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Effects of Anti A§ Monoclonal Antibodies o n t he Amyloid Beta Peptide Fibrillogenesis a nd t heir Involvement in t he Clearance o f Alzheimer's Disease Plaques by Jeffy Pilar Jimnez A dissertation submitted in partial fulfillment of the requirements for degree of Doctor of Philosophy in Chemical Engineering Department of Chemical & Biomedical Engineering College of Engineering University of South Florida Major Professor: Norma Alcantar, Ph.D. Mark Jaroszeski, Ph.D. Ryan Toomey, Ph.D. David Morgan, Ph.D. Garrett Matthews, Ph.D. Date of Approval: April 2, 2010 Key words: passive immunotherapy, ATR FTIR, AFM, monomer, oligomer, protofibril, fibril, protein folding § sheet, § strand Copyright 2010 Jeffy Pilar Jimnez
DEDICATI ON I entirely dedicate this new achievement to my beloved: mo ther, father sister and brothers, for their support, love, advice and words of encouragement no matter the distance.
ACKNOWLEDGMENTS The culmination of this work would not be po ssible without the guidance, support, and encouraging bestowed by my adviser Dr. Norma Alcantar and main collaborators to the project Dr. David Morgan and Dr. Maj Linda Selenica. I also would like to express my gratitude to: The National Science Founda tion Integrative Graduate Education and Research Training ( NSF IGERT, DGE 0221681) program. Special gratitude goes to Dr. Shekar Bhansali and Mr. Bernard Batson. Alfred P. Sloan Foundation Florida Center of Excellence for Biomolecular Identification and Targeted Therapeutics (FCoE BITT) Seed Grant for the financial support. Ritna Pfizer Co Thanks to Drs. Dione Kobanashi and John Lin D r. Stephen Saddow and his group. My research group and peer graduate students Integrative Biology Electron Microscopy C ore at USF especially to Betty Loraamm and Edward Haller All Faculty, Staff and Minority programs of USF Jose Ignacio Rey and lovely family, and last but not least my dear Sister Dr. Jetty Jimenez.
i TABLE OF CONTENT S LIST OF TABLES v LIST OF FIGURES vi ABSTRACT xi i i CHAPTER 1 INTRODUCTION AND PREMISES 1.1. Alzheimer's Disease Facts and Current Therapeutics 1.2. Hypothesis 1.3. Specific Aims a nd Objectives 1.3.1. In vitro Asses sment o f Physicochemical Changes Endured b y A Peptide during Aggregation 1.3.2. Analysis and Evaluation of Effects of Monoclonal Antibodies on A Peptide Fibrils 1.3.3. Influence of Monoclonal Antibody on the Initial Phase of Fibrillization 1.3.4. Construction and E valuation of a Biomimetic Cell Membrane as a Soft Substrate for Future Studies Involving Peptide Antibody Interactions 1.4. Contribution o f t his Work 1.5. Broader Impacts 1 1 5 5 5 6 6 7 7 7 CHAPTER 2 BACKGROUND 2.1. Amyloid Beta Peptide, Agg regation Process, a nd Plaque Formation 2.2. Amino Acids, Peptide Bond, Peptides, and Levels of Structure 2.2.1. Primary Structure 2.2.2. Secondary Structure 2.2. 3 Tertiary Structure 2.2. 4 Quaternary Structure 2.3. Antibodies 2.4. Immunotherapy Preceding Work 2.5. Antib odies Peptide Interactions Theories 8 8 11 13 13 15 15 15 19 21
ii CHAPTER 3 ANALYTICAL TECHNIQUES A ND MATERIALS 3.1. Physical Characterization 3.1.1. Transmission Electron Microscopy (TEM) 3.1.2. Atomic Force Microscopy (AFM) 188.8.131.52. Contact Mo de 184.108.40.206. Non Contact Mode 220.127.116.11. Intermittent or Tapping Mode 18.104.22.168 General Protocol of Sample Deposition and Data Analysis 3.2. Bioc hemical Characterization 3.2.1. A§ Soluble Species Detection: SDS PAGE a nd Immunoblotting 3. 3 C hemical Characterization 3. 3.1 Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR FTIR) 22.214.171.124. Principles 126.96.36.199. Peptides and Amide Vibration Modes 188.8.131.52. Hydrogen Bonding 184.108.40.206. Transition Dipole Coupling Mechanism (TDC) 220.127.116.11.1. Exciton Transfer 18.104.22.168.2. Exciton Splitting 22.214.171.124. General ATR FTIR Data Analysis Pr otocol 126.96.36.199. Some Relevant Consideration f or t he ATR FTIR A nalysis 188.8.131.52.1. Protonat ion or Deprotonation 184.108.40.206.2. Chemical Properties of Neighboring Groups 220.127.116.11.3. Bond Angles and Conformations 18.104.22.168.4. Hydrogen Bonding 22.214.171.124.5. Conformational Freedom 126.96.36.199.6. Overlap of Conformational Changes in the Spectrum 188.8.131.52.7. Rigid D omain Movement 184.108.40.206.8. Subtle Changes within Secondary Structures 220.127.116.11. General Experimental Methodology 18.104.22.168. General Data Analysis 3.4. Reagents 2 3 2 3 2 4 26 27 27 27 30 30 30 33 33 33 35 38 39 39 40 40 43 43 43 43 44 44 45 45 46 4 6 47 49
iii CHAPTER 4 AGGREGATION PATHWAYS OF AMYLOID BETA PEPTIDE 4.1. Protocols and Methods 4.1.1. Peptide's Homogenization 4.1.2. A§ Monomers under Different Media Conditions 4.1.3. Oligomers Stability and their Characteristic Secondary Structure 4.1.4. A§ Species and Kinetics of Fibril Formation 4.1.5. Peptide Secondary Structure Study: Attenuated Total Reflection Fourier Transform Infrared (ATR FTIR) Spectroscopy 4.2. Results Analysis a nd Discussion 4. 2 .1. Species of A§ Peptides 4.2 1 1. A§ Peptide Monomers 4.2 1 2. Oligomeric Species of the A§ Peptides 22.214.171.124.1. Oligomers of A§ 1 40 Peptide 126.96.36.199.2. Oligomers of A§ 1 42 Peptide 4.2.1. 2.3. Oligomers of A§ 40/42 Peptide 4.2 1 3. Fibrillization of A§ Peptides at 25¡C 188.8.131.52.1. A§ 1 40 Fibrils 184.108.40.206.2. A§ 1 42 Fibrils 220.127.116.11.3. A§ 40/42 Fibrils 4.2 1 4. Effects of Temperature during the Fibrillization of A§ Peptides 18.104.22.168.1. A§ 1 42 Fibrils 22.214.171.124.2. A§ 40/42 Fibrils 4. 3 Chapter Remarks 50 50 51 52 54 55 55 56 56 56 65 66 71 76 80 80 83 87 92 92 95 97 CHAPTER 5 MONOCLONAL ANTIBODIES MODIFY FIBRILLOGENESIS 5 .1. Summary 5 .2. Protocols a nd Methods 5 .2.1. Antibody Peptide Solution 126.96.36.199. Effect of Low Antibody Concentration on A§ 40/42 Fibrils 188.8.131.52. Targeti ng A§ 40/42 Monomers and Oligomers with Low Concentration of Different Monoclonal Antibodies 5 .2.2. AFM and TEM Characterization 5 .2.3. ATR FTIR Analysis 5.2.4. SDS PAGE and Western Blot Analysis 101 101 103 103 103 104 105 106 106
iv 5 .3. Result s Analysis and Discussions 5 .3.1. Effects o f Antibodies o n Species o f A§ 40/42 Targeting t he Peptides C Terminus o r N Terminus 184.108.40.206 Effects o f 6E10 a nd 2H6 o n A§ 40/42 Pre Formed Fibrils 5.3.1. 2 Effects o f 6E10 a nd 2H6 o n A§ 40/42 Monomers and Oligomers 5 .3. 2 Proving Suitability of Results 5.3.3. Effects of Antibodies on A§ 40/42 Monomers and Oligomers 5.4. Chapter Remarks 108 108 109 113 117 119 122 CHAPTER 7 MIMICKING T HE NEURON MEMBRANE SURFACE 6.1. Lipid Bilayer Membranes and their Relevance to A Plaque Formation 6 .2. Soft Support Layer U sing Polyethylene Glycol 6.2.1. Constructi on of the Soft Support Layer U sing PEG 6.3. Lipid Bilayer Construction Techniques 6.3.1. Langmuir Blodgett Deposition T echnique 220.127.116.11. Layer by Layer Lipid Membrane Construction 18.104.22.168. Soft Supported Lipid Bilayer Characterization: Surface Topography 6.3.2. Vesicle Based Self Assembled Lipid Bilayers 22.214.171.124. Self Assembled Lipid Bilayers from Vesicles 126.96.36.199. Self Assembled Soft Supported Lipid Bilayer Characterization 6.4. Chapter Remarks 124 124 125 126 128 128 131 132 134 134 135 137 C HAPTER 7 CONCLUSIONS 138 CHAPTER 8. FUTURE DIRECTIONS 143 LIST OF REFERENCES 145 APPENDICES Appendix A. Isoelectric Point of A§ Peptides Appendix B. ATR Crystals 156 157 158 ABOUT THE AUTHOR End Page
v LIST OF TABLES Table 1. Peak Assignment o f A§ Secondary Structures 38 Table 2. Fitting Parameters a nd B oundary Conditions 41 Table 3 Reagents Used in this Work 49 Table 4 General Specifications o f Amyloid Beta P eptides Used in t his Study 51 Table 5. Dominant Structures and Stability Table 98 Table 6. Specificity a nd Binding of Antibodies Tested 102 Table 7 Summary of Observations for the Antibodies Tested in this Chapter 123 Table 8 Dynamic Light S cattering 136 Table 9. Limitations and General Specifications of ATR Crystals 158
vi LIST OF FIGURES Figure 1. Alzheimer's Disease Hallmarks 2 Figure 2. Amyloid Beta Peptide Enzymatic Cleavage 9 Figure 3. General Amino Acid's Structure 12 Figure 4. Amino Acids t o Peptide a nd t he Peptide Bond 13 Figure 5. Peptide's Secondary Structures 14 Figure 6. Sketch of an IgG Monoclonal Antibody Molecule 16 Figure 7. Heavy Chain Isotypes o f Mammalian Antibodies 17 Figure 8. Monoclonal Antibo dy Production Diagram 18 Figure 9. Active v s. Passive Immunization 20 Figure 10. Transmission Electron Microscope 24 Figure 11. TEM Grids Used i n t his Work 2 5 Figure 12. AFM a nd Scanning Process 27
vii Figure 13. SDS PAGE a nd Western Blot Schematic 32 Figure 14. ATR FTIR Spectroscopy Principles 34 Figure 15. Electromagnetic Spectrum 35 Figure 16. Amide Bands 36 Figure 17. Sample's ATR FTIR Data Analysis 42 Figure 18. IR Spectrum Analysis a nd Rigid Domain Movement 46 Figure 19. Sys tematic Study of A§ Monomers u nder Different Media Conditions 54 Figure 20. Amide I and II Regions of A§ 1 40 Monomers i n HFIP 57 Figure 21. Secondary Structures of A§ 1 40 Monomers i n HFIP 58 Figure 22. Secondary Structure Content o f A§ 1 40 Peptide Monomeric Film 59 Figure 23. Secondary Structure o f Amyloid Peptides Monomers i n HFIP 61 Figure 24. Change i n Content o f Secondary Structure for Amyloid Peptides d uring Monomeric Film Formation 62 Figure 25. Morphology o f Monomeric Pe ptides Film 64
viii Figure 26. Spectroscopy Analysis for A§ Peptide Monomers i n DMSO 65 Figure 27. Spectroscopy Study of A§ Peptide during the First 60 Minutes of Incubation in DMSO+TRIS Solution a t 25¡C 67 Figure 28. Kinetics o f A§ 1 40 Oligomers i n DMSO+TRIS Solution at 25¡C 68 Figure 29. A§ 1 40 Average Spectra and Variance Percentage 69 Figure 30. Analysis o f the Amide I and II f or A§ 1 40 Oligomers i n DMSO+TRIS Solution 70 Figure 31. Morphology o f A§ 1 40 Oligomers o ver 48 Hours o f Incubation 71 Figure 32. Change o f Secondary Structures o f A§ 1 42 Dissolved in DMSO after Diluting w ith TRIS Buffer Solution t o 100 M 72 Figure 33. Amide I and II of A§ 1 42 Oligomers during Oligomerization 73 Figure 34. Kinetics of A§ 1 42 Oligomers i n DMSO + TRIS Solution 74 Figure 35. Morphology o f A§ 1 42 Oligomers 75 Figure 36. Change o f Secondary Structures o f A§ 40/42 Incubated i n DMSO a nd TRIS a t 25¡C 76 Figure 37. Kinetics o f A§ 40/42 Oligomers i n DMSO+TRIS Solution 77
ix Figure 38. K inetics a nd Change o f Secondary Structure Content o f A§ 40/42 Oligomers Prepared i n DMSO+TRIS Solution 78 Figure 39. Kinetics o f A§ Oligomeric Species Adsorption 79 Figure 40. Change o f Secondary Structures o f A§ 1 40 Prepared i n DMSO+PBS Solution a nd Incubated a t 25¡C 81 Figure 41. Fibrillization of A§ 1 40 i n DMSO+PBS 82 Figure 42. Fibrillization o f A§ 1 40 a nd Secondary Structures Change 83 Figure 43. Change o f Secondary Structures o f A§ 1 42 Prepared i n DMSO+PBS Buffer Solution t o 100 M a nd Incubated a t 25¡C 84 Figure 44. Fibrillization o f A§ 1 42 a t 25¡C 85 Figure 45. Secondary Structure Change d uring Fibrillization of A§ 1 42 a t 25¡C 86 Figure 46. Change o f Secondary Structures o f A§ 40/42 Dissolved in DMSO a fter Diluting w ith PB S Buffer Solution t o 100 M 87 Figure 47. Fibrillization o f A§ 40/42 a t 25¡C 88 Figure 48. Fibrillization o f A§ 40/42 a t 25¡C 89
x Figure 49. Comparing t he Morphological Evolution of A§ Peptides d uring Fibril Formation at 25¡C 90 Figure 50. Morph ology o f A§ Peptides Films Prepared in DMSO + TRIS a nd DMSO + PBS Solution 91 Figure 51. A§ 1 42 Peptide Fibrillization a t 37¡C 93 Figure 52. A§ 1 42 Secondary Structure Change Effects of Temperature during Fibrilliza tio n 94 Figure 53. Fibrilization of A§ 40/42 at 37 ¡ C 95 Figure 54. Temperature Effect during the Fibrilization Process of A§ 40/42 96 Figure 55. Comparing between A§ 40/42 Fibrils Prepared at 25 ¡C and 37¡C 97 Figure 56. Influence o f Low Concentration Antibodies Targeting A§ 40/42 Fibrils 110 Figure 57. Secondary Structure and Morphological Change s o f A§ 40/42 Fibrils a fter Combined w ith 6E10 or 2H6 Antibodies 112 Figure 58. Monitoring A§40/42 Fibrillization Process from the Beginning of the Incubation Process with N Terminus and C Terminus Antibodies 114
