. The liter ature has previously present ed methods to obtain pure monocyte populations from bone marrow, by allowing these cells to culture and adhere for 7 days (Chu et al., 2005). Howeve r in recent years, studies have suggested that with increasing time in culture the phenotypes of monocytes isolated from mouse, rat, or human brain change considerably from the phenotypes observed immediately ex vivo (Ford et al., 1995; Carson et al.,1998; 66

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Carson et al., 1999). Consequently, we elected to use the cell surface expression of CD11b as a selection mark er for monocytes to sepa rate a purified population of monocytic cells from total bone marro w in mice through the use of Miltenyi Biotecs LS columns and MidiMacs Us ing a magnetic cell separation kit we can enrich our monocyte population and increase the number of monocytes injected into the brain inst ead of using total bone marro w. We have also used a Mouse Macrophage Nucleofector II kit from Amaxa which allows us to transfect about 50% of monocytes within a matter of seconds. The NEP plasmids are also tagged with HA and post transfe ction we have shown that only the transfected cells are positive for HA when compar ed to control transfected cells. We have shown that the secreted fo rm of NEP was extrem ely effective at clearing both diffuse and compact A from the brain compared to the NEP-M plasmid which lacked any enzyme activity There was a significant reduction of both A and Congo Red near the injection site in the NEP-S mice in the cortex and the hippocampus compared to the N EP-M injected group. Thus removal of A appears to occur through the diffusion of the protease throughout the injection site. While upregulating NEP ma y affect other substrates since it is involved in the degradation of other species besides A, we did not examine any up regulation of microglia or astrocytes as a result of the secreted protein. NEP expression is most likely regulated by many factor s that have been implicated in AD with the most notable being aging. While there is no clear understanding what mechanisms cause the age decline in NEP brain levels, it has been shown that there is a definitive decrease in NEP mRNA in the brains 67

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and CSF of AD patients (Wang et al., 2005, Russo et al, 2005). While NEP is a critical protease involved in A clearance, there are several other A degrading enzymes that are likely to play a pivotal role in the removal of A in the brain. IDE and ECE have also been shown to degrade A in vivo, suggesting that each protease may target a different cell or region in the brain and work together through a synergistic effect by targeting different A fragments (Farris et al., 2003, Eckman et al., 2003, Eckman and Eckm an, 2005). It is therefore possible that in addition to NEP, other peptidases may contribute to the degradation of A (Chesneau et al, 2000). Regardless, the data presented in this study support the hypothesis that NEP plays a significant role in A clearance. In conclusion, our study establis hes a systematic procedure for the isolation and transfection of primary mouse monocyt es. We also demonstrate proof of principle t hat NEP secreting monocytes are us eful for transplantation into the brain and delivery of reco mbinant NEP in degrading A plaques. Such monocytes may be useful to deliver therapeutic NEP into the brain in neurodegenerative diseases such as AD. Thus we think that transmigration of NEP-secreting monocytes through the BB B into the brain could offer a novel alternative to transplantation studies. REFERENCES Agger, R., Petersen, M.S., Toldbod, H.E., Holtz, S., Dagnes-Hansen, F., Johnsen, B.W. 2000. Characterization of murine dendritic cells derived from adherent blood mononuclear cells in vitro. Scand. J. Immunol 52, 138-147. 68

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Burke, B., Sumner, S., Maitland, N ., Lewis, C.E. 2002. Macrophages in gene therapy: cellular delivery vehi cles and in vivo targets J. Leukoc. Biol. 72, 417. Carson, M. J., C. R. Reilly, et al. ( 1998). "Mature microglia resemble immature antigen-presenting cells." Glia 22(1): 72-85. Carson, M. J., J. G. Sutcliffe, et al. (1999). "Microglia st imulate naive T-cell differentiation without stimulat ing T-cell proliferation." J Neurosci Res 55(1): 127-34. Cavalho, K.M., Franca, M.S. Camarao, G.C., Ruchon, A.F. (1997). A new brain metalloendopeptidase which degrades the Alzheimer beta-amyloid 1-40 peptide producing solu ble fragments without neurotoxic effects. Brazilian Journal of Medical and Biological Research. 30: 1153-1156. Chesneau, V., Vekrellis, K., Rosner M.R., Selkoe, D.J. (2000) Purified recombinant insulin degrading enzyme degrades amyloid protein but does not promote its oligomerization. J. Biochem 351 509-516. Chu, X. Y., L. B. Chen, et al. (2005). "Effect of bone marrow-derived monocytes transfected with RNA of mouse col on carcinoma on specific antitumor immunity." World J Gastroenterol 11(5): 760-3. Eckman, E. A., and Eckman, C. B. (2005) Abeta-degrding enzymes: modulators of Alzheimers disease pathogenes is and targets for therapeutic intervention. Biochem. Soc. Trans. 33, 1101-1105. Eckman, E.A., Reed, D.K., Eckman, C.B. (2001) Degradation of the Alzheimers amyloid beta peptide by endothelin-converting enzyme. J. Biol. Chem. 276 24540-24548. Eckman, E.A., Watson, M., Marlow, L., Sambamurti, K., Eckm an, C.B. (2003) Alzheimers disease amyloid peptide is increased in mice deficient in endothelin-converting enzyme. J Biol Chem 278 2081-2084 Farris W, Mansourian S, Chang Y., Li ndsley L., Eckman EA., Frosch, M.P., Eckman, C.B., Tanzi, R.E., Selkoe, D.J., Guenette, S. (2003). Insulindegrading enzyme regulates the levels of insulin, amyloid protein, and the amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci USA. 100 4162-4167. 69

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Farris, W., Schutz, S. G., Cirrito, J. R., Shankar, G. M., Sun, X., George, A., Leissring, M. A., Walsh, D. M., Qiu, W. Q., Holtzman, D. M., and Selkoe, D. J. (2007). Loss of neprilysin function prom otes amyloid plaque formation and causes cerebral amyloid angiopathy. Am. J. Pathol. 171, 241-251 Freed, W.J., Poltorak, M., Takashima, H., LaMarca, M.E., Ginns, E.I. (1991). Brain grafts and Parkinsons disease. J. Cell. Biochem 45, 261-267. Ford, A. L., A. L. Goodsall, et al (1995). "Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basi c protein-reactive CD4+ T cells compared." J Immunol 154 (9): 4309-21. Gandy, S. (2005). The role of cerebral amyloid beta accumulation in common forms of Alzheimers disease. J Clin Invest 115 1121-1129. Grace, S., Guthrie, L.A ., Johnstron Jr., R.B. (1988) The use of mouse serum and the presence of non-adherent cells for the culture of mouse macrophages. J. Immunol. Methods 114 21-26. Hemming, M. L., Patterson, M., ReskeNielsen, C., Lin, L., Isacson, O., and Selkoe, D. J. (2007). Reducing amyl oid plaque burden via ex vivo gene delivery of an Abeta-degradi ng protease: a novel therapeutic approach to Alzheimers disease. PLoS. Med. 4, e262 Horwood, N.J., Smith, C., Andreakos, E., Quattrocchi, E., Brennan, F.M., Feldmann, M., Foxwell, B.M.J. (2002). High efficiency gene transfer into nontransformed cells: utility for studyi ng gene regulation and analysis of potential therapeutic targets. Arthritis Res 4 S215. Huang, S. M., Mouri, A., Kokubo, H., Nakajima, R., Suemoto, T., Higuchi, M., Staufenbiel, M., Noda, Y., Yamaguchi, H., Nabeshima, T., Saido, T. C., and Iwata, N. (2006). N eprilysin sensitive synaps e associated amyloid beta peptide oligomers impair neuronal pl asticity and cognitive function. J. Biol. Chem. 281, 17941-17951 Iwata, N., Mizukami, H., Sh irotani, K., Takaki, Y., Mura matsu, S., Lu, B., Gerard, N. P., Gerard, C., Ozawa, K., and Saido, T. C. (2004). Presynaptic localization of neprilysin contributes to efficient clearance of amyloid beta peptide in mouse brain. J. Neurosci. 24, 991-998. Iwata N, Tsubuki, S., Takaki, Y. (2000). Identification of the major Abeta 1-42 degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nature Medicine 6 143-150. 70

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Iwata, N., Tsubuki, S., Takaki Y., Shirotani, K., Lu, B., Gerard, N. P., Gerard, C., Hama, E., Lee, H. J., and Saido, T. C. (2001). Metabolic regulation of brain Abeta by neprilysin. Science 292, 1550-1552. Johnson, G. D., T. Stevenson, et al. ( 1999). Hydrolysis of peptide hormones by endothelin-converting enzyme-1. A comparison with neprilysin. J Biol Chem. 274 (7): 4053-8. Kennedy, D.W., Abkowitz, J.L. (1998). Matu re monocytic cells enter tissues and engraft. Proc. Natl. Acad. Sci. U.S.A. 95, 14944-14949. Kokovay, E. and L. A. Cunningham (2005). "Bone marrow-derived microglia contribute to the neuroinflammatory response and express iNOS in the MPTP mouse model of Parkinson's disease." Neurobiol Dis 19(3): 471-8. Leissring, M.A., Farris, W., Chang, A.Y., Walsh, D.M., Wu, X. Sun, X., Frosch, M.P., Selkoe, D.J. (2003). Enhanced proteolysis of amyloid in APP transgenic mice prevents plaque fo rmation, secondary pathology, and premature death. Neuron 40, 1087-1093. Lindvall, O. (1991). Prospects of transplantation in human neurodegenerative diseases. Trends Neurosci 14, 376-384. Makar, T. K., D. Trisler, et al. (2004) "Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marro w cells as vehicles in mice." Neurosci Lett 356 (3): 215-9. Makar, T. K., S. Wilt, et al. (2002). "IFN-beta gene transfer into the central nervous system using bone marrow cells as a delivery system." J Interferon Cytokine Res 22(7): 783-91. Malm, T. M., M. Koistinaho, et al. (2005). "Bone-marrow-derived cells contribute to the recruitment of microglial ce lls in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice." Neurobiol Dis 18(1): 134-42. Marr, R. A., Rockenstein, E., Mukherjee, A. Kindy, M. S., Hersh, L. B., Gage, F. H., Verma, I. M., and Masliah, E. (2003) Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23, 19921996. Marr, R.A., Rockenstein, E., Mukherjee, A. Kindy, M.S., Hersh, L.B., Gage, F.H., Verma, I.M., Masliah, E. (2003). N eprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23, 1992-1996. 71

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McDermott, J.R., Gibson, A. M. (1997). Degradation of Alzheimers beta amyloid protein by human and rat brain peptidases: involvement of insulin degrading enzyme. Neurochem. Research. 22, 49-56. Mentlein, R., Ludwig, R. and Martensen, I. (1998) Proteolytic degradation of Alzheimers disease amyloid pepbtide by a metalloproteinase from microglia cells. J. Neurochem 70, 721-726. Mills, J. and Reiner, P.B. (1999) Regulation of am yloid precursor protein cleavage. J. Neurochem 72, 443-460. Park, K. W., M. A. Eglitis, et al. (2001) "Protection of nigral neurons by GDNFengineered marrow cell transplantation." Neurosci Res 40(4): 315-23. Priller, J., A. Flugel, et al. (2001). "Tar geting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment." Nat Med 7 (12): 1356-61. Qiu, W.Q., Walsh, D.M., Ye, Z. (1998) Insulin degrading enzyme regulates extracellular levels of amyl oid beta protein by degradation. J. Biol. Chem. 273 32730-32738. Qiu, W. Q., Ye, Z. Kholodenko, D., Seubert, P. and Selkoe, D.J. (1997) Degradation of amyloid protein by metalloprotease secreted by microglia and other neural and non-neural cells. J. Biol. Chem 272 6641-6646. Raghaven R., Brady M.L., Rodriguez-P once M.I., Hartlep A., Pedain C., Sampson J.H. (2006). Convection enhanc ed delivery of therapeutics for brain disease, and its optimization. Neurosurg Focus 20: 4. Russo, R., Borghi, R., Ma rkesbery, W., Tabaton, M., Piccini, A. (2005). Neprylisin decreases uniformly in Alzheimers disease and in normal aging. FEBS Let 579 6027-6030. Saido, TC. (1998). Alzheimers disease as proteolytic disorders: anabolism and catabolism of beta-amyloid. Neurobiol Aging 19:S69-75. Sakurada, C., S. Sakurada, et al. ( 2002). "Degradation of nociceptin (orphanin FQ) by mouse spinal cord synaptic membranes is triggered by endopeptidase-24.11: an in vi tro and in vivo study." Biochem Pharmacol 64(8): 1293-303. Selkoe, DJ (1999) Translating cell biology into therapeutic advances in Alzheimers disease. Nature 399 72

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Simard, A. R., D. Soule t, et al. (2006). "Bone marrowderived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease." Neuron 49(4): 489-502. Tanzi, R.E., and Bertram, L. (2005). Tw enty years of the Al zheimers disease amyloid hypothesis: a genetic perspective. Cell 120 545-555. Tuszynski, M.H., Thal, L., Pay, M., Salmon, D.P., Sang U, H., Bakay, R., Patel, P., Blesch, A., Vahl sing, H.L., Ho, G., Tong, G., Potkin, S.G., Fallon, J., Hansen, L., Mufson, E.J., Kordower, J. H., Gall, C., Conn er, J. (2005) A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat. Med. Vallieres, L. and P. E. Sawchenko. (2003). "Bone ma rrow-derived cells that populate the adult mouse brain preserve their hematopoietic identity." J Neurosci 23 (12): 5197-207. Wang, D.S., Lipton, R.B., Katz, M.J., Davies, P., Buschke, H., Kuslansky, G., Verghese, J., Younkin S.G., Eckm an, C., Dickson, D.W. (2005). Decreased neprilysin immunoreactivity in Alzheimers disease, but not in pathological aging. J. Neuropathol Exp Neuro 64: 78-385. Zassler, B., Humpel, C. (2006). Transplantation of NGF secreting primary monocytes counteracts NMDA-induc ed cell death of rat cholinergic neurons in vivo. Exper. Neurol 198 391-400. Zeng, L., Yang, S., Wu, C., Ye, L., Lu, Y. (2006). Effective transduction of primary mouse blood and bone marro w derived monocytes/macrophages by HIV based defective lentiviral vectors. J. Virol Methods. 134 66-73. 73

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Figure 1: CHARACTERIZATION OF NEP CONSTRUCTS: (A) Schematic representation of the c onstructs expressing NEP-S (NEPs) and NEP-M (NEPm). The plasmi ds express the NEP gene under the control of the chicken -actin promoter. Note the HA t ag present on each construct. The NEPS construct also has a signaling peptide inserted to direct secretion of the enzyme. The NEP-M construct is identical to the NEP-N construct with the exception of a single point mutation at the amino acid point E585V. (B) HEK cells electroporated with the plas mids discussed in (A). Lysates were collected and probed for HA on an immunoblot. Due to the deletion of the membrane binding domain, NEP-S runs lower on the gel as seen on the blot. (A) 74

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(B) 115 kDa 82 kDa 75

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Figure 2: STAINING AND TRANSFECTION EFFICIENCIES OF NEP CONSTRUCTS (A) Bivariate plots of FACS analysis to analyze cell viability with propidium iodide (PI) and transfection efficiency wit h GFP fluorescence after electroporation of a GFP-expressing construct. A minimu m of 10,000 viable cells were acquired and GFP expression was quantified by gatin g out the (PI+) dead cells. Data are presented as density plots. The second pl ot shows that 54% of the live cells expressed the transfected GFP plasmid. (B) Cells transfected with GFP are shown in the top row of images (Panel a), while cells transfected with a control plasmid do not express GFP as seen in (Panel b). Cells transfected with NEP constr uct are positively stained for HA as seen in the bottom row of images (Panel c). 76

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GFP transfected monocytes GFP Brightfield Non transfected monocytes Non transfected Brightfield Non transfected monocytes HA Transfected monocytesA B C 77

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Figure 3: TOTAL A IS SIGNIFICANTLY REDUCED IN THE FRONTAL CORTEX AND HIPPOCAMPUS FOLLO WING NEP-S INJECTION APP transgenic mice were injected with monocyt es expressing either NEP-S-HA or NEP-M-HA into the right cortex and hippocampus. Brains were harvested 7 days post injection. Images showing staining of plaque burden at the sites of injection, with the uninjected, contralateral left cortex (top row) and hippocampus (lower row) serving as control. (Panel A). T he graph shows the quantif ication of percent area of total A staining in the frontal cortex and hippocampus after a single injection of either NEP-S or NEP-M construct (B). indicates P<0.05 and ** indicates P<0.01; when compared to mice treated with NEP-M construct. 78

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(B) 79

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Figure 4: COMPACT CONGOPHILIC AMYLOID DEPOSITS ARE REDUCED BY NEP-S CONSTRUCT. 18 month old APP transgenic mice were injected with monocytes expressing either NEP-S-HA or NEP-M-HA. Cells were stereotaxically injected into the right cortex and hippocampus, with the uninj ected left cortex and hippocampus serving as a control. Brains were harve sted 7 days post injection. Representative micrographs show a qualitative reduction in staining in the NEP-S-HA treated group (A). The graph shows the quantific ation of percent area of total A staining in the frontal cortex and hippocampus afte r a single CED injection of either NEPS or NEP-M construct (B). indicate s P<0.05 and ** indicates P<0.01; when compared to mice treated with NEP-M construct. (A) 80

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(B) 81

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Figure 5: THERE IS NO CHANGE IN GFAP STAINING IN THE FRONTAL CORTEX AND HIPPOCAMPUS FOLLO WING NEP-S INJECTION Brain sections showing GFAP staining from both the contralateral and ipsilateral sides of the cortex and hippocampus in both the NEP-M and NEP-S mice (A). The graph shows the quantificati on of percent area of total GFAP staining in the frontal cortex and hippocampus after a sing le injection of either NEP-S or NEP-M construct (B). (A) 82

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(B) 83

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Figure 6: THERE IS NO CHANGE IN MICROGLIAL STAINING IN THE FRONTAL CORTEX AND HIPPOCAMPUS FOLLOWING NEP-S INJECTION Brain sections showing IBA-1 staining from both the contralateral and ipsilateral sides of the cortex and hippocampus in both the NEP-M and NEP-S mice (A). The graph shows the quantificat ion of percent area of to tal IBA-1 staining in the frontal cortex and hippocampus after a sing le injection of either NEP-S or NEP-M construct (B). (A) 84

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(B) 85

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PAPER 3: TRAFFICKING MONOCYTES DELIVER THERAPEUTIC GENES TO THE BRAIN OF AMYLOID DEPOSITING APP+PS1 TRANSGENIC MICE Lebson L1, Nash K1, Kamath S2, Morgan D1, Gordon MN1 aAlzheimer's Research Laboratory, University of South Florida, School of Basic Biomedical Sciences, Depar tment of Molecular Pharmacology and Physiology, 12901 Bruce B Downs Blvd, Tampa, Florida 33612, USA. ACKNOWLEDGEMENTS: This work was supported by grants to MNG from the Alzheimers Association (IIRG-07-58446) and the National Institute on Aging (AG15490). LL is the Benjamin Sc holar in Alzheimer Research. 86