xi Figure 59. Effects o f 2H6 a nd 6E10 Antibodies o n A § 40/42 Monomers + Oligomers 115 Figure 60. Effect of 2H6 a nd 6E10 o n Soluble Species of A§ 40/42 Peptide Equimolar Mixture d uring Fibrillogenesis 117 Figure 61. Comparison of Predicted Secondary Structures vs. Experimental Structures 118 Figure 62. Monitoring A§ 40/42 F ibrillization Process from the B eginning of the Incubation Process with N Terminus and C Terminus Antibodies 120 Figure 63. Effects of 2H6, 6E10, and 4G8 Antibod ies on A§ 40/42 Monomers + Oligomers 121 Figure 64. PEG Cushion Layer Construction 127 Figure 65. Langmuir Blodgett Deposition Technique 129 Figure 66. Langmuir Blodgett Trough 130 Figure 67. Schematic o f Lipid Bilayer Construction 132 Figu re 68. Characterization o f Soft Supported Lipid Bilayer Membrane 133
xii Figure 69. Titration Curve of A§ 1 40 Indicating i ts Isoelectric Point 157 Figure 70. Titration Curve of A§ 1 42 Indicating i ts Isoelectric Point 157
xiii EFFECTS OF ANTI A § MON OCLONAL ANTIBODIES ON THE AMYLOID BETA PEPTIDE FIBRILLOGENESIS AND T HEIR INVOLVEMENT IN THE CLEARANCE OF ALZHEIMER'S DISEASE PLAQUES Jeffy Pilar Jimnez ABSTRACT Alzheimer's disease (AD) is the most common cause of senile dementia worldwide. AD is a n eurodegenerative disorder characterized by the loss of memory and language skill, collapse of the cognitive function, and distortion of social behavior. As of today, the onset mechanisms of AD and cure are unknown; however, three hallmarks are commonly e ncountered: extra and intracellular accumulation of amyloid beta (A ) peptide plaques, formation of intracellular neurofibrillary tangles, and inevitable neuronal death. Hypothetically, a possible scenario provoking or involved in the onset of AD is a cas cade effect that starts with an imbalance in the production and clearance of A§ peptide that consequently leads to its accumulation, formation of tau protein tangles and neuronal death. This work studied and characterized the mechanisms governing A peptid e aggregation and the effects of using anti A§ monoclonal antibodie s to modify this process. These mechanisms play an important role in the formation
xiv of AD plaques and are critical in the search for therapies involving A§ peptide plaque clearance. Yet, an tibody based therapies for plaque clearance are not well understood, adding to the existing concerns about side effects in humans, hence there is a necessity of knowledge in this matter. In this work different N terminus, C terminus, and Mid domain antibod ies were used against A§ peptide species (monomers, oligomers, and fibrils) to probe peptide aggregates modification and disruption. Additionally, construction of a soft supported lipid bilayer membrane was proposed to study the adhesion mechanisms of A§ p eptide and interactions with antibodies, mimicking the neuronal cell surface. The main characterization techniques used in this work were: atomic force microscopy (AFM) and transmission electron microscopy that allowed the physical exploration and visuali zation of the different processes of aggregation in terms of adhesion, size evolution, and distribution of the peptide; and attenuated total reflectance Fourier spectroscopy (ATR/FTIR) which allowed monitoring the change of secondary structures for the pep tide during the processes studied. It is endeavored that this work will help to elucidate the effects attributed to the molecular interactions between A peptide species and antibodies to target A§ plaque's clearance in the brain of AD patients. Ultimatel y, this study provides novel information critical for the formulation of effective therapies to prevent and treat AD with less collateral effects. It also represents a contribution to the basic scientific knowledge regarding peptide antibody interactions w ith application to other diseases related to protein misfolding.
1 CHAPTER 1 INTRODUCTION AND PREMISES 1.1. Alzheimer's Disease Facts a nd Current Therapeutics Alzheimer is an irreversible neurodegenerative disease characterized by the progressive loss of memory, coordination, social skills, and reasoning. In 2006 Alz heimer's disease (AD) was reported as the sixth leading cause of death across all ages in the United States [1, 2] Furthermore, AD significantly reduces the life expectancy and quality of those afflicted by it  Statistics show that before a person's 65 th birthday 1 person in every 1000 is diagnosed with AD, and after this age this rate increas es to 1 person in every 8 T he chance raises to 1 person in every 5 after the age of 80  As of today, nearly five million Americans live with A D  This number is expected to quadruple in the next four decades  Worldwide AD cases have reached 26 million people and it is expected to i ncrease to 106 million by 2050. In addition, reports show that in the United States alone t he government and affected families spend approximately 148 billion dollars annually to treat patients with AD [1, 5]
2 The onset mechanisms of AD and the cur e are still unknown; however, three pathological hallmarks are commonly observed among patients with AD: the presence of extracellular deposits of amyloid beta (A§) plaques, intracellular neurofibrillary tangles (NFTs), and neuronal apoptosis. Figure 1 (a) and (b) shows the first two major microscopic hallmarks mentioned above  In this work special attention is rendered to understanding the process of A§ plaque formation and clearance. A§ peptide deposits (also known as senile plaques) are mainly composed of amyloid beta peptides containing 1 40 and 1 42 amino acids residues (A§ 1 40 and A§ 1 42 respectively) [7 9] Figure 1. Alzheimer's Disease Hallmarks. (a) Left: a normal neuron. Right: depicting the three hallmarks of AD (A§ plaques, NTF's, and neuron's apoptosis)  (b) Classical histopathology of AD's brain showing plaq ues and neurofibrillary tangles (arrows), taken from Cordell, 1994 
3 Despite the fact that A§ peptide does not have a known function in the brain, it is present in healthy brains. Studies show that insoluble A§ peptides along with a soluble sub product are proteolytically cleaved from a larger transmembrane protein known as a myloid p recursor p rotein (APP) [12, 13] Soluble forms of APP are believed to have neuroprotective and neurotrophic functions. However, A is considered an unwanted by product of t he APP processing  The A peptides are found as globular and non fibrillar forms in small concentrations of pico to nano molar and can be traced in the extracell ular and cytoplasmic (inside the cell) regions in both normal and AD brain tissues. Nevertheless, significantly small oligomeric and protofibril species of A peptides are enough to cause profound cytoskeletal degeneration and cell death by plaque formati on through underlying mechanisms still not understood [14 20] It is also known that plaques and tangles are accumulated in neurons, mainly throughout the cortical and limbic brain regions  The cortical region is responsible for the abilities and activities related to thinking, basic aspects of perception, movement, and adaptive response to the outside world. The limbic region is primarily responsible for emotional behavior and the formation of memories  In the past decade there have been significant advances in the field of AD; however, an effective treatment for its cure is not available yet  Currently, some common drugs containing active ingredients such as cholinesterase
4 inhibitors 1 or glutamine regulators 2 are not able to cure the disease. Rather, these drugs only serve to "delay the progression of some of AD's symptoms"  There is a tremendous need to find an effective treatment that radically stops and prevents the brain's cognitive degeneration and the irreversible loss of memory A feasible and promising treatment is immunotherapy. In the past active immunization (vaccination) was tested in a phase II clinical trial [25, 26] which was suspended after observing severe undesired immune response to the treatment (7% of the patients out of 312 subjects presented encephalitis) Nevertheless patients monitored afterward showed less cognitive function degeneration in compari son to the control individuals and this study gave important insights into immunotherapeutic treatments [26, 27] 1 Razadyne¨ (galantamine) also known as Reminyl¨, Aricept¨ (donepezil), Cognex¨ (tacrine, with safety concerns), and Exelon¨ (rivastigmine); these are usually prescribed to patients with mild to moderate AD to prevent the cholinesterase's breakdown 2 Namenda¨ (memantine), an N methyl D aspartate (NMDA) antagonist; prescribe to patients with moderate to severe AD, to r egulate production of glutamate (in large am ounts leads to brain cell death ).
5 A possible therapy currently under study is passive immunization, which consists of the administration of monoclonal antibodies as a mean s to assist the dissolution and clearance of A peptide plaques from the brain in patients with A D 1.2. Hypothesis It is possible to asse ss the effects of different monoclonal anti A§ antibodies directed against aggregates of A§ peptides in vitro 1.3. Specific Aims a nd Objectives With the purpose of proving the current hypothesis, the work was d ivided into four specific aims, explained below. 1.3.1. In v itro Assessment o f Physicochemical Changes Endured b y A Peptide d uring Aggregation The aggregation process of A§ peptides was first monitored and characterized chemically by attenuated total r eflection Fourier transform infrared spectroscopy (ATR FTIR); this technique allowed the identification of the peptide's secondary str uctures involved in such event. The chemical characterization results were then checked by the physical characterization of the species encountered at different collection times using atomic force microscopy (AFM).
6 Two isoforms of A§ peptide, A§ 1 40 (with 40 amino acid sequences) and A§ 1 42 (with 42 amino acid sequences), were studied individually and in combination (1:1 A§ 1 40 to A§ 1 42 equimolar mixture, A§ 40/42 ). The main objective was to create a baseline understanding of the kinetic and structural process es during fibrillation and to adapt an analytical method for posterior studies. 1.3.2. Analysis a nd Evaluation o f Effects o f Monoclonal Antibodies o n A§ Peptide Fibrils The purpose was to determine the effects of antibodies on mature fibrils. The antibodies used for this study had different affinity and specificity. The substoichiometric molar ratio of antibody to p eptide used in this study was 1:1000 1.3.3. Influence of Monoclonal Antibody o n the Initial Phase of Fibrillization Physical, chemical, and biochemical methods were used to analyze the action of different antibodies on A§ peptide species. This aim foc used on understand ing the exten t to which the antibodies were influencing the aggregation process and to evaluate the possible effects on plaque clearance by targeting monomers + oligomers, being this the initial phase of aggregation.
7 1.3.4. Construc tion a nd Evaluation o f a Biomimetic Cell Membrane a s a Soft Substrate f or Future Studies Involving Peptide Antibody Interactions The main objective was to produce a stable soft support that could serve to mimic the role played by the cell membrane as a su bstrate for the AD plaques and to test the action of monoclonal antibodies on A§ peptide aggregates. 1.4. Contribution o f t his Work The principal contribution of this work was to provide an in vitro method to evaluate the effects of antibodies on amylo id beta peptide species, probing their effectiveness to destabilize neurotoxic forms of A§ and disrupt their natural aggregation pathway. This work provided basic scientific knowledge to help understand and improve antibody based therapies for AD with ex tension to other protein misfolding related diseases. 1.5. Broader Impacts The methods and findings from this work opened paths to evaluate critical biomolecular interactions to understand protein misfolding related diseases and possible therapies.
8 CHAPTER 2 BACKGROUND This chapter gives a general review of concepts and principles that provide the bases to later discuss our results and explain the phenomena observed during the different studies performed in this work. 2.1. Amyloid Beta Pept ide, Aggregation Process, a nd Plaque Formation The name amyloid comes from the Latin "amylum" and Greek amylon" meaning starch in both languages. Rudolf Virchow in 1854 used this name to refer to the material deposited in the brain as macroscopic abnorm alities, due to its reactivity to iodine sulfuric acid, a reagent used to identify starch and cellulose derivatives  It was later in 1859 when Friedreich and Kekule demonstrated that the material identified as cellulose was protein  Amyloid peptides are not exclusive to Alzheimer's disease (AD) T hey are also associated with diseases in other organs such as the liver, spleen, and kidney  The amyloid peptide found in neuritic plaques in AD patients is kno wn as amyloid beta (A ) peptide due to its natural tendency to form sheet secondary structures 
9 As mentioned in Chapter 1, AD's neuritic plaques are mainly composed of two isoforms of A peptide A 1 40 and A 1 42  These peptides are fragments produced by the proteolytic cleavage of a larger transmembrane molecule (APP), a process briefly sketched in Figure 2. The pro d ucts of this proteolytic cleavage are soluble neuroprotective secreted ectodomain sAPP (via secretase cleavage) and extracellular A 40/42 peptides (via consequent and # secretase cleavage) [32, 33] It is worth mentioning th at even though the concentration of secreted A§ 1 42 is near 10% of the A§ 1 40 concentration, A§ 1 42 peptide is the main component of neuritic plaques deposits [13, 34] Figure 2. Amyloid Beta Peptide Enzymatic Cleavage Schematic of A§ 1 40 and A§ 1 42 production by c leavage of the APP proteins by the intervention of specialized enzymes
10 The mechanisms of neuritic plaque formation are still unclear. However, it has been suggested that soluble species of A peptide undergo a series of molecular rearrangements (misfoldi ng) triggered by environmental stress that lead to the formation of fibrillar deposits. U nder normal conditions, cells with elaborate quality control machinery guarantee that proteins fold ed im properly are eliminated. This machinery includes proteins s pecialized organelles and cell s that act as chaperones and activate processes to selectively eradicate misfolded proteins. In the case of the central nervous system (CNS) the specialized quality control cells are the microglial cells. P roteasomes and lys osomes structures also contribute to the degradation and elimination of misfolded proteins inside cells  However, a particular characteristic that distinguishes a myloid and a few other misfolded proteins (such as alpha synuclein, cystatin c and tau among others) is the ability to aggregate into higher order species such as oligomer s protofibril s and fibril s. These species cannot be transported into the central c atalytic pore of the proteasome enzyme complex for degradation. This characteristic allows amyloid peptides to evade proteasome and lysosome quality control machineries, which lead s to accumulation of m isfolded amyloidogenic peptide. This system grows an d propagates offering resistance to degradation for the amyloid toxic  In addition, a small increase in the concentration of misfolded protei n can dramatically a ccelerate the process of plaque formation 
11 In vitro a myloid beta peptide fibrillization can be recreated and studied at different levels of aggregation by manipulating the peptide's concentration, temperature, pH, and ionic strength of the media [37 38] Similarly, it i s possible to alter and destabilize production of A§ species (oligomers, and protofibrils) critical during fibrillogenesis by introducing an external agen t (i.e. beta sheet breakers). Although active and passive immunotherapy have shed light into the possible mechanism involved in the activation of the immune system and clearance of A§ from the brain, little is known about the impact on protein folding produced by monoclonal antibodies binding to A§ peptide. This work evaluates the anti A§ monoclonal antibodies molecules as external agents used to alter peptide's aggregation and promote peptide solubility 2.2. Amino Acids, Peptide Bond, Peptides, a nd Levels of Structure Amino acids are the building blocks of peptides and polypep tides T hey are formed by two main functional groups: an amino and a carboxyl group which are both linked through a chiral central carbon atom (C ) that also holds hydrogen bonding and a distinctive R group (side chain) as shown in Figure 3 T he arrangement of these constituents around the central carbon atom makes them chiral, meaning they have two isomers, L isomer and D isomer. However, the L isomer is found only in peptides and polypeptides. Naturally, the amino acid residues present in a peptide or polypeptide molecule influence the properties and adopt ed conformations.
12 Figure 3. General Amino Acid's Structure Peptides and polypeptid es are sequential and unbranched polymers of amino acids linked from head to tail, with less than 50 and more than 50 amino acid residues, respectively  The union between a carboxyl group and an amino group through a covalent amide linkage is referred to as a peptide bond  which is s hown in Figure 4 The peptide bond is essentially planar, and it has what is considered a double bond strength that prevents free rotation around it H owever, vibration and translation are possible I n this study though, only vibration was quantifi able with ATR FTIR spectroscopy ; as it will be explained in Chapter 3 It is important to mention that the peptide bond is uncharged, and this absence of charge allows the amino acid chains to tightly pack together linked by peptide bonds into globular struc tures  The main event responsible for the change in conformation will be the los s or gain of protons depending on the me dia's pH. The molecular interactions among peptides are also affected by temperature changes and the media's ionic strength.
13 Figure 4. Amino Acids t o Peptide a nd t he Peptide Bond A final point pertinent to this section is the presentation of the fo ur different levels of structure adopted by peptides and polypeptides, which are explained as follows. 2.2.1. Primary Structure They are s imply f ormed by an amino acid sequence in a linear fashion and without taking into account three dimensional rearrang ements of the residues. 2.2.2. Secondary Structure They represent a three dimensional arrangement of the polypeptide in the space This is conceived by the disposition of the hydrogen bonds between
14 adjacent amino acid residues in a molecule and between m olecules, which allow s the peptide molecules to arrange themselve s into characteristic patterns. The following were the main secondary structures or patterns investigated in this work: parallel § sheet, antiparallel § sheet helix, unordered (or random coils), and § turns structures, shown as part of the short peptide in Figure 5 Figure 5. Peptide's Secondary Structures T he most important and known secondary structures are the helical and pleated segments ( formed by parallel or antiparallel § strands ). These segments constitute regular structures that extend along one dimension. Most proteins exhibit th ese types of structures, which allow peptides to play specific roles in the body.
15 2.2.3. Tertiary Structure Th is structure is present when polypeptide chains assume a tight three dimensional shape, adopting a globular form. This type of structure gives to the polypeptides a lower surface to volume ratio allowing the molecule s to shield from the solvent  2.2.4. Quaternary Structure This structure is composed of several folded peptides or polypeptides that are gathered in a more complex molec ule, as in the case of enzymes. 2.3. Antibodies Antibodies also known as immunoglobulins are natural bioengineered proteins with high binding specificity to a wide range of molecules including peptides, carbohydrates, and nucleic acids  Figure 6 shows the characteristic parts of an antibody molecule. Antibodies have a particular "Y" sh ape, which is formed by two heavy chains and two light chains T he type of heavy chain determine s the antib ody isotype (Figure 7) to which they belong. The Fab (fragment antigen binding) region of the antibody is composed of domains from both heavy and li ght chains of the antibody, and plays an important role in the binding and specificity to a particular site of the ant i gen known as the epitope. I n the case of this work, the antibodies used were specific to A§ peptides, either 1 40, 1 42 or both, with ep itopes variable depending on the antibody.
16 Figure 6 Sketch of an IgG Monoclonal Antibody Molecule The region of light chains is generally recognized as Fab and promotes specific biding characteristics The distinction between the different antibody isotypes is based on their heavy chain, which in turn is related to the source where they are produced; for instance, IgA is found in the mucous secretions, while the IgG is mainly found in the blood and in tissue liquids. A functional difference between the various isotypes is based on the part of the immunoresponse's cascade of which they are part. Between the 5 main isotypes shown in Figure 6 (IgG, IgA, IgE, IgD, and IgM) the most common isotype in the human body is the IgG which was also used in thi s work.
17 Figure 7. Heavy Chain Isotypes o f Mammalian Antibodies. IgG provides the majority of antibody based immunity against invading pathogens In general, antibodies tha t are synthetically produced can be classified in two large groups: monoclonal and polyclonal. Polyclonal antibodies are obtained from the sera of immunized animals and they are a mixture of different immunoglobulin types binding to multiple sites on the antigen used for immunization  On the other hand, monoclonal antibodies have a single immunoglobulin type binding to a single specific site of a molecule (antigen, protein, carbohydrate or nucleic acid) used for immunization.
18 Figure 8. Monoclonal Ant ibody Production Diagram Monoclonal antibodies are synthetically produced by the fusion of antibody producing mouse B cells, which have a finite life span, with "immortalized" cells of a mouse myeloma (cancer cells) These fused cells are cultivated an d successful immortalized antibody producing cells are selected  T his process is pictured in Figure 8. Each cell or clone obtained by this method is capable of producing large amounts of a single type of antibody, which binds to specific epitopes of the antigen molecule (A§ 1 40 A§ 1 42 or both).
19 2.4. Immunotherapy Preceding Work Immunotherapy for treating AD is a subject that has been investigated since the late 1990's Beka Solomon (1996) and her group were the first who reported the effectiveness of N terminus antibodies to block fibril formation in vitro  After this publication, many other studies involving immunotherapy for AD were published from Solomon's group and from other scientists all over the world  In 2003, preliminary results were published of th e first clinical trial (Elan Wyeth clinical trial)  in which patients with AD where vaccinated with AN 1792 (purified A§ 1 42 ), and after advancing to a Phase II (372 patients with mild to moderated AD), the study was stopped after finding 7% of the patien ts suffering from encephalitis. Neverthe less, the immunized patients were monitor ed after the treatment was stopped and a reduction in cognitive decline in comparison to the controls was found This has been the bas is to continue in the search for an effective but safe immunotherapy treatment for patients with AD  It is important to no te t hat immuno therapy can be classified in two types: active and passive immunization (Figure 9 ). Briefly, active immunization can take place either by injecting into the individual the whole antigen or part of it, whichever way provokes an immune response resulting in the production of specific anti bodies to the injected antigen. T he other type of immunization consists of the injection of specific antibodies directed to the antigen of interest (either monoclonal or polyclonal).
20 Figure 9. Active v s. Passive Immuni zation. Adapted from Schenk, 2002  An ideal scenario for using immunotherapy to target a specific event (clear ance of A§ deposits) would be decreasing the body's immun e response to a mild level so that the external agent (antibody in the case of passive immunization) could mediate the desire d event in a controlled manner F rom here derives the idea of using mono clonal antibodies to trigger the clearance of A§ deposits. Indeed, this idea has been tested in mice, finding activation of the clearing plaque mechanisms and cognitive recovery to a certain level [46 54] But sti ll the key questions remain: W hat is the mechanism involved in this clearing event? I s it possible to determine what antibody will activate and help the body to clear th ese deposits in the most effective way? What about the quantities?
21 The present study pr ovides insights that help to understand from a molecular interaction point of view the effect of the antibody on the plaques and help to identify the species at different stages of the plaques formation and clearance. 2. 5 Antibodies Peptides Interact ions Theories For the purpose of this study it is important to consider the affinity between the antibody and the A peptide from a thermodynamic point of view. The affinity is related to the type of interactions that occur at the interface between the tw o molecules. In terms of energy, the affinity between two molecules will depend on two thermodynamic factors, the enthalpy and entropy at the interface  When antibodies and A peptide molecules are free in aqueous solution they have a consid erable configurational (translation and rotational energy, measurable with ATR FTIR spectroscopy) entropy that is lost when the association (binding) occurs T his effect is observed in the analysis to be presented in Chapters 4 to 6 Moreover, the noncov alent interactions (H bonds, salt bridges and van der Waals forces) between each molecule's surface residues with the water are lost when the antibody and A § peptide enter in contact. Nevertheless when the bound water is released from the two interacting interface s it leads to an increase in entropy of the solvent. Hydrophobic residues that become buried at the interface cause an increase in entropy of the solvent in a similar way.
22 However, at the same time, new hydrogen bonds, salt bridges, and v an der Waals interactions a re formed between the antibody and the A peptide [40, 56] Energy los s es and gains due to molecular interactions driving aggregation can be indirectly detected as a function of integrated absorbanc e with ATR FTIR spectrometric analysis. In addition, there are other factors that might also affect the molecular interactions between the antibody and the A peptide such as the presence of salts (ions in solution) and the posttranslational modification s involving morphological cha nges, as is the case of monomers oligomer s and fibril s species adopted by the A peptide 
23 CHAPTER 3 ANALYTICAL TECHNIQUES AND MATERIALS In this study different analytical methods are combined to provide a comprehensive physicochemical characterization and ex planation of the processes of peptide aggregation, as wel l as the effects produced by different anti A§ monoclonal antibodies when they are tested against A§ peptide aggregates. This chapter explains the techniques used in a systematic way by dividing the m into physical, biochemical and chemical characterization. The techniques explained here are brought into play in the subsequent chapters. 3.1. Physical Characterization These techniques served to evaluate the morphology of the sample s by studying their dimensions as well as dispersion and distribution of the different peptide aggregates across the substrate's deposition area. T wo physical characterization techniques were utilized: transmission electron microscopy (TEM) and atomic force microscopy (AFM) The primary aggregates of interest for the study were monomers, oligomers or protofibrils, and fibrils.