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ABSTRACT The accumulation of amyloidprotein (A ) in Alzheimers disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of A is therefore thought to serv e as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of A and defects in the degradation of A could contribute to AD disease progression. Additionally, studies implicating the hom ing of monocytes to regions of CNS damage have led to the idea that it may be possible to us e genetically modified monocytes to carry exogenous genes of interest into the brai n or other organs for the purposes of gene therapy. As an approach to increase expressi on of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. Thes e monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. M onocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS In addition, we found significant reductions in both A and Congo red staining in the NEP-S injected mice only. These studies show that putting mono cytes together with an amyloid degrading enzyme such as neprilysin offers a pow erful novel therapeutic tool for the treatment of AD. 87

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INTRODUCTION The senile plaque is a pathological hallmark of Alzheimers Disease (AD) and is composed of A activated microglia, astrocytes, and degenerating neurons (Dickson et al., 1988; Selkoe, 2000; Mott and Hulette, 2005). Microglia play a key role as the resident m ononuclear phagocyte population in the CNS (Perry and Gordon, 1988). These cells shar e many phenotypical and functional characteristics with other tissue macrophages and with peripheral blood monocytes, suggesting that microglia parti cipate in many immune reactions of the brain. Microglia originate from the bone ma rrow and emigrate to the blood as monocytes (Review: Gordon and Taylor, 2005). Despite tremendous efforts in the past, monocyte derived and resident CNS microglia remain almost indistinguishable on the basis of know n immunophenotypic markers (Simard et al., 2006). However, using chimeric mice in which bone marrow cells were marked with GFP, MHCII, Y chromosome, congenic CD45,1/CD45.2 molecules, and others, bone marrow derived cells were shown to enter the brain and differentiate into microglia (Tacke et al ., 2006; Imari et al., 2002; Rivier et al., 2004; Hickey et al., 1992; Priller et al., 2001 ). Subsequent studies in mice have even shown an increase in microglial engr aftment in several disease models such as experimental autoimmune encepha lomyelitis (EAE) (Heppner et al., 2005), Parkinsons Disease (PD) (Kokovay and Cunningham, 2005) and AD (Malm et al., 2005; Stalder et al., 2005) These observations have provided the basis for work aimed at using autol ogous bone marrow to deliver therapeutic 88

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genes to damaged regions of the CNS (Par k et al., 2001; Makar et al., 2002). Bone marrow cells have been used as a delivery system to express transgenes in the CNS such as GDNF and IFN(Makar et al., 2004). Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of A and defects in the degradation of A might play a role in AD disease progr ession. Neprilysin (NEP) is a membrane bound zinc metallopeptidase that has implicated in degradation of both extracellular and intracellular A (Iwata et al., 2001). The location of NEP at the presynapses and on axons indicates that this protease may play a key role in A catabolism at and around neuronal synapses Consequently a reduction of NEP activity may cause a local increase in the concentration of A at these regions. An aging dependent decline in NEP level o ccurs naturally in regions which are generally affected in AD (Fukami et al ., 2002; Maruyama et al., 2005; Wang et al., 2005). Thus, a proposed potential therapeutic strategy to prevent the accumulation of A would be to re-establish NEP activity in the brain. The blood brain barrier (BBB) strict ly limits the transport of soluble materials, pathogens and large moleculess into the brain and thereby helps to maintain a protected environment within the CNS (Krol and Neuwelt, 198; Zhang and Pardridge, 2001). The BBB is vital for normal brain function but represents a considerable complication in the devel opment of gene therapeutic interventions for neurodegenerative diseases. Current CNS gene therapy studies therefore rely on the direct injection of vectors or cells that contain a therapeutic gene of interest (Bloch et al., 2004; Crystal et al., 2004; Janson et al., 2002). This 89

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generates limitations which include t he risk for CNS damage as a consequence of drilling holes into the skull and intr oducing catheters to deliver genes. Therefore, noninvasive and safer gene delivery methods are needed. As an approach to increase expressi on of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. Thes e monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. M onocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS In addition, we found significant reductions in both A and Congo red staining in the NEP-S injected mice only. The GFP-positive monocytes visualized in the brain associated with amyloid plaques and retained the hem atopoietic marker CD11b. Cells of amoeboid shape also expressed a macrophage marker, CD68, indicating a dual role for these cells. These studies show that putti ng monocytes together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatment of AD. 90

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METHODS Neprilysin Constructs Endogenous neprilysin is a membranebound ectoprotease. Because A accumulates extracellularly in AD, we hypothesized that an extracellular localization of protease would be most effective in degrading A Consequently, the membrane binding domain was delet ed, and a sequence triggering secretion was added to the construct. As a control, a construct containing neprilysin with a single-point mutation at the catalytic si te precluding proteolysis was also generated. An HA tag was appended to each construct. Bone Marrow Collection Transgenic GFP mice obtained from Jackson Laboratories (C57BL/6-Tg(UBCGFP)30Scha/J [Stock #004353]) were over dosed with pentobarbital. The femurs and tibias were removed aseptically on a cold plate, and bone marrow was flushed from the bone using a 25G 5/8 gauge needle attached to a syringe. Single cell suspensions were prepared by repetitive pipetting and the cell suspension was then passed through a 70 m nylon mesh to remove particulate matter. Cells were then collected by c entrifugation for 5 minutes at 300xg (4C). Then cells were then incubat ed with RBC lysis buffer (Milt enyi Biotech, CA) for 5 minutes at room temperat ure. Equal volumes of PBS were added and the cells were collected by centrifugation. Cell numbers were counted and estimated using a hemocytometer after resuspending in RPMI 1640 medium with L-glutamine 91

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(Mediatech Inc., VA) supplemented with 10 % heat inactivated fetal bovine serum (FBS), streptomycin (100 g/ml), and gentamicin (100 g/ml). Magnetic Cell Separation of CD11b+ Cells After the removal of bone ma rrow from mice, CD11b+ cells were separated using Miltenyi Biotecs LS columns and MidiMa cs magnet following the manufacturers instructions. In summary, 107 freshly harvested bone marrow cells were resuspended in 90 l of medium (PBS + 0.5% BSA), mixed with 10 l of CD11b antibody conjugated to magnetic microbeads (Miltenyi Biotec, Auburn, CA), and incubated for 15 minutes at 4C. These beads can isolate cells while binding only a fraction of the antigenic sites. The cell suspension was then applied to the column in a magnetic field and washed 3 mL of buffer 3 times. The column was then separated from the magnet, and CD11b+ cells were eluted in 500 l of medium and stored at 4C. Five X 106 freshly isolated CD1 1b+ (GFP) cells were then resuspended in 200 l of RPMI 1640 medi um and subjected to transfection. Monocyte Transfections Monocytes were transfected using t he Amaxa Mouse Macrophage Nucleofector Kit. In brief, cells were centrifuged fo r ten minutes at 1000g, resuspended in 100 l of Nucleofector Media containing 10 g plasmid, and transferred to a cuvette for nucleofection, Program Y-001. After tr ansfection, cells were transferred to a cell culture dish and incubated at 37 for 1 hour before use. 92

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Implantation of Microvascular Port The microvascular ports (Kent Scientific ) are 9x9x3 mm polyurethane ports that were placed subcutaneously in the nape of the neck of a mouse and are connected via catheter to the right jugular vein. A skin incision was made over the area of the right jugular a sterile trocar tunneled to the soft tissue between the scapula and an incision placed at the tip of the trocar. The vessel was dissected free from surrounding tissue. A proximal and distal ligature were placed around the vessel. The distal ligature was ligated, and a venotomy was performed and the catheter advanced into t he jugular vein 5 mm. The proximal suture was placed around the catheter and secured. A subcutaneous pocket was made between the scapulae, and the port was placed into the pocket. Both incisions were closed with suture. The catheter was flushed with 100unit per ml heparin in saline solution, approximately three times the dead space of catheter (30 l). On the day of injection, mi ce were anesthetized with isoflurane, the catheter was cleared by inserting a 27 g needle through the skin into the port and injecting 30 l of 100U/ml heparin in sali ne, followed by 100 l of monocyte cell suspension, followed by another 30 l of the heparin solution. Transgenic mice We used APP Tg2576 derived mice that we re bred at the Univ ersity of South Florida as previously described (Holco mb et al., 1998) (Gordon et al 2002). Tg2576 mice 9 months of age, at the start of the expe riment were assigned to one of three groups. The first group rece ived the Nep-S transfected monocytes 93

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injections via the microvascular port and the second group received the Nep-M transfected monocyte injections. These mi ce were injected twice a week for two months. The third group did not receive any injections of monocytes and served as a baseline control. Tissue Collection and Histochemical Procedures Mice were overdosed with pentobar bital (100mg/kg). The aorta was clamped and the heart was perfused with saline. The brain was removed, bisected sagittally and the left half wa s immersed in freshly prepared 4% paraformaldehyde in 100 mM PO4 buffer (pH 7.4). The brains were postfixed in paraformaldehyde for 24 hours. The tissue was cryoprotected in a series of sucrose solutions, frozen, sectioned in the horizontal plane at 25 m using a sliding microtome and stor ed at 4C in Dulbeccos phosphate buffered saline for immunocytochemistry and histology. Immunohistochemistry was perform ed on free floating sections as described in detail previ ously (Gordon et al, 1997). Antibodies used for immunohistochemistry: CD11b (rat monoclonal anti CD11b, Serotec, Raleigh, NC); GFP (rabbit monoclonal anti-GFP; Chemicon, Temecula, CA), 6E10 (Covance, Emeryville, CA); CD68 (Serotec, Raleigh, NC). Single and multiple immunofluorescent la beling: after incubation with t he primary antibody, the free floating sections were then incubated fo r 2 hours with the appropriate fluorophore coupled secondary antibodies [Alexa Fluor 594 (1:1500), AlexaFluor 488 (1:1500), AlexaFluor 350 (1:1500) (Mole cular Probes, Eugene, OR]. Sections 94