24 3.1.1. Transmission Electron Microscopy (TEM) This is a microscopy technique that consists of a focused beam of primary electrons that is directed through the sample under study. The electron beam can be generated by ther mionic emission from a filament, commonly tungsten However, the electron s beam can also be produced by a lanthanum hexaboride (LaB 6 ) source. The electrons are accelerated using electrostatic and electromagnetic fields to focus the beam and deflect it to a constant angle  The instrument used was a Morgagni 268D transmission electron microscope from FEI Corporation (NE, US) which is shown in Figure 10 The source of primary electrons in this instrument comes from the thermionic em i ssion of a tungsten filament. T he instrument provides a point to point resolution of 0.45 nm and a resolution between lines of 0.34 nm. It has a Soft Imaging Mega 3 digital camera with an ultimate resolution of 1280x1024 pixels per image All processes of the specimen's imaging takes place in a vacuum chamber (10 6 Torr). Figure 10. Transmission Electron Microscope
25 The accelerating voltage chosen for the analysis was either 100kV or 60kV depending on the thickness of the sample and it was adjusted to get the best contrast. The sample preparation depends on the specimen to be studied. For instance, in the case of amyloid peptide, a drop (5 # L) of the sample is simply placed on a special grid (rinsed with 5 # L of Millipore water) and dried with ultrapure nitrogen. In this study two different type s of grids were used: Formvar coated grid stabilized with evaporated c arbon film (150 mesh, FCF150 Cu ) and inert s ilicon nitride grids ( DuraSiN TM ). The last one used to characterize the sample not only with TEM but also with AFM. Both grids were purchase d from Electron Microscopy Science (PA, US) and they are shown in Figure 11 below (a) Formvar/Carbon (b) DuraSiN TM (150 mesh) Figure 11. TEM Grids Used i n t his Work (a ) Carbon grid (150 mesh) coated with Formvar film, a transparent and inert polymer. (b) DuraSiN TM showing window covered with silicon nitrate Diameter =3.05mm Window 0.5x0.5 mm x y
26 3.1.2. Atomic Force Microscopy (AFM) This technique is also known as scanning force microscopy (SFM) or scannin g probe microscopy (SPM). I t allows for the visualization of the topographical structure of conductive and non conductive materials. Commonly, an AFM consists of a piezoelectric scanner, on which the sample rests; this scanner allows movements in a preci se fashion in the x, y, and z directions. However, having the three dimensional movement freedom in the same scanner makes th e instrument very sensitive to disturbances, resulting in long collection times. In this work, the AFM used (XE 100, Park Systems Inc., CA, US) consists of two piezoelectric scanners; one scanner accounts for the x y movements only, and another piezoelectric scanner located on the head where the cantilever rests (Figure 12( a ) ) separately allows movement on the z direction. This n ew design increases imaging flexibility and reduces the collection time. Another important component in the AFM is a photodiode, which senses the movements of the scanning tip when a laser positioned on it deflects due to its interaction with the sample. This deflection is then translated by the photodiode into either a current or a voltage, and ultimately into a digital image (Figure 12( b) ) With AFM, it is possible to image and characterize samples on a solid gas interface as well as solid liquid inter face. In this work images were collected in both edia; however, primarily images were collected on solid gas interfaces. Atomic force microscopy allows characterization of the sample's s urface by three different modes, as explained below.
27 3.1.2. 1. Contact Mode I n which the tip of the cantilever gets in touch with the sample (hard materials) 188.8.131.52. Non Contact Mode Wh ere the tip does not contact the sample The non contact mode is used in characterization of soft materials (biological, i.e. cells). (a) AFM, XE 100 Schematic (b) Imaging Process Figure 12. AFM a nd Scanning Process. The sample's topology can be studied by dragging a micro scale cantilever across its surface. (a) Shows a full diagram of the instrument; (b) illustrates a detail of the cantilever and detection system on A fibrils 184.108.40.206. Intermittent or Tapping Mode This mode can be used to prevent the cantilever from being trapped by capillary forces caused by the presence of an extremely thin film of water
28 surrounding the sample in air. Also this mode is use to pres erve the sample, especially when biological samples are analyzed. Tapping mode was used to analyze the A§ peptides deposited on mica. Therefore, in this study tapping was the mode of preference for the samples' characterization. During the tapping mode, the cantilever is deliberately excited by an electrical oscillator to amplitudes of up to approximately 100 nm, creating an effective tapping modality as the cantilever tra vels over the sample (Figure 12( b ) ). In addition, because the contact time of the t ip with the sample is relatively short, it is possible to reduce lateral force (that could cause distortion of the image) and also preserve the sample's morphology [59 ] There are a wide variety of cantilevers commercially available; the selection depends on the application and media. In this work, the cantilever used was the type Tap300 Al (Budget Sensors, Bulgaria). These cantilevers were purchased with an aluminum coating to inc rease the laser's reflectivity. This type of cantilever has a reso nant frequency of 300 100 kHz and a force constant of 40 N/m. The substrate material used to image the peptide's s amples was mica. This is a preferred material when wor king with biological specimens  Mica was selected because it is atomically smooth (0.2 to 0.6 ), and at the same time, an easy to clean (sheets cleavage) and i nert material. In this study natural mica of the type ASTM V 1, purchased from Axim Mica (NY, US) was used The physical characterization methods used in this study facilitated check ing the physical size of the aggregates during time and determin ing the
29 kind of secondary structures dominating at each stage of the aggregation process of samples with anti A§ monoclonal antibodies and alone 3.1.2 4. General Protocol of Sample Deposition and Data Analysis In this work all the samples were deposited under a laminar flow cabinet (Class 100) to avoid contamination by settling of undesirable particles. Each aliquot collected at specific times was diluted with Millipore water to 20 # M. Then, 5 L of the diluted solution was deposited on freshly cleaved mica and incubated for 5 min. A small Teflon O ring (external $ 7mm, internal $ 5mm) placed on the mica served to confine the peptide solutions. The excess of peptide solution was flushed with 1 ml of Millipore water, dried with ultrapure nitrogen, and stored in a vacuum dessicator at 30 in Hg for later AFM imaging. As mentioned in Section 3.1.2, the images were taken with a XE 100 a tomic f orce m icroscope from Park Systems Corp. (Santa Clara, CA) at 25¡C. The scans were performed with TAP300Al cantilevers (Budge t Sensors, Bulgaria) using tapping mode, to preserve the sample. The image resolution was 512x512 pixels, and the scan rate was 0.75 Hz, the set point of the cantilever was variably moved to satisfy the image quality but never lowered beyond 0.260 # m. Im ages were collected at 5 random points on the mica surface, and each point was imaged using three different scan sizes, first 35x35 # m x y, then zooming to 10x10 # m x y, and finally to 2x2 # m x y; this was done to assure a fair exploration of the sample di stribution across the mica.
30 The processing and analysis of the images was performed with WSxM software (version 5.0)  3.2 Biochemical Characterization 3.2.1. A§ Soluble Species Detection: SDS PAGE and Immunoblotting The combination of these two methods, SDS PAGE ( sodium dodecyl su lfate polyacrylamide gel electrophoresis ) and Western Immunoblotting a llow for discrimi nating the different soluble peptide species in the incubation solution when separated by their differen ce in molecular w eight In this sense, the sample after being diluted in water and centrifuged is first analyze d with SDS PAGE, in which the peptide is loaded on a 4 12% Bis Tris gel, and then exposed to an electrical current that allows the diverse aggregates species to migrate and distribute on the gel at different speeds depending on their molecular weight Normally, the gel used is polyacryamide T his is an inert material, and serves only as a medium for the peptides to move (under buffered conditions). The peptide's species recognition takes place once the peptide (distributed on the gel) is carefully transferred to a polyvinylidene difluoride m embrane where it is likely to bind. A step prior to recognition is the "blocking" step, in which either Bovine s erum albumin (BSA) or non fat dry milk are used to prevent the interaction of the antibody with the unbounded sites of the membrane, and theref ore create s "n oise".
31 The membrane is incubated with a primary antibody specific to N terminal of the peptide (A§) by gentle agitation for 12 hours. Then, the gel is rinsed to remove any unbounded primary antibody and at this point, a second antibody that targets the primary antibody is added to enhance recognition. Finally, the immunostained peptides can be visualized usin g chemiluminescence techniques. For the effect of this work, this step was performed in accordance with the manufacturer's recommenda tions (ECL, Amersham, Denmark). Western blots images were captured with a Fujifilm Lass 300 Intelligent dark box scanner and quantified with a linear range of detection using Alpha Ease software (AlphaEaseFC, Alpha Innotech Corporation, San Leandro, CA). This analysis allows one to quantify the intensity of the bands corresponding to the following : monomers species (4.5 KDa), Low Molecular Weight (LMW) oligomers or protofibrils (14 to 21 KDa), High Molecular Weight (HMW) oligomers or protofibrils (38 to 9 8 KDa) and larger soluble aggregates (188 to 250 KDa). Figure 13 shows the schematic representation of the analysis explained above
32 Figure 13. SDS PAGE a nd Western Blot Schematic It shows the steps involved in the biochemical characterization of A aggregates. The sample is diluted and centrifuged, then exposed to an electrical current through a gel separating aggregates by molecular weight, then the aggregates are transferred to a membrane and immunotagged. The membrane is digitized and the resu lts are quantified with computer software It is important to mention that during the peptide aggregation, monomers associate as a paranuclei specie s (pseudostable) at the initial times of incubation  T his paranuclei can be detected from the monomeric through the LMW region. In the next chapter, this technique is linked to the physical view of the different species and i s also investigated with atomic force microscopy.
33 3 3 Chemical Characterization Vibrational spectroscopy was used in this work to analyze and monitor the chemical configuration changes of amyloid peptides. This technique also allowed to determine and understand the effects of antibody molecules targeting A§ peptide. While the experimental setup is relatively simple, the analysis of the resulting data is rather complex Hence, this section explains the most important details to be considered for the a nalysis and further understanding of the results. 3.3.1. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR FTIR) 220.127.116.11 Principles This technique is based on the combination of two main principles: the first one is the detectio n of the vibrational energy emitted by the molecules when excited by an i nfrared (IR) light; and the second principle is based on enhancing the interaction between the molecules analyzed and the IR beam, therefore improving the detection sensitivity of th e method. The last principle, occurs by using a crystal with high refractive index that allows the IR beam to undergo multipl e internal bouncing when entering the crystal in a 45¡ angle of incidence, the beam creates an evanescent field at each point of c on tact with the sample's interface (Figure 14). The evanescent field created by the beam, approximately penetrates th e sample 1.66 # m in depth; distance measure d at 1000 cm 1 for a ZnSe crystal. This depth of penetration varies with the crystal used (ZnS e, Ge,
34 diamond, etc), angle of incidence, and refractive index of the sample. In this work all the experiments were performed using a ZnSe crystal as the ATR element. Bending Stretching Enhancement of the signal with an ATR crystal The evane scent field penetrates the sample 1.66 m approximately for a ZnSe Crystal (a) (b) Figure 14. ATR FTIR Spectroscopy Principles. (a) T wo main and general vibrational energies. (b) S chema tic representation of an ATR accesso ry, where the main element is a ZnSe crystal used to enhance the sensitivity of detection T he crystal is beveled in the two edges where the beam enters and exists in the crystal, allowing 12 points of contact with the sample. The equation explains the depth of penetration that the beam has inside the media under study, which depends on the wavelength of the energy, the angle of incidence on the crystal, and the refractive index of both the sample and crystal The enha ncement of the detection is of particular importance for this s tudy in which the concentration used is r elatively small (100 # M).
35 It is important to mention that ATR FTIR spectroscopy works in the Mid Infrared region of the electromagnetic spectrum ( Figur e 15), going from 400 through 4000 wavenumbers. Figure 15. Electromagnetic Spectrum. The Mid IR region is located between 400 and 4000 wavenumbers The range can be slightly modified by the ATR crystal used. For instance, in the case of ZnSe crys tal the range goes from 650 to 4000 wavenumbers. 18.104.22.168 Peptides and Amide Vibration Modes As explained in Chapter 2 section 2.2, amino acid residues formed the peptides. The functional group of amino acids is the amide group, C(=O)NH Therefore, cen tral vibrational energy characteristic of the peptides is the amide type [6, 62, 63] T he Mid IR region of the electromagnetic spectrum shows 5 regions correspond to the amide groups (Figure 16( a ) )
36 Figure 16. Amide Bands. Showing the mid IR range going from higher vibrational energy to lower: (a) All the amide bands encountered in the mid IR range, (b) Amide I, the most straightforward band holding the secondary structure information of the peptide, (c) NH stretching and bending energies are shown (NH stretching also is presen t in the Amides A and B). Amide II can be related to protein absorption to the crystal, (d) Amide III region. The example spectrum shown here corresponds to 5 days of incubation A§ 40/42 Peptid e
37 From the amide bands or regions shown in Figure 16 the mor e relevant are the amide I and amide II regions; r ang ing from 1600 to 1700 cm 1 and 1500 to 1600 cm 1 respectively. The amide I region, reveals the changes suffered by the backbone of the peptide without neglecting the side group's effects  Because, interactions of the type NH and NH 2 allow the peptide to anchor to the crystal's surface an d this interactions are detected in the amide II region, this interactions serve to investigate and understand the process of peptide's surface adsorption and desorption; simply by calculating the area under the amide II region. The amide I region is analy zed by its deconvolution in different peaks, each corresponding to an existing secondary structures of the peptide. Table 1 summarizes approximately the ranges in which the secondary structures of amyloid peptides are localized. The amide I band is sens itive to the secondary structure change in peptide and polypeptides  However, explaining the large splitting into two bands of antiparallel § strands structures, has become a challenge for researchers. In this sense, some light on this issue has been shown by the use of the transition dipole couplin g mechanism theory  This mechanism serves to explain how the A§ structures split in two bands, one positioned with center at 1630 cm 1 and the other center at 1690 cm 1 Other effect such as hydrogen bonding also modifies the amide I frequen cy of the polypeptide.