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were rinsed in Delbeccos PBS and co verslipped with VECTASHIELD Mounting Medium with DAPI. Congo Red Histology: Hydrated sect ions were incubated in alkaline alcoholic saturated NaCl (2.5mM NaOH in 80% alcohol) for 20 minutes and then incubated in 0.2% Congo red in alkaline alcoholic satu rated NaCl solution for 30 minutes. Sections were rinsed through th ree changes of 100% ethanol, cleared in xylene, then coverslipped with DPX. Immunoblotting Brain homogenates were prepar ed according to (Cardona et al., 2006) and were loaded on SDS polyacrylamide gel elec trophoresis and transferred to a nitrocellulose membrane for Western blot analyses. The blots were washed three times using Tris-buffered saline with Tween20 (TBST), incubated in milk solution (TBST with 3% non fat dry milk) for 1 hour, and washed three times with TBST. The blots were incubated in the primary antibody (rat anti-HA; Roche, Indianapolis, IN) in milk solution (diluti on 1:1000) overnight, washed 3 times with TBST, incubated with streptavadin horser adish peroxidase (1:1000, Burlingame, CA) in milk solution (dilution 1:1000) fo r two hours, and washed three times with TBST. The blots were enhanced with LumiGOLD ECL Western Blot Detection Kit (SignaGen Labs; Gaithersburg, MD) and visualized on film. 95

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Image Analysis GFP fluorescence, immunohistochemis try and Congo red staining were quantified with Image Pro Plus (Media Cybe rnetics) image software. To quantify the staining of t he sections, we randomly sampled every 8th section from the mouse brain. All values from each br ain region obtained from a single mouse were then averaged together to represent a single value for that animal. Statistical analysis was performed using ANOVA followed by Fischers LSD post hoc means comparison test (S tatview software from SAS). RESULTS NEP-S-HA Transfected Monocytes Reduce A and Congo Red After Direct Injection: Total A is comprised of both compac t and diffuse amyloid deposits that can be quantified using immunohi stochemical methods. Tg2576 mice 9 months of age, at the start of the ex periment were assigned to one of three groups. The first group receiv ed the NEP-S transfected monocyte injections via the microvascular port and the sec ond group received the NEP-M transfected monocyte injections. The third group did no t receive any injections of monocytes and served as a baseline control. Repr esentative immunostain ing micrographs in the frontal cortex and hippoc ampal regions from each group are shown in Figure 1A. A qualitative reduction in A can be seen in both the cortex and the hippocampus in the NEP-S tr eated group with le vels of A comparable to the 9 month old untreated mice. Q uantification of the percent area of positive staining 96

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showed a significant reduction in A staining in the NEP-S treated mice in both the cortex and hippocampus (Figure 1B). The removal of compact plaques through NEP mediated degradation has remained speculative. Congo red binds to the beta-pleated sheet conformation of fibrillar A and allows us to detect compact am yloid plaques. Following peripheral injections of neprilysin transfected m onocytes, we observed a significant reduction of Congo red staining in the NEP-S treated mice compared to the NEPM and the untreated control mice (Figure 2A). Quantification of the total area occupied by Congo red stain revealed a sign ificant reduction in the frontal cortex and hippocampus with the NEP-S treated mice (Figure 2B). These levels again were comparable to 9 month old treated mi ce, suggesting that the treatment of NEP-S halted the progression of am yloid deposition of the mice. HA is detected in brain tissue but not in plasma. To determine the effects that the N EP-S might have on the periphery and the CNS, we probed the plasma and brains of the mice for HA 24 hours following the last injection. Western blot analysis (Figure 3) using an antibody against HA detected the presence of t he HA tag in the NEP-S and NEP-M brain and not the uninjected control mice proving we coul d correctly identify the presence of HA. We did not detect the presence of HA in the plasma of any of the mice implying that NEP expression was onl y affecting the CNS. 97

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Transfected GFP+ monocytes migrated to the brain vicinity of amyloid plaques. Imaging of GFP-positive cells was done using confocal microscopy. Brain sections were stained with A (blue), CD11b (red), and GFP (green) to detect the location and phenotype of t he injected monocytes (Figure 4). Approximately 95% of GFP positive cells were positive for CD11b confirming their hematopoietic origins. Cells were located throughout the brain and revealed an association with amyloid plaques. These amyloid plaques were also surrounded by resident microglia (i.e. GF P negative and CD11b positive cells). We also know that peripheral monocytes can enter the brain and serve as CNS macrophages. To analyze this we st ained brain sections with CD68, a macrophage marker and found that GFP-positive cells also co-localized with this marker (Figure 5). It is in teresting to note that the cells that were labeled with CD68 remained in an amoeboid st ate, while those cells that had begun to display ramifications did not posses this marker. DISCUSSION NEP expression in the brain has been shown to decrease during aging and in the beginning stages of AD progre ssion (Iwata et al., 2002; Caccamo et al., 2005). Therefore finding a way to sele ctively up regulate brain NEP activity has been proposed as a therapeutic treatment to stop the progression of AD. The results reported here confirm that inje cted GFP-positive monocytes derived from the bone marrow migrate from the peri phery into the brains of APP+PS1 98

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transgenic mice. Furthermore, we have found that this migration is rapid, with numerous cells present twenty four hours af ter transplant. These new cells were distributed throughout the brain and appear to reside within the parenchyma, because following perfusion with PBS thes e cells remained in the brain. Double labeling analyses show that almost all GFP+ monocytes retained the hematopoietic marker CD11b. These finding are in agr eement with others who have reported that BMDCs retain a hematopoietic phenotype in the brain (Vallieres and Sawchenko, 2003; Wehner et al., 2003). In addition, we also observed cells positively labeled for CD 68, a marker for macrophages. While we did not actually determine if the GF P-positive cells were phagocytosing A increasing evidence has hypothesized that microglia may play a protective role in AD by mediating clearance of A Indeed, when we com pared the amount of amyloid in the three treatment groups we saw a small trend in the NEP-M treated group that may indicate peripheral derived monocytes alone could delay the progression of AD like pathology. However several groups have shown that during the aging process, the expression of microglial A receptors and A degrading enzymes declines, resulting in decreased A uptake and degradation and increased A accumulation. Fiala et al., also found that monocytes and macrophages from AD patients displa yed ineffective phagocytosis of A when compared with monocytes and macrophages from age matched control non AD patients. While it is not certain w hat mechanisms are responsible for the decreased expression of degrading enzym es, reactive oxygen species and proinflammatory cytokines have been found to increase in transgenic mice with AD 99

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pathology and may play a role in this proc ess (El Khoury et al., 2008; Hickman, 2008). We have shown that the secreted fo rm of NEP was extrem ely effective at clearing both diffuse and compact A from the brain compared to the NEP-M plasmid which lacked any enzyme activity There was a significant reduction of both A and Congo Red staining in the NEP-S mice in the cortex and the hippocampus compared to the NEP-M and control group. The levels of A were comparable to that of a 9 month of APP+PS1 mouse indicating that the NEP-S transfected monocytes halted the progr ession of amyloid deposition by up regulating NEP in the brains of these animals. While increasing NEP in the brain may affect other substances, since it is involved in the degradation of other species besides A during the two months of injections we found no signs of weight loss or deteriorating health. Ex vivo gene therapy represents a novel method of treatm ent for a variety of neurodegenerative diseases. However, in order to develop a successful ex vivo gene therapy it is essential to est ablish an effective method for the delivery of bone marrow derived monocytes into t he CNS of APP+PS1 transgenic mice. In current studies of bone marrow monocyte transplantation, we as well as other groups have demonstrated the inability to deliver significant numbers of monocytes into the brain following intraperitoneal or tail vein infusion (Malm et al., 2005; Djukic et al., 2006; Andreesen et al., 1990). In addition, the feasibility of long term tail vein injections is limited by the fact that scar tissue can form and decrease the available area fo r an injection site. In this study, the use of the 100

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mouse microvascular port was a novel and critical method t hat aided in the significant number of GFP positive cells seen in the brain. This route of cell injection will be critical for all future cells therapies tested over a long term study and provide a reliable induction of cells into the blood stream. While many neurotrophic factors show promise in the treatment of neurodegenerative disorders, t heir use has been limited due to their inability to cross the blood brain barrier and by thei r limited diffusion throughout the brain (Verall, 1994). The use of bone marrow derived monocytes to deliver therapeutic genes directly to brain should result in a low probability of eliciting an immune reaction as well as concentrating the therapeutic gene at the site of brain injury. However the development of reversible or controlled expres sion systems will be critical to avoid unwanted side effects t hat may in the long term alter the immune system or result in the increase of secondary side effects. NEP expression is most likely regulat ed by many factors that have been implicated in AD with the most notable bei ng aging. In support of this Fukami et al., reported significant age related reduc tions in NEP expression in amyloid vulnerable regions of tr ansgenic mouse brains. Simila r results were obtained by Caccamo et al., who showed that the le vels NEP decrease with age in the mouse hippocampus but not in the cerebellum, which is resistant to amyloid deposition. Recently, in addition to neprilysin, several peptidases such as endothelin converting enzyme 1 (ECE1), endothel in converting enzyme 2 (ECE2), and insulin degrading enzyme (IDE) have been identified as A degrading enzymes through the use of transgenic knockout mouse models (Eckman et al., 2003; 101