38 Table 1 Peak A ssignmen t o f A§ Secondary Structures The assigned peaks have been selected by gathering data from related literature [66, 67] and adapted from the current study observations during secondary s tructure study Structure Range [cm 1 ] Antiparallel § Strands Low frequency, Intermolecular High frequency, Intramolecular 1 597 16 30 16 80 1 710 Parallel § Sheets 162 8 1640 Unordered 1640 1651 Helix 1650 1657 § Turns Low frequency, Intermolecular High frequency, Intramolecular 1657 1675 1680 1700 22.214.171.124. Hydrogen Bonding The nature of hydrogen bonding has an evident effect on shifting the position of peaks for different stru ctures  as explained below. The peak absorbance for structures in the a mide I region correlates to the strength of the hydrogen bonding; this is true for most cases except in the case of the main frequency band of sheets. In descending order of hydrogen bonding, the secondary structure bands are the following: intramolecular antiparallel § strands extended chains (1610 1628 cm 1 ), intermolecular § sheets (1630 1640 cm 1 ), helices (1648 1658 cm 1 ), 3 10 helices ( 1660 1666 cm 1 ) and non hydrogen bonded amide groups in DMSO (1660 1665 cm 1 )  T his range is slightly varied from those shown in T able 1. H owever at the moment of
39 t he analysis both are considered. The peak frequency of the a mide I group is lowered by 20 to 25 cm 1 and is influenced by th e two possible hydrogen bonds connecting to the C=0 group. In the case of polypepti des, experiments indicate that a mide I main absorption band can vary by 30 cm 1 because of different hydrogen bond strength s among peptide residues. Alpha helices hydrogen b onding to solvent influence s absorption frequency by approximately 20 cm 1 ; solvated helices induce a 20 cm 1 lower absorbance maximum for a mide I. 126.96.36.199. Transition Dipole Coupling Mechanism (TDC) It is a resonant mechanism that causes secondary structures to influence amide I absorbance frequencies. Transition Dipole Coupling (T DC) is dependent on distance between amide groups in a peptide chain, and it is caused by resonance interaction due to oscillation of dipoles of such neighboring amide groups. TDC manifests itself in two ways on the FTIR spectra [70, 71] and is explained as follows. 188.8.131.52.1. Exciton T ransfer In an analogous way to fluorescence ( Frster ) resonance energy transfer (FRET), energy is absorbed and transmitted to nearby structures delocalizing excitation states over a length of 8 ; typically exciton transfer energy for helix has a constant of 0.5 ps (pico seconds).
40 184.108.40.206.2. Exciton S plitting Additionally TDC results in the splitting of bands since coupled oscillators result in different excitation energy levels in phase and out of phase This, r esult s in the splitting of the absorbent contribution on amide I for the case of antiparallel sheet structures with an approximately separation of 70 cm 1 220.127.116.11 General ATR FTIR Data Analysis Protocol Each spectrum collected with ATR FTIR spectroscopy was smoothed and baseline c orrected in Omnic (version 7.2a, Thermo Fisher Scientific). The spectroscopical regions of interest were the amide I (1600 1700 cm 1 ) and amide II region (1500 1600 cm 1 ). To the amide II region the area under the curve was simply calculated and this val ue represented the absorption or desorption of the peptide to or from the crystal respectively. The amide I region (1597 1710cm 1 ) was selected and saved in Comma Separated Values (CSV) format. The file was then analyzed with OriginPro (version 8 SR5, Or iginLab). In OriginPro the analysis was perfomed by peak fitting using the parameters shown in T able 2 This operation is known as spectrum's deconvolution by interactive least squares curve fitting technique, using a Gaussian distribution model.
41 Tabl e 2. Fitting Parameters and Boundary Conditions N¡ Meaning Value LB Value LB Param UB UB Value 0 offset 0 y0 1 center 1614.8125 1597 <= xc_1 <= 1630 1 width 8 1 <= w_1 <= 8 1 amplitude 0.0097 0 <= A_1 2 center 1628 1628 <= xc_2 <= 1640 2 width 8 1 <= w_2 <= 8 2 amplitude 0.01229 0 <= A_2 3 center 1640 1640 <= xc_3 <= 1651 3 width 6.11721 1 <= w_3 <= 8 3 amplitude 0.00483 0 <= A_3 4 center 1651.05348 1650 <= xc_4 <= 1657 4 width 6.35162 1 <= w_4 <= 8 4 amplitude 0.0033 0 <= A_4 5 c enter 1664.41119 1657 <= xc_5 <= 1675 5 width 6.93525 1 <= w_5 <= 8 5 amplitude 0.00224 0 <= A_5 6 center 1676.44412 1670 <= xc_6 <= 1690 6 width 7.39482 1 <= w_6 <= 8 6 amplitude 0.001 0 <= A_6 7 center 1685.63946 1680 <= xc_7 <= 1710 7 width 5 .59086 1 <= w_7 <= 8 7 amplitude 5.19289E 4 0 <= A_7 The resulting peaks after the curve fitting represent the contribution of each secondary structure, this quantity is calculated by determine the area under each fitted peak, and consequently the add ition of these peaks represent the amide I region. Each peak is assigned as a particular structure depending on the position. In this way it is possible to monitor the secondary structure percentage (as a normalized value) and the raw value. The latest v alue can be related to the kinetic of each particular secondary structure followed with time. An example of this fitting is shown in Figure 17
42 Figure 17 Sample's ATR FTIR Data Analysis It Depicts A 1 4 2 after 48 hours of incubation. The colored area s correspond to the estimated contribution of each specific structure to the total absorbance calculated by deconvolution of the amide I region. Also the amide II region is shown and it represents the surface protein adsorption I t is important to mentio n, that in previous studies (Jimenez, 2005  ) the investigation of the a mide I has been performed considering the band assignment in a more general fashion. Assuming only one frequency of antiparallel § sheets (called intermolecular interactions, 1610 1625) and considering the high frequency ra nge (1670 1700 cm 1 ) as § turns. In this new study to give to the antiparallel § sheets structure the importance they have and to understand the role they play during aggregation/dissolution of the peptide the bands assignment in the a mide I region has be en judiciously modified assigning
43 § turn to a range of 1670 1690cm 1 and a introducing a new band between 1680 1710cm 1 corresponding to high frequency antiparallel § s heets [73, 74] 18.104.22.168. Some Relevant Consideration for the ATR FTIR Analysis In order to understand the possible molecular interactions taking place in a solution and related to the absorption energies present at determined points of the spectra, it is important to consider the following important phenomena. 22.214.171.124.1. Protonation or D eprotonation For instance, at pH 7.4 the A§ 1 40 and A§ 1 42 are deprotonated (see Appendix A), having a net charge (theoretically) of 2 and 3 respectively. This unbalance of net charge contributes to the conformational change of the peptide molecules as well as their absorption energies. 126.96.36.199.2. Chemical Properties of Neighboring Groups For instance amide group allows for localizing a particular absorption band and differentiat ing it from a C=O originated from an aldeh y de or ketone group. In this work this is not relevant because it is known t hat only peptides would be showing this C=O bonding. 188.8.131.52.3. Bond Angles and Conformations Under the effect of Coulomb interactions, proteins modify their bonding angles; this affects the three dimensional structure of the ir backbone originating
44 changes (conform ational changes). This combination is responsible for the amide I band sensitivity to secondary structure. 184.108.40.206.4. Hydrogen Bonding Another important interaction encounter ed in peptides is the hydrogen bonding; this type of interaction stabilizes the peptide mole cules in their lower energy state depending on the media conditions The strength of hydrogen bonding can be directly observed as mentioned earlier in section 220.127.116.11 18.104.22.168.5. Conformational Freedom A very important aspect of FTIR spectroscopy is the fact that a s structures are more flexible, the band that they represent in the vibrational spectrum is wider. Heterogeneity of structures caused by conformational freedom results in broader spectrum bands defining structures O n the other hand, more rigid structures have less conformational freedom and result in tighter spectrum bands. When structures interact through binding, conformational freedom is usually reduced; this is also the case for molecules binding peptides and proteins and for peptide peptide binding, r esulting in the reduction of bandwidth by a factor of two  The incremented rigidity and reduced conformational freedom sometimes in creases rigidity in distant parts of large molecules such as proteins as well.
45 22.214.171.124.6. Overlap of C onformational C hanges in the S pectrum The fact that different structures overlap in the spectrum makes analysis of signals more complex. However, spectra can be subtracted, with the limitation that the infrared difference spectrum reveals only the net change of secondary structure. In this work, by amide I deconvolution it is possible to distinguish the structures during overlapping of conformational changes. 126.96.36.199.7. Rig id Domain M ovement Movements of relative large structures are not dete cted in near infrared spectra. Hinge regions of proteins are an interesting example in which the larger rigid domains are not detected while the hinge flexible regio n is prevalent in th e spectra. An example of two rigid domains moving about a flexi ble domain is shown in Figure 18 ( black line ) I n this case, the detection of the flexible domain would be prevalent established by compari ng it to the larger and more rigid domains attached t o it T he detection would be limited to the conformation of the relative orientation of neighboring amide group s in the flexible domain. However t he re is increased sensitivity in the detection of conformational changes where the backbone portion of a mol ecule is intimately linked to flexible domains; f or example, if an helix shorten s there are changes not only in the amide modes of the unwinding backbone portion, but also in others amides related to the remaining portion of the helix.
46 188.8.131.52.8. Subtle Changes within Secondary Structures Finally, subtle changes within secondary structures such as twists in § sheet, bending of an helix or hydrogen bonding to backbone carbons, are detectable in the amide I of the mid infrared spectrum. Figure 18 IR Spec trum Analysis and Rigid Domain Movement. Adapted from Barth 2007  184.108.40.206 General Experimental Methodology The experiments were performed with a 6700 Nicolet spectrometer (Thermo Fisher Scientific, US). Two different accessories were used: an ATR trough plate for the study of monomeric solutions (with more flexibility to evaporate and redissolve the peptide's in different solvents), and a closed ATR flow cell accessory for the peptide solution in HFIP, and t he oligomeric and fibrillar solutions (hermetically closed to avoid evaporation during long incubation times, and with temperature controller).
47 The IR beam was focused at normal incidence onto one of the 45¡ beveled faces of the ATR zinc selenide (ZnSe, n s = 2.4) trapezoidal crystal, where the IR beam undergoes multiple internal reflections resulting in signal enhancement. A crystal made of ZnSe was the preferable used because of its low reflection losses in the infrared  wide infrared range (between 20,000 and 650 cm 1 ), compatibility to aqueous solutions, and chemical inertness ( Appendix B ) After each measurement, the flow cell was dismantled and thoroughly cleaned with isoprop yl and Millipore water; in addition the crystal was exposed 5 minutes to UV to eliminate organics, and rinsed again with Millipore water. All the flow cell parts w ere finally dried with an ultrapure nitrogen stream and re assembled. 220.127.116.11 General Data Analysis For the case s of long incubation time s (i.e., 24h, 48h, or longer) and with several spectra collected by hour (i.e., 6 spectra per hour) high vari ability is recorded from spectrum to spe ctrum For these cases, set of spectra with time ranges of 4 hours were average d a nd the vari ance among them was calculated. To calculate the average absorbance (Eq.1) for a time frame (for example 4 to 8 hours ) all curv es under that timeframe are added and averaged by the number of curves recorded during that time; this yields an average spectrum:
48 Average Absorbance(w) = Absorbance ( t ) t 1 t n n (Eq. 1) The software calculates then the variance for the curves during that time frame as Eq.2 shows: Variance(w) = Absorbance(t) Average Absorbance( )2 t 1 t n # n (Eq. 2) Finally, the number to calculate the Relative Spectral Variance (Eq.3), the variances (Eq.2) for each wavelength in the region are integrated to calculate the area under the variance curve, divided by the average absor bance (Eq.1), and multiplied by 100. Relative Spectral Variance = Variance(w).dw w 1 w n Average Absorbance(w).dw w 1 w n 100 (Eq. 3) This relative spectral variance or %Variance is a good indicator of stability of the processes undergone by A peptides in their stabilization at different conformations; typical values for stable Amide I and Amide II regions are around 10%; as shown in Figure 30.