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Farris et al., 2003; Miller et al., 2003). While neprilysin has been shown to have the greatest effect in the reduction of A each of these peptidases is likely to play a different role in the total A clearance in the brain. Their differences in enzymatic and cellular locations are likely to play additive, or synergistic roles in the removal of A (Eckman et al., 2001; Saido and Nakahara, 2003). Developing a cell therapy that can incorporate t he degrading properties of each of these enzymes may therefore result in an effe ctive combination therapy that could result in total A clearance. In conclusion, our results demonstrate that ex vivo gene delivery of an A degrading enzyme halted the progression of amyloid deposition. However, there are still many questions that still need to be addressed to further develop new gene/cell therapeutic treatm ents. Defining the impact of the donor and recipient age as well as the signaling interaction between A and peripheral monocytes will be critical for the development of new gene delivery approaches and may reveal new therapeutic targets. Har nessing the peripheral immune system to pharmacologically control brain A levels may serve as a powerful new tool to improve recovery and repair brain damage for the treatment of AD. REFERENCES Andreesen R., Scheibenbogen C., Brugger W., Krause S., Meerpohl H.G., Leser H.G., Engler H., Lohr G.W (1990). Adoptive transfe r of tumor cytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Res. 50: 7450-7456. 102

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Bloch J., Bachoud-Levi A.C., Deglon N., Lefaucheur J.P., Winkel L., Palfi S., Nguyen J.P., Bourdet C., Gaura V., Re my P., Brugieres P., Boisse M.F., Baudic S., Cesaro P., Hantraye P., Aebischer P., Peschanski M. (2004). Neuroprotective gene therapy for Huntingtons disease, using polymerencapssulated cells engi neered to secrete human cilary neurotrophic factor: result s of a phase I study. Hum. Gene Ther 15: 968975. Bondolfi L., Calhoun M., Ermini F., Kuhn H.G., Wiederhold K.H., Walter L., Staufenbiel M., Jucker M. (2002). A myloid associated neuron loss and gliogenesis in the neocortex of amyloid precursor protein transgenic mice. J. Neurosci 22: 515-522. Cardona A.E., DeRen H., Sasse M.E., Ransohoff R.M. (2006). Isolation of murine microglial cells for RNA analysis of flow cytometry. Nature Protocols. Coraci I.S., Husemann J., Berman J.W ., Hulette C., Dufour J.H., Campanellea G.K., Luster A.D., Silverstein S.C., El-Khoury J.B. (2002) CD36, a class B scavenger receptor, is expressed on microglia in Alzheimers disease brains and can mediate production of r eactive oxygen species in response to beta-amyloid fibrils. Am J. Pathol 160 : 101-112. Crystal R.G., Sondhi D., Ha ckett N.R., Kaminsky S.M. Worgall S., Steig P., Souweidane M., Hosain S ., Heier L., Ballon D., Di nner M., Wisniewski K., Kaplott M., Greenwald B.M., Howell J. D., Strybing K., Dyke J., Voss H. (2004). Clinical protocol. Administra tion of a replication-deficient adenoassociated virus gene transfer vector expressing the human CLN2 cDNA to the brain of children with late in fantile neuronal ceroid lipofuscinosis. Hum. Gene Ther. 15: 1131-1154. Davalos D. (2005). ATP mediates rapid mi croglial response to local brain injury in vivo. Nat. Neurosci 8 : 752-758. Dickson D.W. (1999(. Microglia in Alz heimers disease and transgenic models. How close the fit? Am J Pathol 132 : 86-101. El Khoury J., Hickman S.E., Thomas C.A ., Cao L.,Silverstein S.C., Loike J.D. (1996). Scavenger receptor mediat ed adhesion of microglia to betaamyloid fibrils. Nature 382 : 716-719. El Khoury J., Hickman S.E., Thomas C.A ., Loike J.D., Silverstein S.C. (1998). Microglia, scavenger receptors, and the pathogenesis of Alzheimers disease. Neurobiology of Aging 19: S81-S84. 103

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El Khoury J.B., Moore K.J., Means T.K. Leung J., Terada K., Toft M., Freeman M.W., Luster A.D. (2003). CD36 mediates the innate host response to beta amyloid. J. Exp. Med 197 : 1657-1666. Geissmann F., Jung S., Littman D.R. (2003) Blood monocytes consist of two principal subsets with dist inct migratory properties. Immunity. 19: 71-82. Gordon S., Taylor P.R. (2005). Monocyte and macrophage heterogeneity. Nat Rev. Immunol 5 : 953-964. Grage-Griebenow. E., Flad H.D., Ernst M. (2001). Heterogeneity of human peripheral blood monocytes subsets. J. Leuko Biol 69 : 11-20. Heppner F.L. (2005). Exper imental autoimmune encephalom yelitis repressed by microglial paralysis. Nature Medicine 11: 146-152. Hickey W.F., Vass K., Lassmann H. (1992) Bone marrow derived elements in the central nervous system: an immunohi stochemical and ultrastructural survey of rat chimeras. J. Neuropathol. Exp. Neurol 51: 246-256. Janson C., McPhee S., Bilaniuk L., Haselg rove J., Testaiuti M., Freese A., Wang D.J., Shera D., Hrh P., Rupin J., Saslow E., Goldfarb O., Goldberg M., Larijani G., Sharrar W., Liouterman L., Camp A., Kolodny E., Samulski J., Leone P. (2002). Clinical protocol. G ene therapy of Canavan disease: AAV-2 vector for neurosurgical deliver y of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Ther 13: 1391-1412, Kokovay E., Cunningham L.A. (2005). Bone marrow derived microglia contribute to the neuroinflammatory response and express iNOS in the MPTP mouse model of Parkinsons disease. Neurobiology of Disease 19: 471-478. Kroll R.A., Neuwelt E.A. ( 1998). Outwitting the blood brain barrier for therapeutic purposes: osmotic opening and other means. Neurosurgery 42: 10831099. Lawson L.J., Perry V.H., Gordon S. (1992). Turnover of resident microglia in the normal adult mouse brain. Neuroscience 48 : 405-415. Leissring M.A., Farris W., Chang A.Y., Wals h D.M., Wu X., Sun X., Frosch M.P., Selkoe D.J. (2003). Enhanced proteol ysis of beta-amyloid in APP transgenic mice prevents plaque fo rmation, secondary pathology and premature death. Neuron : 40: 1087-1093. Malm T.M. (2005). Bone ma rrow derived cells contribute to the recruitment of microglial cells in response to bet a-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiology of Disease 18: 134-142. 104

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Massengale M., Wagers A.J., Vogel H., Weissman I.L. (2005). Hematopoietic cells maintain hematopoietic fates upon entering the brain. J. Exp. Med 201 : 1579-1589. McGeer P.L., Itagaki S., Tago H., McGeer E.G. (1987). Reactive microglia in patients with senile dementia of t he Alzheimer type are positive for the histocompatability glycoprotein HLA-DR. Neuroscience Letters. 79: 195200. Meda L., Cassatella M.A., Sz endrei G.I., Otvos L. Jr., Baron P., Villalba M., Ferrari D., Rossi F. (1995) Activation of microglia l cells by beta-amyloid protein and interferon-gamma. Nature 374 : 647-650. Nimmerjahn A., Kirchoff F., Helmchen F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308 : 1314-1318. Perlmutter L.S., Barro n E., Chui H.C. (1990). Mor phologic association between microglia and senile plaque amyloid in Alzheimers disease. Neuroscience Letters 119 : 32-36. Priller J. (2001). Targeti ng gene modified hematopoietic cells to the central nervous system: use of green fluore scent protein uncovers microglial engraftment. Nature Medicine 7 : 1356-1361. Selkoe D.J. (2000). The origins of Alz heimer disease: a is for amyloid. JAMA 283 : 1615-1617. Simard A.R., Soulet D., Gowing G., Ju lien J.P., Rivest S. (2006). Bone marrow derived microglia play a critical role in restricting senile plaque formation in Alzheimers disease. Neuron 49 : 489-502. Stalder A.K. (2005). Invasi on of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. Journal of Neuroscience 25: 1112511132. Stalder M., Phinney A., Probst A., Sommer B., Staufenbiel M., Jucker M. (1999). Association of microglia with amyloi d plaques in the brains of APP23 transgenic mice. Am J Pathol. 154 : 1673-1684. Streit W.J., Walter S.A., Pennell N. A. (1999). Reactive microgliosis. Prog. Neurobiol. 57: 563-581. Sunderkotter C. (2004). Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J. Immunol 172 : 44104417. 105

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Tanaka R., Komine-Kobayashi M., Mochizuk i H, Yamada M., Furuya T., Migita M., Shimada T., Mizuno Y., Urabe T. (2003). Migration of enhanced green fluorescent protein expressing bone marrow derived microglia/macrophages into the mous e brain following permanent focal ischemia. Neuroscience 117 : 531-539. Vallieres L., Sawchenko P.E. (2003). Bone marrow derived cells that populate the adult mouse brain preserve their hematopoietic identity. Journal of Neuroscience. 23: 5197-5207. Van Furth R., Cohn Z.A. (1968). The origin and kinetics of mononuclear phagocytes. J. Exp. Med. 128: 415-435. Verrall M. (1994). Nature 370 6. Wehner T., Bintert M., Eyupog lu I., Prass K., Prinz M., Klett F.F., Heinze M., Bechmann I., Nitsch R., Kirchoff F., Ke ttenmann H., Dirnagl U., PRiller J. (2003). Bone marrow derived cells expr essing green florescent protein under the control of the glial fibril ary acidic protein promoter do not differentiate into astrocyt es in vitro and in vivo. Journal of Neuroscience. 23: 5004-5011. Yan P., Hu X., Song H., Yin K., Bateman R.J., Cirrito J.R., Xiao Q., Hsu F.F., Turk J.W., Xu J., Hsu C.Y., Holt zman D.M., Lee J.M. (2006). Matrix metalloproteinase-9 degrades amyloid beta fibrils in vitro and compact plaques in situ. J. Biol. Chem 281 : 24566-24574. Yan S.D., Chen X., Fu J., Chen M., Zhu H., Roher A., Slattery T., Zhao L., Nagashima M., Morser J. Migheli A., Nawroth P., Stern D., Schmidt A.M. (1996). RAGE and amyloid-beta peptide neurotoxicity in Alzheimers disease. Nature 382 : 685-691. Zhang Y., Pardridge W.M. (2001). Rapid tr ansferring efflux from brain to blood across the blood brain barrier. J. Neurochem 76: 1597-1600. 106