49 3 4 Reagents Table 3 lists all reagents used in this work. Table 3. Reagents Used in this Work Reagent Manufacturer Description 1,1,1,3,3,3 Hexafluro 2 Propano l (p a : >99%), HFIP Fisher Sci. Very volatile transparent liquid Methyl Sulfoxide Anhydrous (p:>99%), DMSO or Me 2 SO Acros Viscous transparent liquid Tris (Crystallized) p:>99%) Fisher Sci. BP152 500 White powder Phosphate buffered saline, PBS Sigma Aldr ich, P3813 White powder Hydrochloric Acid, HCl Acros 124630010 Nitrogen Airgas NI UHP300, Ultrapure gas
50 CHAPTER 4 AGGREGATION PATHWAYS OF AMYLOID BETA PEPTIDE Amyloid beta (A§) peptides and proteins in general modify their molecular con figuration ( i.e., secondary structures) influenced by the media conditions in order to minimize their overall energetic state [78, 79] T his chapter will analyze and discuss how media conditions affect the secondary structure of A§ peptides Th e conditions evaluated here have been used to identify features corresponding to each A§ species during aggregation. The species studied are: monomers, which are the single molecules of A§ peptide characterized by their hydrophobic character (A§ 1 42 in pa rticular) ; oligomers which are found during the nucleation or seeding state and that are also known as the intermediate species during fibrill ization  ; and finally the fibrils the main components of neuritic plaques in A lzheimer's disease brain tissue. In later chapters the findings from this chapter will be used as guidelines to elucidate changes in aggregation and effects o f antibodies on the A§ peptide species. 4.1. Protocols a nd Methods Each peptide in this study, A§ 1 40 A§ 1 42 and their equimolar mixture A§ 40/42 were prepared following a common protocol explained below.
51 4.1.1. Peptide's Homogenization Amyloid Bet a (A§) peptides were purchased from American Peptide INC. (Sunnyvale, CA). Table 4 below shows the general specifications of A§ peptides used in this study. The peptides were homogenized and aliquoted in HFIP as described by Stine et al. 2003  Briefly, HFIP was injected into the peptide's flask using a gas tight Hamilton glass sy ringe with a Teflon plunger, at a peptide's concentration of 1 mM. Table 4 General Specifications o f Amyloid Beta Peptides Used i n t his Study Characteristic A§ 1 40 (Amyloid Beta 1 40) A§ 1 42 (Amyloid Beta 1 42) Sequence Asp Ala Glu Phe Arg His Asp Se r Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Alabh Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala MW (a.m.u.) 4,329.9 4,514.1 Formula C 194 H 295 N 53 O 58 S 1 C 199 H 307 N 53 O 59 S 1 Purity (HPLC Analysis) 97.2% 87.2% Solubility 1mg/ml in water 1 mg/ml in 0.05M Tris buffer Counter ion Trifluoroacetate T rifluoroacetate American Peptide Cat. No. 62 0 78 62 0 80
52 T he peptide immediately dissolves b ecaus e of the high polarity of the HFIP, that help s to break down the peptide's pre existing tertiary structures  After 10 minutes in HFIP at 25¡C, the peptide was aliquoted in sterile microcentrifuge tubes and left overnight in a lamin ar flow cabinet (Class 100) allowing the HFIP to evaporat e. This protocol created a clear peptide film in the bottom of each vial. Each film was also exposed to 30 in Hg of vacuum for 8 hours to eliminate traces of HFIP and humidity. Each vial was close d with nitrogen (gas) and sealed with Parafilm M. Finally all the vials were stored at 20¡C in a jar containing molecular sieves and closed with nitrogen to maintain a low humidity environment. 4.1.2. A§ Monomers u nder Different Media Conditions Figur e 19 illustrates the protocol followed t o understand and identify the initial molecular conformation and morphology of A§ peptide s under media conditions that at different incubation times promote defined monomers, oligomers, and fibrils. A§ 1 40 A§ 1 42 a nd A§ 40/42 peptide s were exposed to the conditions explained below and monitored until stability was reach ed. That is, the peptides were monitored 30 minutes at conditions (a) (c); and for 1 hour at conditions (d) and (e).
53 Figure 19 explains the detai ls of this study as follows: Figure 19(a): e ach peptide was dissolve d in HFIP at 1mM concentration, as described in the pre treatment step (section 4.1.1 ), and allowed to equilibrate for 30 minut es in a gas tight ATR flow cell. Continuous spectra were coll ected during this period. Figure 19(b): Once the HFIP evaporates a film is formed, it was determined if monomeric species were maintained. Thus, i n a second experiment each peptide dissolved in HFIP as described in Figure 19 (a) was deposited on a n open ATR trough plate. Then, the HFIP was removed by evaporation assisted by a dry nitrogen (gas) stream. Continuous spectra were collected for each peptide film during 30 minutes. In addition a 5 # l drop of this solution was deposited on freshly cleaved mic a and the film was later imaged with atomic force microscopy (AFM) to relate peptide's structure to its morphology. Figure 19(c): When oligomers or fibrils are prepared, the peptide's film is always re dissolved in DMSO to 5 mM concentration. Figure 19( d): The above solution (c) is taken to 100 # M (0.1 mM) using 20 mM Tris buffer solution (2.42g Tris Base pH 7.4 w/HCl, prepared in 1L of solution) to prepared oligomers. Figure 19(e): To prepare fibrils, solution (c) is diluted to 100 # M with 0.01 M phos phate buffered saline solution. AFM was used to analyze the morphology of A§ 1 40 A§ 1 42 and A§ 40/4 2 ,as prepared in Figures 19 (d) and 19 (e)
54 Figure 19 Systematic Study of A§ Monomers u nder Different Media Conditions. (a) A§ peptide is dissolved in HFI P and monitored with ATR FTIR spectroscopy; (b) the HFIP is evaporated from the peptides' solution leaving a film; (c) the peptide's film is redissolved with DMSO; (d) The peptide dissolved in DMSO is diluted to 100 # M with TR IS solution to obtain stable ol igomers ; (e) The peptide dissolved in DMSO is diluted to 100 # M with 0.01M PBS solution to obtain fibrils 4.1.3. Oligomers Stability a nd t heir Characteristic Secondary Structure T he morphology and secondary structure dynamics of the oligomeric intermediat e species of the A§ peptides was characterized The oligomers were prepared follow ing a method established in a preliminary study  Briefl y, the peptide film was dissolved in DMSO (5 mM) and diluted to 100 # M (0.1 mM) with a 20 mM Tris buffer solution (2.42 g /L Tris Base at pH 7.4 adjusted with HCl).
55 The solution prepared to form oligomers was split in two portions, one portion was inject ed in an ATR flow cell to follow in situ the evolution in secondary structures at 25 ¡ C; the second portion of the oligomeric solution was aliquoted and incubated at the same temperature (25 ¡C); each aliquot was retrieved at different times during 48 hours for later A FM analysis 4.1.4. A§ Species and Kinetics of Fibril Formation The first step to promote fibril formation was to re dissolve the peptide film with DMSO until it reached concentration of 5 mM, the dissolution process was assisted by 5 minutes o f sonication. This was followed by the addition of PBS (0.01 M, pH 7.4), diluting the peptide solution to 100 # M. After 1 minute of sonication the solution was split in two portions, the first one was injected in the ATR flow cell for spectroscopy analysi s ( incubated at 25¡C) and the second portion was aliquoted for AFM analysis. Aliquots retrieved at different time points. 4.1.5 Peptide Secondary Structure Study: Attenuated Total Reflection Fourier Transform Infrared (ATR FTIR) Spectroscopy Once the acc essory was assembled and prior to injecting the peptide solution, a portion of fresh peptide free incubation media solution was flushed through the cell in each new experiment. Spectra and background of this media in contact with the crystal were obtained. The peptide free solution background was used to eliminate the solvent contribution in the peptides solutions. The experiments were run for 30 minutes for monomer characterization, and for 48
56 hours for oligomeric and fibril formation. Oligomers and fibril s solutions were set up to collect a new spectrum every 10 minutes. 4.2. Results, Analysis a nd Discussion This section shows and discuses the particular characteristics of the A§ species, beginning with the monomeric species of the two isoforms A§ peptid es (A§ 1 40 and A§ 1 42 ) and their equimolar mixture; moving on to the oligomers and fibrils characterization for each peptide. 4.2.1. Species of A§ Peptides During peptide aggregation different species can be found and define d. H owever referred here as monomer, oligomers, and fibrils as species of the A§ peptide. 18.104.22.168. A§ Peptide Monomers This first study was designed to determine the characteristic content of secondary structure present in the monomeric specie of A§ peptides.
57 Figure 2 0 Ami de I and II Regions of A§ 1 40 Monomers i n HFIP. The spectra displayed here were collected for A§ 1 40 during 30 minutes of incubation in HFIP at 1mM. T he amide I region shows that the antiparallel § sheet are the dominant structures within the monomeric spe cies T he lack of absorbance in the amide II region is evidence of the absence of protein adsorption, hence all the monomeric species are in s olution A§ 1 40 peptide dissolved in HFIP was injected in a hermetic ATR flow cell accessory and monitor over 30 minutes with ATR FTIR spectrometry. Figure 21 shows that monomers are unequivocally detected in HFIP confirmed by the lack of protein adsorption revealed by the amide II region. After deconvolution of the amide I region for each time shown in Figure 20 wi th clear center in the antiparallel § sheet band (1624 cm 1 ), it is possible to obtain the information plotted in Figures 21 (a) and 21 (b) show ing the evolution of secondary structures during 30 minutes of incubation.
5 8 Figure 2 1 Secondary Structures of A§ 1 40 Monomers i n HFIP. (a) Secondary structures evolution of the monomers in solution. (b) Secondary structure content c hange for the monomeric species. E ach point in this plot was calculated by integrating the area under the curve corresponding to t he different specific IR bands defining each secondary structure (Shown in Table 1) at the times shown in (a). O bserve in that after 10 minutes the structures start reaching equilibrium and between 27 and 30 minutes stabilize Figure 21 (a) shows the incre ase of antiparallel and parallel § sheets secondary structures during 30 minutes of incubation in HFIP, this figure suggests that the monomers are constantly evolving but they do not aggregate. However the dominant configuration of the monomeric species is the antiparallel § sheet type, Figure 21(b).
59 Figure 2 2 Secondary Structure Content o f A§ 1 40 Peptide Monomeric Film. (a) The shift of a mide I and the appearance of amide II show a significant difference when protein adsorption occurs. (b) Comparis on of secondary structure content change before (light color bars) and after (solid bars) HFIP has evaporated Main differences are observed, as the switch from antiparallel § sheet structures to parallel § sheets with a larger influence of helix structures Continuing with the study of A§ 1 40 peptide, once the HFIP solvent evaporates, it leaves a film with characteristic secondary structures, shown in Figure 22. The shift of the amide I region in this figure confirms that when the peptide s absorb to the crystal, parallel § sheet structures are highly involved in this process. Protein adsorption (film formation) is verified by analyzing the amide II region (Figure 22(a)). Figure 22(b) compares the content of secondary structures of the pe ptide before and after the HFIP is evaporated (film formation). The monomeric film is characterized by the dominant content of parallel § sheets and helix structures, with decrease of antiparallel § sheet structures, this confirms that the peptide is
60 not in solution anymore. It is important to note that the antiparallel § sheet structure was significantly reduced as the film wa s formed. The results for A§ 1 40 shows that 30 minutes (Figure 21 ( b ) ) is a reasonable incubation time in HFIP for the p eptide (also found for the cases of A§ 1 42 and A§ 40/42 ) to have its secondary str ucture in equilibrium. The two peptide s and their 1:1 co mbination under study (A§ 1 40 A§ 1 42 and A§ 40/42 ) were incubated for 30 minutes in HFIP as an initial practice for all experiments involving these peptides Comparison of the monomeric species in HFIP for A§ 1 40 A§ 1 42 and their equimolar combina tion is shown in Figure 23. It is important to no te that A§ 1 42 peptide shows an antiparallel § sheet to helix ratio close to 1 (0.85) in its monomeric state. In previous work (Jimenez, 2005  ) helix structures were found to play an important role during fibril formation of A§ 1 42 peptide. Hence it is not surprising to find antiparallel § sheet structures competing with helix structures in its monomeric state. Prion proteins capable of am yloid formation exhibit similar characteristics when forming fibrils; with similar roles played by antiparallel and helix structures This is evidenced in a NMR study performed to the prion protein domain PrP(121 231) This domain contains most of the point mutation sites known to infectious prion proteins that form two stranded antiparallel § sheet and three helices 
61 Figure 2 3 Secondary Structure o f Amyloid Peptides Monomers i n HFIP. (a) A mi de I region spectra corresponding to 30 minutes of incubation in HFIP for A§ 1 40 A§ 1 42 and A§ 40/42 (b) Secondary structure content corresponding to the monomers of A§ 1 40 A§ 1 42 and A§ 40/42 in HFIP T he values shown in this plot were obtained by ave raging the spectra shown in (a) and the error bars are the standard deviations Notice that, a ntiparallel § sheet (blue) is the dominant structure in A§ 1 40 and A§ 40/42 monomeric species W hen in the case of A§ 1 42 both antiparallel and helix (green) st ructures dominate the monomeric species (c) Schematic approximation of two A§ monomer s mainly show ing antiparallel § sheet and small regions of helix structures (image representing the dimer of transthyretin, shown w ith permission of Landry, 1996) The res ults found in this study, reveale d the importance of antiparallel structures to stabilize folded monomers in solution Note that one would find parallel § sheet structures as well as part of the overall structure. However, the dominant secondary structu re in all 3 cases is the antiparallel § sheet. This observation, is in agreement with the name given to the Alzheimer's disease amyloid peptide, that is amyloid "Beta" peptide, due to the high content in § sheet structures in its monomeric folded state [83, 84] Figure 23(c) is an schematic
62 approximation of two amyloid forming monomer s, with permission of Landry, 1996, this picture represents the antiparallel beta sheet in e quilibrium with small domain of helix structures. This model is in ag reement with our results. The study performed to A§ 1 40 of HFIP evaporation (Figure 22) and film formation, was repeated for A§ 1 42 and the peptide equimolar mixture A§ 40/42 the results comparing the secondary structure content for each case are shown in Figure 24. Figure 2 4 Change i n Content of Secondary Structure for Amyloid Peptides d uring Monomeric Film Formation. (a) Amide I and II regions for A§ 1 40 A§ 1 42 and A§ 40/42 (b) C omparison of secondary structure content before and after the HFIP evaporates A§ peptides dissolved in HFIP (light color bars) versus their films (solid bars)
63 In general, Figure 24(a) shows that when a film is formed, there is a significant shift in the secondary structures (amide I), and a prominent absorption in th e amide II region. Figure 24(b) shows that the deposition is mainly dominated by parallel § sheet structures, while the monomeric state in solution is dominated by antiparallel § sheet structures. Figure 25(a) shows the characteristic morphology of A§ 1 40 and A§ 1 42 monomeric films; which were obtained by depositing the peptide dissolved in HFIP on freshly cleaved mica as explained in 22.214.171.124. Figure 25(b) shows the height distribution analysis for the films from Figure 25(a) corresponding to A§ 1 40 and A§ 1 42 peptides. A § 1 42 peptide formed more dense film s than A§ 1 40 this can be explained by the hydrophobic nature of A§ 1 42 peptide which reduces steric interaction promoting a denser film. Two other studies (Harper in 1997, and Shao in 1999) are in agr eement with the hydrophobic nature and ability to pack conferred to A § 1 42 peptide [85, 86] In addition to these studies with HFIP, Figure 26 shows the results after the pep tide films are redissolved with DMSO, since this is the protocol followed prior to oligomeric or fibrillar preparations. As Figure 26 shows the action of DMSO is to redissolve the peptide and stabilize the secondary structure content. However, when oligom eric and fibrillar solutions are prepared, sonication is an important step to ensure the redissolution of the film. Notice that the film after DMSO addition shown in Figure 26 was not sonicated.