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Figure 1: TOTAL A IMMUNOHISTOCHEMISTRY IS SIGNIFICANTLY REDUCED IN NEP-S TREATED MICE. 9 month old APP+PS1 transgenic mice were injected peripherally with monocytes expressing either NEP-S (n=8 ) or NEP-M (n=9) once a week for two months. The control mice (n=7) did not re ceive any injection over the two month period. Brains were harve sted 24 hours post last inject ion. Panel A shows 6E10 A immunohistochemistry in the frontal co rtex and hippocampus of mice. Panel B shows quantification of percent area of tota l A staining in t he frontal cortex and hippocampus. Statistical analysis wa s performed using one-way ANOVA with Fishers LSD multiple comparison test. Brackets between bars signify statistical significance (p<0.05). Values are mean SEM (standard error of mean). Magnification = 40X. Scale bar = 120 m for all panels. 107

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(A) 108

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(B) 109

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Figure 2: CONGO RED STAINING IS SIGNIFICANTLY REDUCED IN NEP-S TREATED MICE. Brain sections from each group were st ained with Congo Red to quantify fibrillar plaques. Micrographs in panel A show Cong o Red staining in the frontal cortex and hippocampus of mice. Panel B shows q uantification of perce nt area of total Congo Red staining in the frontal cort ex and hippocampus. Statistical analysis was performed using one-way ANOVA with Fishers LSD multiple comparison test. Brackets between bars signify statis tical significance (p<0.05). Values are mean SEM (standard error of mean). Ma gnification = 40X. Scale bar = 120 m for all panels. 110

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(A) 111

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Figure 3: HA is detected in brain tissue but not in plasma. Lysates were loaded for Western blot anal ysis performed using antibody to HA. Nep-S transfected HEK cells (lane 1), brai n lysates (lanes 2, 4, 6) and plasma lysates (lanes 3,5,7). These blots are r epresentative of larger blots showing similar results. 113

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Figure 4. GFP positive monocytes migrate to plaques in APP+PS1 transgenic animals and retain hematopoietic marker. Imaging of GFP-positive cells was done using confocal microscopy. Brain sections were stained with fluorescent markers to detect the location and phenotype of the injected monocytes. Almo st all GFP cells co localized with CD11b (a-c and d-f). No GFP positive cells were detected in uninjected control mice (g-i). Pictures were ta ken at 63X and scale bar = 20 m. 114

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Figure 5. A subpopulation of GFP+ cells express a macrophage phenotype. Ameboid cells expressed both GFP and CD 68, while cells which displayed ramifications expressed only GFP (a-c and d-f). No GFP positive cells were detected in uninjected control mice (g-i). Pictures were taken at 63X and scale bar = 20 m. 115

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CONCLUSIONS Alzheimers disease (AD) is a progressive age related neurodegenerative disorder that afflicts more than 13 m illion people worldwide. Clinically, AD patients exhibit an inability to assimila te new information, and as the disease progresses both declarative and non declarative memory become even more profoundly impaired. The pathological features of AD include senile plaques, neurofibrillary tangles and neuron loss. According to the amyloid cascade hypothesis, aggregation of -amyloid in senile plaques is an early and necessary event in AD (Hardy and Higgins, 1992; Hardy and Selkoe 2002). There is substantial evidence in favor of the amyloid hypothesis which has recently been the subject of a number of reviews (Sisodia, 1999; St. GeorgeHyslop, 1999). The strongest evidence in favo r of it is the finding that multiple point mutations in the amyloid precursor protein (APP), presenilin I and presenilin II, all result in overproduction of the 42 amino acid form of A Each of these mutations results in the familial form of the disease which neuropathologically looks very similar to that of sporad ic AD. For this reason, a number of laboratories and pharmaceutical companies are attempting to develop treatments that reduce the production of A with the hope that this will reduce the symptoms and clinical progression of the disease. Several potential methods have been proposed for removal of A deposits, including vaccination (Schenk et al., 1999), gene therapy (Marr et al., 2004), and cell therapy (Bard et al., 2000; Wyss Coray et al., 2003). 116

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Traditionally thought of as an immunolog ically privileged organ, today the CNS is known to have an endogenous immune system that works through the interaction of immunocompetent cells such as astrocytes and microglia. Microglia are found throughout the CNS; comprising between 5-15% of the cells (Federoff et al., 1995; Kreutzberg et al., 1996; Stre it et al., 1995). Their activation is an early response to CNS injury and is a feature of several brain pathologies including AIDS dementia, Multiple Sclero sis, Alzheimers and Downs Syndrome. Unclear in these neuropathologies are t he relative contributions of resident microglia versus infiltrating macrophages toward neurodegeneration on one hand and toward neuroprotecti on on the other. It is now generally accepted that adul t mouse microglia originate from monocytes/macrophage precursor cells migr ating from the yolk sac into the developing CNS (Pessac et al., 2001; Alli ot et al., 1999). Once CNS residents, these newly migratory cells actively proliferate during dev elopment, thereby giving rise to the resident CNS microglia l population. More re cently however, it has been shown that bone marrow derived cells can continuously enter the CNS and become cells that phenotypically resemb le resident microglia in the adult mouse (Eglitis and Mezey, 1997; Brazelt on et al., 2000; Mezey et al., 2000). Because peripheral and resident microglia have similar phenotypic markers it has been difficult to identify wh ich cells play a role in AD disease pathology. However, since we are now able to track the migration of GFP labeled cells, it is possible to identify the locati on from which these cells are originating. The migratory route monocytes take onc e leaving the bone marrow is currently 117

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unclear and previous work has suggest ed that hematopoietic cells may migrate into the brain parenchyma to give rise to microglia (Massengale et al 2005, Priller et al., 2001, Vallieres et al., 2003). To approach the quest ion of how long bone marrow derived monocytes stay in the blood we us ed transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. Using flow cytometry we were able to identify the GFP monocytes in the blood and determine their relative numbers at each of the time points (Figure 1). As we hypothesized, the injected monocyt es spent very little time in the blood stream. Given the data we were able to det ermine the half life of these cells to be 1.5 hours following the injection. By 24 ho urs we were not able to find any GFP labeled monocytes in t he blood circulation. Although previous studie s have demonstrated infiltration of peripheral monocytes following bone marrow irradiatio n and GFP reconstitution, the percent contribution of circulating monocytes to the microglial reaction in brains has not been investigated. To examine the effects of the APP+PS1 transgene on the recruitment on peripherally injected monocytes, we compared the migration route to various organs in both non transgenic and 16 month old APP+PS1 mice. Quantification of cell numbers in each or gan was analyzed at 1, 3, and 7 days post cell injection using both stereolog ical (Paper 1: Figure 2A) and flow cytometry analysis (Paper 1: Figure 2B). The APP+PS1 mice recruited a significant number of GFP positive cells to the brain following a single injection (Paper 1: Figure 3). These cells were found mainly in the cortex and 118

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hippocampal locations, regions that exhi bit high amyloid load. In comparison, nontransgenic control mice recruited only a few cells to the brain and the few that were present were associated with the blood vessels. Stereological analysis detected a significant difference in recr uitment in the liver, lung and spleens between the non transgenic and the APP+PS1 transgenic mice as. While not significant, these trends were also conf irmed using flow cytometry analysis (Figure 2B). This significant increase in the organs of the nontransgenic mice is most likely due to competitive recruitment of these cells to the brain tissue in the APP+PS1 transgenic mice. An important factor in t he recruitment of monocytes is to determine which population is recruited to the brain. In our study we isolated monocytes from bone marrow using a magnetic cell separation kit that used CD11b as the marker to isolate the cells. However, recent effo rts have used combinations of markers in an attempt to distinguish monocytes from macrophages, neutrophils and other leukocytes, to identify functional subs ets of monocytes (Tacke et. Al, 2006; Djukic et al., 2006; Kato et al., 1996). CCR2-CX3CR1high monocytes that home constitutively to tissues appear to belong to a different group than CCR2+CX3CR1low monocytes that home only when the tissue is inflamed. The two subsets of monocytes may exhibit di fferent responses to pathogens, and it will be important to determine whether they differ in expression of lectins and tolllike receptors. CCR2+CX3CR1low monocytes are hypothesized to be involved in innate inflammatory responses, contribut ing to clearing debris as well as in triggering of the adaptive response toward pathogens. In contrast cells derived 119

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from resident CCR2-CX3CR1high monocytes may be involved in tissue homeostasis as populations of resi dent macrophages include specialized subsets such as osteoclasts, Kupffer cells, and microglia. Thus it will be important for us to determine exactl y which population is attracted to A plaques in our transgenic mouse model. By ta rgeting molecules restricted to CCR2+CX3CR1low monocytes such as chemokine receptors, it may be possible to manipulate their function in infla mmatory diseases, without affecting the potential homeostatic role of the CCR2-CX3CR1high monocytes in the brain. Many studies have provided evidence t hat microglial cells are attracted to amyloid deposits both in hum an samples and in rodent transgenic models that develop this disease (Weigel et al., 2003; Weigel et al., 2004; Malm et al., 2005). It is therefore important to study the development of these deposits, as well as the effects they have on their cellular environment. The precise role of microglia in AD is still under debate. The idea that an immune reaction in AD is the cause of neurodegneration comes from many reports demonstrating that amyloid plaques are often surrounded by microglial cells (Weigel et al., 2003; Weigel et al., 2004; Malm et al., 2005). Some indicate that microglial activation is induced by aggregated amyloid (Casal et al., 2002), w hereas others report that diffuse forms may also be responsible for the im mune response seen in this disease (Takata et al., 2003; Hashioka et al., 2005). While we have demonstrated that per ipheral monocytes can be recruited to the brains in APP+PS1 transgenic mice the exact mechanisms that mediate these cells to the brain parenchyma are still unclear. We and others have 120