64 Figure 2 5 Morphology o f Monomeric Peptides Film. (a) AFM image of A§ 1 40 (top) and A§ 1 42 (bottom) peptide monomers on mica (scan size: 0.5x0.5 # m x y); (b) Gaussian distribution of the heights found in each case (topography), please observe that the A§ 1 42 film has a less dispersed distribution (red line, centered at 1nm) In general, this study has proven that when peptides are pre treated with HFIP, their conformations in solution are reduced to their monomeric form. This results in a homogeneous (monomeric) film when the HFIP is allowed to evaporate. This is the rationale for pre treating the peptides with HFIP as a homogenizing point prior to prepare any peptide solution.
65 Figure 2 6 Spectroscopy Analysis for A§ Peptide Monomers in DMSO. (a) Amide I and II regions for A§ 1 40 A§ 1 42 and A§ 40/42 the amide I region shows that the peptides dissolved in DMSO are structurally stable (no shift), the absorption observed in the amide II region shows that there is peptide remaining as a film (b) Secondary structure content by peptides, comparison between the dry peptide film (light color bars) and the peptide redissolved in DMSO solid bars) Note that in all three cases antiparallel § sheet structures increased in DMSO 4.2.1 2. Oligomeric Species of the A§ Peptides Oligomers are the intermediat e species in the formation of fibrils  Understanding the stability and secondary structure characteristics of these species is key to explain fibril formation and disruption (which will be discussed in chapters 5 and 6 in detail). This study also characterized the kinetics of secondary structures responsible for the forma tion and stability of oligomeric species of A§ peptides. The study is presented by peptide type.
66 126.96.36.199.1. Oligomers of A§ 1 40 Peptide Two stages of the oligomerization process were monitored in this study. First, the peptide dissolution and hydration taking place in the initial 60 minutes of incubation (Figure 27); and second, the formation and stabilizatio n of the oligomeric species during 48 hour of incubation (Figure 28). Molecular rearrangement is observed in the first 60 minutes of incubation (Figure 27). During the first 10 minutes antiparallel § sheets and helix are the dominant structures; at this point monomers are mainly in solution. After 10 minutes of incubation the structures shift with increase in parallel § sheets structures, this is the moment that marks the beginning of the oligomerization process (Figure 27(c)). Figure 28 (a) shows the spectra corresponding to 48 hour of total incubation, represented by t he multicolored lines. Figure 28(b) shows the same 48 hour of total incubation in grey but representing time ranges of 4 hours in red. This visualization technique provides a quick way to observe progressive spectral changes, and assess variance visually ; for the whole amide spectra, or for the amides I and II independently.
67 Figure 2 7 S pectroscopy Study of A§ 1 40 Peptide during the First 60 Minutes of Incubation i n DMSO+TRIS Solution a t 25¡C. (a) Amide I and II regions, spectra from 0 to 60 mi nutes, amide II shows protein adsorption, on amide I region shifting can be observed showing secondary structure instability. (b) Comparison of the total integrated absorbance of amide I vs. amide II from 0 to 60 minutes. (c) Reorganization of the secondar y structure that assists the process of aggregation into stable oligomers. (d) Evolution of s econdary structure content of A§ 1 40 the first 60 minutes of incubation
68 Figure 2 8 Kinetics o f A§ 1 40 Oligomers i n DMSO+TRIS Solution at 25¡C. (a) Spectra of the Amide I and II regions for A§ 1 40 peptide during 48 hours of incubation at 25¡C. (b) Sets of spectra selected each 4 hours (red spectra), during 0 to 48 hour of incubation Figure 29(a) shows the average spectra calculated from the time frames show n in Figure 28(b). The average spectra were calculated using (Eq.1) from Section 188.8.131.52. Figure 29(b) shows the %Variance calculated with (Eq.3) also from Section 184.108.40.206.
69 Figure 29 A§ 1 40 Average Spectra and Variance Percentage. (a) Spectra ob tained from averaging the 4 hours range as shown in (Eq.1); (b) %Variance for each time point corresponding to Amide I and II separately and individually, observe that when values are under 30% of variance (Eq.3) the calculation is considered to be statist ically acceptable, more variability than this values reflects instability of the sample in that range of time The analysis of the amide I and II regions for A§ 1 40 peptide incubated in DMSO+TRIS solution is shown in Figure 30(a), it represents the behavi or of A§ 1 40 during the formation of stable oligomeric species during 48 hours. (b) Shows the changes exhibited by the amide I and II regions each 4 hour time frame (averaged spectra) with overall activity in secondary structure (amide I) and adsorption (amide II). As the deconvolution of amide I spectra reveals in (c), all secondary structures increase for every structure in absolute absorbance values. However, when these values are normalized Figure 30(d) reveals that the
70 overall content of parallel st ructures increases while antiparallel structures decrease during the formation and stabilization of A§ 1 40 oligomers. Figure 3 0 Analysis o f t he Amide I a nd II f or A§ 1 40 Oligomers i n DMSO+TRIS Solution. (a) A verage d spectra using ( Eq.1) from Sec tion 220.127.116.11 (b) Change of integrated absorbance under the amide I and II during the incubation period. (c) Secondary structure kinetics of A§ 1 40 in DMSO+TRIS during 48 hour of incubation. (d) Change in secondary structure content for A§ 1 40 in DMSO+TR IS during 48 hour of incubation
71 Figure 3 1 Morphology of A§ 1 40 Oligomers over 48 Hours o f Incubation. (a) 1x1 # m x y scans of A§ 1 40 from 0.5h to 48h of incubation. (b) Average diameter of the oligomers encountered in solution at different incubatio n times Figure 31(a) shows the morphology of A§ 1 40 oligomers through time, these images have a scan size of 1x1 m x y. Results in Figure 31(b) show the average diameter of the oligomeric species shown in Figure 31(a), and it can be observed that these species maintained their structure and shape over 48 hours incubation in DMSO+Tris (2:98 volume to volume) at 25¡C. 18.104.22.168.2. Oligomers of A§ 1 42 Peptide As in the previous study (22.214.171.124.1), this section investigates the stability of A§ 1 42 oligomeri c species using two timeframes: during the first hour (Figure 26) of incubation (in DMSO and TRIS solution at 25¡C, Figure 32) and during 48 hour to monitor stability.
72 Figure 3 2 Change o f Secondary Structures o f A§ 1 42 Dissolved i n DMSO after Di luting with TRIS Buffer Solution t o 100 # M. (a) Spectra of the Amide I and II regions from 0 to 60 minutes, Amide II shows protein adsorption, on Amide I region shifting can be observed showing instabilities; (b) Comparison between Amide I and Amide II int egrated absorbance from 0 to 60 minutes; (c) Reorganization of the secondary structure that trigger a process of aggregation into oligomers is shown; (d) Change of secondary structure content of A§ 1 42 in the first 60 minutes of incubation Figure 32 sho ws progression of spectra collected during the first hour of A§ 1 42 oligomeric species production in DMSO+TRIS buffer at 25¡C. It is important to indicate that the predominant species are antiparallel sheet and
73 helix as it can be seen in 32(d); this b ehavior was also observed in the case studied in section 126.96.36.199 for A§ 1 42 monomers in HFIP (Figure 23(b)). For the case of 48 hours study, the spectra of the A§ 1 42 oligomers (Figure 33(a)) showed some instability as evidenced by the Figure 33(b) and Fig ure 34(b), not reaching and stable spectra trajectory and reflected on the variability from time to time. However, this was not an impediment for the oligomeric structure to form and be stable (Figure 35). The explanation for this movement among the spectr a might be due to the fact that these oligomers are in a reactive state, therefore the structures and even the adsorption varied from spectrum to spectrum. Even as the stability of A§ 1 42 oligomers lags behind that of A§ 1 40 it is evident that at all tim es helix and antiparallel sheet are the dominant structures contained and stabilizing the oligomeric species (Figures 34(c) and (d)) of A§ 1 42 peptide. Figures 34(c) and (d) are the result of deconvoluting the average spectra shown in Figure 34(a). Figure 3 3 Amide I and II of A§ 1 42 Oligomers during Oligomerization (a) Spectra of the amide I and II regions for A§ 1 42 in DMSO+PBS during 48 hours of incubation at 25¡C. (b) Spectra selected in 4 hours ranges (red spectra)
74 Figure 3 4 Ki netics o f A§ 1 42 Oligomers i n DMSO+TRIS Solution. (a ) Average spectra obtained from the 4 hours range shown in Figure 33 (b). (d) Variances calculated from 33 (b) for the average spectra. (c) Secondary evolution of A§ 1 42 in DMSO+TRIS during 48 hour of incu bation. (d) Change of secondary structure content for A§ 1 42 in DMSO+TRIS during 48 hour of incubation
75 Figure 3 5 Morphology o f A§ 1 42 Oligomers. (a) 1x1 # m x y scans of A§ 1 42 from 0.5h 48h of incubation. (b) Average diameter of the oligomers encountered in solution at different incubation times, even when at 12 hour and 24 fibrils are shown, they are rarely found, this fibrils have diameters in the range of t he oligomeric species (3510nm) The progression of AFM scans of A§ 1 42 oligomeric s pecies in Figure 35 (a), and the analysis of particle sizes in (b) show changes in size of oligomers that stabilizes after 24 hours. Even though fibrils were observed in AFM experiments, these were scarce and did not play an important role in the morpholo gy of aggregates, which were dominated by A§ 1 42 oligomeric species.
76 188.8.131.52.3. Oligomers of A§ 40/42 Peptide The study in this section mirrors the two preceding studies but tests a combination of both peptide A§ 1 40 and A§ 1 42 at a molar ratio of 1:1. Similarly to A§ 1 40 and A§ 1 42 oligomeric study, ATR FTIR spectroscopy and AFM analysis were performed for 1 hour and 48 hour time scopes. Figure 36. Change o f Secondary Structures o f A§ 40/42 Incubated i n DMSO a nd TRIS a t 25¡C. (a) Spectra of the Amide I and II regions from 0 to 60 minutes, Amide II shows protein adsorption, on Amide I region shifting can be observed noting instabilities; (b) Reorganization of the secondary structure that trigger the process of oligomers formation; (c) Change in secondary structure present in the A§ 40/42 peptides during the first 60 minutes of incubation in DMSO+TRIS solution
77 Figure 36(a) shows the spectra (amide I and II regions) corresponding to the initial 60 minutes of incubation, (b) shows t he decreased and stabilization of the evolution for antiparallel structures and the rest, respectively. Figure 36(c) shows that antiparallel § sheet structures are dominant in this first hour of incubation. Figure 37. Kinetics o f A§ 40/42 Oligomers i n DMSO+TRIS Solution. (a) Spectra of the Amide I and II regions for A§ 40/42 peptide spectra during 48 hours of incubation at 25¡C; (b) Sets of spectra selected each 4 hours (red spectra), du ring 0 to 48 hour of incubation Figure 37(a) shows the spectra c ollected from 0 to 48 hours of incubation, representing only the amide I and II regions of the spectrum, (b) explains schematically the process of time sets selection. The stability spectral analysis for A§ 40/42 oligomers shown in Figure 38 (b) and corresp onding to the average spectra in (a), shows that after 12 hours of incubation the oligomers reach stability. This can also be visually observed in figure 37(b).