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hypothesized that A can cause peripherally circul ating macrophages to cross the blood brain barrier via chemokine recrui tment. (Fiala et al., 1998). Alterations in the permeability of the BBB, expression of cell adhesion molecules, and activation of chemoattractants and their receptors are events that control the migration of blood immune cells in t he CNS. It has been proposed that these processes are controlled by pro and ant i inflammatory cytokines that are released after a brain injury from cells normally present in the CNS, such as astrocytes, endothelial cells and microglia (Gimbrone, 1999). It is evident that soluble inflammatory factors are import ant since virtually all cytokines and chemokines that have been studied in AD including IL-1 IL-6, TNF IL-8, TGFand MIP-1 are upregulated (Neuroinflammation Working Group, 2000). In addition to these soluble mediators, ce ll surface molecules are important in regulating the inflammatory response in the brain. Microglial cells cultured in vitro have been shown to display an increased migratory response upon treatment with beta chemokines, incl uding MCP-1, suggesting t hat these molecules may play an important role in the traffi cking of mononuclear phagocytes within the brain (Peterson et al., 1997) We have shown in previo us work that when MCP-1 is injected into the brains of non transgen ic mice, the signal of this chemokine can recruit peripheral monocyt es to enter the brain. El Khourey et al., crossed a CCR2 knockout mouse with an APP trans genic mouse and showed a decreased microglial accumulation in the Alzhei mer disease brain which resulted in increased A deposition. Also, Hofman et al also demonstrated that MCP-1 expression is present in the cerebral micr ocirculation in tissue sections from AD 121

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but not control patients. However, other cell adhesion molecules may play a key role in the recruitment of these cells. In the AD brain there is elevated expression of ICAM-1 in the cerebral microcircula tion (Frohman et al., 1991; Schoning et al., 2002; Merrill and Benveniste, 1996). Severa l groups have also shown that the transmigration of monocytes into the br ain might require LFA-1 and Mac1, which each bind ICAM-1 (Hakkert et al., 1991; Andrew D., 1998). These data, in conjunction with our results, highlight the brain endotheliu m as a key regulator of white blood cell migration across the blood brain barrier as well as an orchestrator of ev ents within the CNS. Similarly, the term monocytes may describe a heterogeneous group of cells with similar appearance but different roles in the immune system. Many different studies have alluded to A as a culprit in AD disease progression because the 40-42 amino acid peptide is the major component of amyloid deposits seen in postmortem AD pathology (Selkoe, 1999, Gandy. S, 2005). However, in the large number of AD cases in whom a genetic risk factor has not yet been identified, the reasons for the excessive accumulation of A in the brain are unknown. A levels are determined by the balance between A production and removal and it is possible that deficiency in the proteolytic degradation of A peptides could contribute to AD, especially late onset sporadic AD (Iwata et al., 2000, Iwata et al, 2001, Eckman et al., 2001). The proteases involved in the degradat ion and clearance of A have received very little attention, and the main mechanisms that regulate the steady state levels of A in the normal brain remain unclear. 122

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The parallel of NEP with A in the location of the brain makes this protease stand out among other A degrading proteases as a key regulator of A degradation (Iwata et al., 2005; Saido et al., 2005; Salto et al., 2003). The active site of neprilysin faces the extracellular side, where A is hypothesized to be released; implicating a mutual interaction between NEP and A in vivo. The synaptic and axonal localiz ation suggests that after NEP is synthesized in the soma it is then axonally transported to the presynaptic terminals where A degradation is likely to take place. This idea was reinforced when a recombinant AAV vector expressing NEP was injected into the dentate gyrus of neprilysin knockout mice. Not only was the pres ence of NEP detected in the injected hippocampus, but NEP staining was presen t in the contralateral hippocampus indicating that NEP was transported ax onally to presynaptic sites through afferent of neuronal circuits (Iwata et al., 2004). APP also undergoes axonal transport to presynaptic terminals, where A is generated through sequential cleavages by and secretases and released to extracellular space (Price et al., 2000; Selkoe 2001). Therefore the sites for the degradation of A and the location of NEP seem to be closely linked to each other highlighting the important role this protease has as a therapeutic target for the tr eatment of AD. Gene therapy by gene transfer to the diseased or injured CNS provides the basis for the development of potentiall y powerful new therap eutic strategies for a broad spectrum of human neurologic disorders. Previously, several viral systems encoding genes of interest have been injected into brains with variable success. The retroviral vectors have show n high integration efficiency however 123

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they demonstrate low and unstable vira l titer and the need for cell division (Mulligan 1993; Dranoff and Mulligan 1995). Adenoviruses have also shown efficient gene transfer and viral stability, but strong antigenicity has resulted in unwanted side effects for patients (Koz arsky and Wilson, 1993). An additional challenge in working with several viral vect ors is that transduction often leads to microglial activation (Bhat and Fan, 2002). Neverthel ess, the literature with respect to microglia transduction is very small. However in our study, we have found a method to harvest, isolate and tr ansfect monocytes and re-inject them back into an animal within hours (Paper 2) While monocytes are available in the circulation, the percentage of monocyte cells is much greater in bone marrow than in the blood. It has also been show n that monocytes isolated from bone marrow were more receptive to viral vector mediated transduction than monocytes from mouse blood (Zeng et al, 2006). As an approach to increase expression of NEP in a transgenic mouse model of AD we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. Based on previous studies transfection e fficiencies less than 10% have been accomplished using traditional transfecti on techniques, including electroporation, liposomal transfection reagents and DEAE-dextran (Mayne, Borowicz et al. 2003). We have however developed a procedure that allowed us to transfect about 50% of monocytes within a matter of seconds (Figure 2A). These monocytes were transfect ed with a NEP construct desi gned to express either a secreted form of NEP or a form which lacks any enzyme activity. The NEP-M construct was used because it allowed us to develop a control for the presence 124

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of an endogenous enzyme as well as explore the ability of monocytes to degrade A We also determined that the secreted form of NEP was extremely effective at clearing both diffuse and compact A from the brain compared to the NEP-M plasmid which lacked any enzyme activity. There was a significant reduction of both A and Congo Red near t he injection site in the NEP-S mice in the cortex and the hippocam pus compared to the NEPM injected group (Paper 2; Figures 3 and 4). Thus removal of A appears to occur through the diffusion of the protease throughout the injection site. The recent discovery of enhanced lesion site specific infiltration of bone marrow derived microglial progeni tors into the CNS in irradiated mice (Asheuer et al., 2004; Hess et al., 2004; Priller et al., 2001; Tanaka et al., 2003) has raised the possibility of using gene manipulat ed bone marrow cells, hematopoietic stem cells or other types of micr oglial cell progenitors for site specific deliv ery of gene therapy to the injured or diseased CN S (Biffi et al., 2004). Therefore we combined the results from the time cour se study and the intracranial study to develop a peripheral ex vivo gene therapy study. We dec ided to inject the mice twice a week for two months based on t he time course study results that demonstrated almost all cells were gone from brain after a week. The results from the two month study once again confirmed that injected GFP-positive monocytes derived from the bone marrow mi grate from the periphery into the brains of APP+PS1 transgenic mice. Double labeling anal yses also showed that almost all GFP+ monocytes retai ned the hematopoietic marker CD11b (Paper 3; 125

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Figure 4). These finding ar e in agreement with others who have reported that BMDCs retain a hematopoietic phenotype in the brain (Vallieres and Sawchenko, 2003; Wehner et al., 2003). In addition, we also observed cells positively labeled for CD68, a marker for macrophages (Paper 3; Figure 5). We have shown that the secreted fo rm of NEP was extrem ely effective at clearing both diffuse and compact A from the brain compared to the NEP-M plasmid which lacked any enzyme activity There was a significant reduction of both A and Congo Red staining in the NEP-S mice in the cortex and the hippocampus compared to the NEP-M and c ontrol group (Paper 3; Figures 1 and 2). The levels of A were comparable to that of a 9 month old APP+PS1 mouse indicating that the NEPS transfected monocytes halted the progression of amyloid deposition by up r egulating NEP in the brains of these animals. Neprilysin may play a role in degrading a variety of neuropeptides, such as enkephalin, somatostatin, atrial natri uetic peptide, subst ance P, neurokinins, cholecystokinin, nociceptin, and corticotr poin-releasing factor, based on in vitro experiments (Roques et al., 1993; Barnes et al., 1995; OCuinn et al., 1995; Johnson et al., 1999; Sakurada et al., 2002; Turner, 2004). However, whether these neuropeptides are in vivo substrates is unclear. Fo r instance, the levels of enkephalins were not increased in the brains of neprilysin knockout mice (Saria et al., 1997), despite the fact that neprily sin is a potent enkephalin degrader, indicating that neprilysin deficiency is co mpensated for by other proteases. While increasing NEP in the brain may affect other substances, since it is involved in the degradation of othe r species besides A during the two months of injections 126

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we found no signs of weight loss or deteriorating health. Also when we examined the plasma and brain regions from each mouse, we were unable to detect the presence of the HA transgene in the plasma indicating that the effects seen were restricted to the brain region (Paper 3; Figure 3). While we did not actually determi ne if the GFP-positive cells were phagocytosing A increasing evidence has hypothesiz ed that microglia may play a protective role in AD by mediating clearance of A Indeed, when we compared the amount of amyloid in the three treatm ent groups, we saw a small trend in the NEP-M treated group that may indicate peripheral derived monocytes alone could delay the progression of AD like patho logy. There is evidence from several groups that microglia are necessary for effective removal of amyloid through activation of various treatments. Her ber et al., 2004, confirmed that LPS treatment reduces A deposition in APP/PS1 transgenic AD mice. The crossing of APP transgenic mice with mice that overexpress TGF produced offspring that had increased microglial cell activati on and reduced amyloid loads (Wyss-Coray et al., 2001). Also, blocking of complement activati on reduced microglial cell activity in APP tg mice and resulted in an increase of A (Maier et al., 2008). Imaging of mouse amyloid deposits in vi vo by two photon confocal microscopy has shown that local clearance of amyloid plaques after direct application of an A specific antibody occured in connection with the local increase of activated microglial cells (Lombardo et al., 2003). This interaction has been hypothesized to occur as a result of FcR mediated phagocytosis of A antibody immune complexes by microglial cells. Also, it has been reported that increased cell 127

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surface expression of the receptor M-CSF by microglial cell s can accelearate phagocytosis of aggregated A through increasing microg lial cell expression of FcRs for IgG (Mitrasinovic and Murphy, 2 002). Each of these studies supports the concept that in the mous e model of AD, activation of microglial cells may play a beneficial role in reducing A without causing significant neuronal damage. The data supporting a role for microglia in A clearance are convincing, but these data also raise an important question. Why does A continue to accumulate and why does AD neurop athology develop despite continual microglia recruitment? One possible hypothesis for the failure of microglia to stop AD progression is that these cells bec ome inundated by the ex cess amount of A produced and canno t degrade A as fast as it is being generated. Another hypothesis is that as AD progresses, the phenotype of peripheral and resident microglia shifts towards a more pro-infla mmatory state and as a result these cells lose their A clearing capabilities, resulting in increased A accumulation. Also A might not continue to be seen as an ant igen or the signals released from the deposits decrease so that peripheral cells are no longer attracted to the deposits. A final hypothesis is that monocyte inf iltration across the BBB might occur less often in older animals due to the fact that the immune response weakens with age in AD patients (Richartz et al., 2005). In support of th is possibility, Fiala et al., 2005 found that monocytes and macr ophages from AD patients displayed ineffective phagocytosis of A when compared with monocytes and macrophages from age matched c ontrol non-AD patients. It is not clear which of the above mechanism accouts for reduced clearance of A by microglia, but 128

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determining a method to increase the num ber of effective clearing monocytes might serve as a neuroprotective therapy to eliminate or halt the progression of A development. There has been significant interest to identity the cell surface receptors that mediate microglial and A interaction. A number of receptors have been shown to bind A including scavenger receptor cla ss A (SR-A; El Khoury et al., 1996; Paresce et al., 1996), SR-B1 (H usemann et al., 2001), the neuronal 7 nicotinic acetocholine receptor (Dineley et al., 2001; Pettit et al., 2001), the receptor for advanced glycation end pr oducts (RAGE; Yan et al., 1996), the formyl peptide chemotactic receptor (FPR ; Lorton, 1997; Le et al., 2001), heparin sulfate proteoglycans (Giulian et al ., 1998; Scharnagl et al., 1999) and the 5 1integrin (Matter et al., 1998). Howeve r, only SR-A and RAGE have been shown to interact with fibrillar forms of A (Yan et al., 1996; El Khoury et al., 1996). It has been hypothesized that both RAGE and SR-A can interact with A resulting in stimulation of intracellular signal transduction pathw ays involved in production of proinflammatory cytokines. Another proposed mechanism for A activation of microglia is through the receptor for advanced glycation end products (RAGE) and macrophage colony stimulating factor (M-CSF). (Lue et al.,2001). These receptors have been shown to bind A and trigger signals leading to cellular activation. After degradation of A M-CSF over expressed by the microglia in the vicinity of A deposits has resulted in enhanced phagocytosis of A resulting in clearance of the deposits (Mitras inovic and Murphy, 2003). 129

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Aging of adaptive immunity has been greatly studied in recent years (Grebeck-Loebenstein and Wick, 2002). Howeve r, considerably less information is available on how aging affects the innate immune system. While there are indications that age related changes within the innate immune system may influence the development of age rela ted neurodegenerative di sorders such as AD, how old age changes the reaction of microglia to stimuli such as A is not yet known. The first studies on the effe ct of healthy aging on microglial cells have recently appeared. Microglial cells fr om non demented elderly donors showed several abnormalities within their cytoplasmi c structure, includ ing deramification, gnarling and fragmentation of processes (Streit et al., 2004). These changes were interpreted to be different from physical changes that are typically seen during microglial activation in neurodegenerative diso rders. Also the down-regulation of various proteases is implicated in the A deposition observed in human brains durin g the aging process. In addition to neprilysin, several peptidases such as endothelin converting enzyme (ECE1), ECE2, and insulin degrading enzyme (IDE) have been identified as A degrading enzymes though the use of genetic knock out and knock in mice (Eckman et al., 2003; Farris et al., 2003; Miller et al., 2003). These proteases are likely to contribute to overall A clearance in the brain by complementing each other in a cell or brain region specific location. NEP degrades A inside secretory vesicles and on the extracellular surf ace, while ECEs degrade A in acidic compartments (Eckman et al., 2001; Saido and Nakahara, 2003). Also, IDE which is primarily a cytosolic and peroxisomal enzyme (Duckw orkth et al., 1998) can be found in the 130

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plasma membrane (Vekrellis et al., 2000). As a result these poteases may be collaborating to effectively remove A and prevent AD. Developing a combination therapy which encompasses each proteases actions may then be the next step in the development of an even more effective ex vivo gene therapy for the removal of A A better knowledge of the role of in flammation and protease regulation in the development of many neurodegenerative diseases is required before we can safely develop new therapeutics designed at preventing or reversing neuronal damage. While an amplified immune respon se could be adverse to the CNS, increasing evidence reveals that a controlled inflammatory response in the brain might be beneficial to the maintenance and proper function of the CNS. In the case of AD, bone marrow or blood deriv ed monocytes would serve as an ideal alternative to other cells employed in gene therapy because of their natural homing abilities to the CNS under inflamma tory conditions. In addition, the self donation of bone marrow would have the advantage of being safe, widely applicable and ethically acceptable. Since the majority of AD cases are sporadic and do not display any clear genetic causes, multiple factors are likely involved in the accumulation of A in AD. There is mounting data now available to link A degradation in AD pathogenesis. A is a substrate of a wide range of proteases, which are likely to contribute to the accumulation of A in AD. Decreases in protease degradation through genetic mutations and nongenetic fa ctors, such as oxidative damage may contribute to reduced A catabolism. Current results from in vitro and animal 131

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models support NEP, IDE, and ECE as possible enzymes for A degradation, but data from humans remains largely missing. Measurement of brain A levels and amyloid histopathology in correlation with enzyme activity, location and expression will help identify which of these enzymes ar e significant players in AD. Information about monocytes subsets and their functions will also impact our understanding of diseases and the design of therapeutic strategies. Whether monocytes are predetermined to home to specific tissues, and if so, when and where they gain the chemotactic receptor s and adhesion molecules that facilitate their entry into these tissues will represent critical questions for future research. We have presented new evidence that the peripheral monocyte population may be more complex than previously thought. There seems to be a scenario beginning to develop, where resident CNS mi croglia can interact with functionally different populations of per ipheral monocytes. Harnessi ng the peripheral immune system to pharmacologically control brain A levels may serve as a powerful new tool to improve recovery and repair brain damage for the treatment of AD. REFERECES Akiyama H., Barger S., Barnum S., Bradt B. Bauer J., Cole G.M., et. al. (2000). Inflammation and Alzheimers Disease. Neurobiology of Aging 21: 383421. Akiyama H., Arai T., Kondo H., Tanno E., Haga C., Ikeda K. (2000). Cell mediators of inflammation in the Alzheimers disease brain. Alzheimer Disease and Associated Disorders 14: S47-S53. 132

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146 ABOUT THE AUTHOR Lori Ann Lebson received her Bachelor s degree in Biomedical Science from the University of South Florida in 2004 and her Masters degree in Medical Sciences from the University of South Florida in 2006. Before entering the Ph.D program at USF, Lori had conducted research at t he H. Lee Moffitt Cancer Center, Johns Hopkins University and the National Cancer Institute as an undergraduate research associate. En thralled by the exciting scientific discoveries made through molecular and hi stological work, her interest in scientific research intensified. In November 2004, Lori entered the Alzheimers disease Research Laborator y under the mentorship of Marcia N. Gordon Ph.D. and Dave Morgan Ph.D. Loris research focused on developing a novel cell and gene therapy method to be used for the treatment of Alzheimers disease. She successfully defended her doctoral disse rtation in November 2008 at the University of South Florida.


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Monocytes as gene therapy vectors for the treatment of Alzheimer's disease
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ABSTRACT: The accumulation of amyloid-§; protein (A§) in Alzheimer's disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of A§; is therefore thought to serve as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of A§; and defects in the degradation of A§; could contribute to AD disease progression. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genetically modified monocytes to carry exogenous genes of interest into the brain or other organs for the purposes of gene therapy.To determine the time course of monocyte recruitment into the brain during the neurodegenerative damage characteristic of Alzheimer's disease, we used transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. We determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found this time to be 1.5 hours post injection. We also examined the effects of the APP+PS1 transgene on the recruitment of peripheral monocytes and showed that these cells are actively recruited to the brains in AD transgenic mouse models compared to non transgenic mice. As an approach to increase expression of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice.These monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. Monocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS. In addition, we found significant reductions in both A§ and Congo red staining in the NEP-S injected mice only. These studies show that putting monocytes together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatment of AD.
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