78 Figure 38. Kinetics a nd Change o f Secondary Structure Content o f A§ 40./42 Oligomers Prepared i n DMSO+TRIS Solution. (a) Spectra obtained from averaging the 4 hours range as shown in Figure 37(b); (b) Percentage of variance calculated for each average spectra in (a) from the data shown in Figure 37(b). (c) Secondary structure evolution of A§ 40/42 in DMSO+TRIS during 48 hour of incubation; (d) Change in secondary structure content for A§ 40/42 in DMSO+TR IS during 48 hour of incubation During the first 60 minutes of the stability study, the oligomeric equimolar mixture of A§ 40./42 seems to reach relative stable conformations (Figure 36(c)), after this short initial time, the peptide mixture shows and increase in antiparallel § sheet dominating over the second structure increasing, the parallel § sheets.
79 AFM analysis was performed to this peptide combination, finding no evidence of adsorption to the mica at any time, suggesting that the oligomers in the ATR crystal are in a dynamic equilibrium of adsorption and desorption, explaining the adsorption shown in Figure 39, and at the same time t he absence in the mica surface (not shown). Figure 39. Kinetics o f A§ Oligomeric Species Adsorption. The values shown in this plot are the integrated areas under each amide II region corresponding to each different time, and as explained in section 3. 3.1.2 it corresponds to the peptide adsorption on the ATR crystal A comparison of the first 48 hours of oligomeric adsorption values from the Amide II region in Figure 39 reveals that A§ 1 40 has the most adsorption tendency to the FTIR crystal while A§ 1 42 has a comparable adsorption to the peptides mixture; still A§ 40./42 has a value in between the individual peptides.
80 4.2. 1 3. Fibrillization of A§ Peptides at 25¡C The main focus of this work is to understand and obtain the necessary knowledge to inte rpret the mechanisms of peptide fibrillization, to then be able to target a dissolution process (this will be discuss in Chapters 5 and 6). In the amyloid hypothesis, it is stated that when an imbalance in the amyloid peptide production and clearance equil ibrium is altered, this drives to A§ plaque formation [88, 89] initiating a cascade of effects that ultimately lead to neuronal death, hence understanding t he process of peptide aggregation at the molecular lever is important to treat the disease. This study parallels the previous sections for monomers and oligomers of A§ 40. A§ 42 and the equimolar mixture of A§ 40./42 In this set of experiments, the pepti de film dissolved in DMSO was diluted to 100 # M with PBS and incubated at 25¡C, protocol that yields to fibril formation  184.108.40.206.1 A§ 1 40 Fibrils A§ 1 40 fibrils were studied for both time scopes of 60 minutes and up to 96 hours, the samples were analyzed with ATR FTIR spectroscopy and AFM. The first 60 minutes of A§ 1 40 fibril formation of process have very little stability as it is evidenced by the changes in spectra shown in Figure 40(a), the integrated absorbance of the Amide I and Amide II in (b), and absolute and relative structure con tent values from (c), and (d).
81 Figure 40. Change o f Secondary Structures o f A§ 1 40 Prepared i n DMSO+PBS Solution a nd Incubated a t 25¡C. (a) Spectra of the Amide I and II regions from 0 to 60 minutes, Amide II shows protein adsorption, on Amide I region shifting can be observed showing the molecular rearranging of the molecules; (b) Comparison between Amide I and Amide II integrated absorbance from 0 to 60 minutes; (c) Reorganization of the secondary structure that trigger the process of peptide ag gregation; (d) Change of secondary structure content of A§ 1 40 in the first 60 mi nutes of incubation
82 Figure 41. Fibrillization of A§ 1 40 i n DMSO+PBS. The fibril are formed at 25¡C and monitored during 96 hours. (a) Amide I (1700 1600 cm 1 ) and amide II (1600 1500 cm 1 ) spectra. (b) Amide I region, spectra selection shown up to 48 hours; this study is performed to evaluate the stability and struct ural change of the peptides. (c) Average spectra calculated from the selected times in (b) The stability analysis presented in Figure 41 allows observing the limited stability of the fibrils formed by A§ 1 40 in PBS until more than 80 hours as average spectra show in (c); time frame not shown in (b).
83 Figure 42. Fibrillization o f A § 1 40 a nd Secondary Structures Change. (a) Morphological change monitored with AFM. (b) Most relevant secondary structures kinetics. (c) Most relevant secondary structure content Figure 42 (a) shows the progression of fibril formation for up to 240 hours for the AFM scans, and 96 hours for FTIR amide I secondary structure content. The analysis shows that fibrils are stable at more than 80 hours and the predominant structures are antiparallel sheet and helix for formed fibrils. 220.127.116.11.2. A§ 1 42 Fibri ls A§ 42 fibrils were studied under FTIR ATR and AFM analysis for both time scopes of 60 minutes and 240 hours, the analyzed data is presented in this section.
84 Figure 43. Change o f Secondary Structures o f A§ 1 42 Prepared i n DMSO+PBS Buffer Soluti on t o 100 # M a nd I ncubated a t 25¡C. (a) Spectra of the Amide I and II regions from 0 to 60 minutes, Amide II shows protein adsorption, on Amide I region shifting can be observed showing instabilities; (b) Comparison between Amide I and Amide II integrated absorbance from 0 to 60 minutes; (c) Reorganization of the secondary structure that trigger a process of aggregation into oligomers is shown; (d) Change of secondary structure content of A§ 1 42 in the first 60 minutes of incubation A§ 1 4 2 process of fi bril formation reaches relative stability at 30 minutes as it is evidenced by the changes in spectra in Figure 43(a), this is further confirmed
85 by the changes in absorbance of the Amide I and Amide II in (b), and absolute and relative structure content val ues from (c), and (d). Figure 44. Fibrillization o f A§ 1 42 at 25 ¡C (a) 0 to 72 hours spectra during the A§ 1 42 peptide aggregation. (b) Aggregation evolutio n by time ranges. (c) Averages s pectra calculated from (b) The stability analysis prese nted in Figure 44 shows the relatively high stability of the fibrils formed by A§ 1 42 in PBS which increases in the 72 hours of the study, as stability visualization in (b), and as average spectra show in (c).
86 Figure 45. Secondary Structure Change d uring Fibrillization o f A§ 1 42 a t 25¡C (a) Rate of aggregation per structure. (b) Percentage change of secondary structure content. (c) AFM morphological characterization of the A§ 1 42 fibril formation Figure 45 shows secondary structure analysis of FTIR ATR data for up to 96 hours shows that the remaining dominant secondary structure over the long term evolution of fibrils is parallel sheets structure. The AFM scans on Figure 45(c) show that the morphology of the fibrils change over the 240h scan which is in agreement with the secondary structure analysis in Figure 45(b) in which only parallel § sheet structure remains dominant among other secondary structures.
87 18.104.22.168.3. A§ 40/42 Fibrils The fibrillation process of A§ 1 40 and A§ 1 42 at a ratio of 1:1 and at 25 ¡ C was investigated in this section. As for the previous cases, the study was done for the first hour and then for up to 96 hours. Figure 46. Change o f Secondary Structures o f A§ 40/42 Dissolved i n DMSO a fter Diluting w ith PBS Buffer Solution t o 100 # M. (a) Spectra of the Amide I and II regions from 0 to 60 minutes, Amide II shows protein adsorption, on Amide I re gion shifting can be observed showing instabilities; (b) Comparison between Amide I and Amide II integrated absorbance from 0 to 60 minutes; (c) Reorganization of the secondary structure that trigger a process of aggregation into oligomers is shown; (d) C hange of secondary structure content of A§ 40/42 in the first 60 minutes of incubation
88 Figure 46 shows the first hour evolution of the fibrillization process the A40/42 equimolar mixture, which initially has as dominant secondary structure, antiparallel !-sheets. Figure 47. Fibrillization o f A 40/42 a t 25C. (a) 0 to 72 hours spectra during the A 1 42 peptide aggregation. (b) Aggrega tion evolution by time ranges The stability analysis of the A40/42 depicted in Figure 47, shows little stability of the fibrils during the 72 hour timeframe.
89 Figure 48. Fibrillization o f A§ 40/42 at 25¡C (a) Spectra average by time range, (b) AFM morphological characterization of the A§ 40/42 fibril formation (c) Most relevant structures changing du ring fibrillization, (b) Percentage change of mos t relevant secondary structures The morphology of the A§ 40/42 fibrils depicted in Figure 48, shows parallel sheets as the dominant structure for most of the process on the long term analysis.
90 Figure 49. Comparing t he Morphological Evolution of A§ Peptides d uring Fibril Formation at 25¡C (a) AFM morphology characterization. (b) TEM images of A§ pepti des at 10 days of incubation The AFM scans and TEM images in Figure 49 provide an opportunity to compare the morphology of fiber formed by either A§ 1 4 0 and A§ 1 42 peptide, as well as their 1:1 combination. AFM scans at 1x1 m reveal that fibrils formed by the mix of peptides A§ 4 0/42 are generally thicker and formed of globular aggregates; this is the case for initial fibril structures formed in A§ 1 4 0
91 Figure 50. Morphology o f A§ Peptides Films Prepared i n DMSO + TRIS a nd DMSO + PBS Soluti on. The Films shown in (a) where obtained 30 minutes after the samples were prepared. (b) Topographical analysis of oligomers in DMSO + TRIS solution; (c) Topographical analysis of oligomers in DMSO + PBS solution
92 The observations of morphology abou t fibrils formed from the peptide equimolar mixture of A§ 4 0/42 is mirrored by the morphology of oligomeric aggregates shown in Figure 50(a). Quantification of such aggregates from AFM scans show that in the case of the TRIS solution, the size of oligomeri c aggregates from A§ 1 4 0 are smaller with smaller average height, A§ 1 4 2 are larger in size, and the size of oligomers from the mix A§ 4 0/42 appear in between those of the peptides. This is not the case for oligomeric aggregates in PBS solution shown in 50 (b); which are considerably larger for the mix A§ 4 0/42 than those for each peptide on their own. This morphological behavior is similar to that shown for fibrils in Figure 49. 22.214.171.124. Effects of Temperature during the Fibrillization of A§ Peptides This section introduces two new studies complementary to the A§ peptides fibrillization assessment. Now, varying the incubation temperature to 37¡C. these studies are only performed for A§ 1 42 and A§ 40/42 peptides. 4.2.1 4.1 A§ 1 42 Fibrils Figure 51 shows the evolution of both amides (I and II) during the process of fibril formation at 37C. Amide II shows adsorption of protein. Amide I region shows molecular rearrangement better appreciate it in Figure 51(b). As previously explained, Figure 51(c) represents th e average spectra for the 4 hours ranges selected from Figure 51(b), and the analysis of secondary structure is (performed to the amide I region) is shown in Figure 52(a) and (b).
93 Figure 51. A§ 1 42 Peptide Fibrillization a t 37¡C. (a) Spectra fr om 0 to 96 hours of incubation. (b) Time selection by ranges. (c) Average spectra calculat ed per time range selected in (b) Figure 52(b) shows the parallel structures as the dominant conformation along with the unordered structures. To assess the effe ct of temperature on the evolution of parallel and antiparallel structures, these structures are compared in Figures 52(c) and (d); it can be observed in these two figures that at 25¡C the fibrillization process is more stable than at 37¡C, however they bo th follow same path of aggregation. In addition the fibrils encountered at 48 hours of incubation have similar morphology in both cases (Figure 52(e).)
94 Figure 52. A§ 1 42 Secondary Structure Change Effects of Temperature d uring Fibrillization (a) Kinetics of the secondary structures at 37C. (b) Content of the secondary structures change. (c) Temperature effect on the change of parallel § sheet structures. (d) Temperature effect on the change of antiparallel § sheet structures. (e) Morphology of the A§ 40/ 42 fibrils at 48 hours of incubation
95 126.96.36.199.2. A§ 40/42 Fibrils Figure 53(a) shows the evolution of amide I and II for A§ 40/42 when the incubation temperature is 37¡C. In Figure 53(b) the changes per 4 hour ranges of the two amides can be e xplored, and the averages of the time ranges are shown in Figure 53(c), the changes in morphology during the aggregation process is shown in Figure 53(d). Figure 5 3 Fibrillization o f A§ 40/42 a t 37¡C. (a) AFM morphological characterization of the A§ 40/42 fibril formation. (b) Secondary structure as function of preferable energetic state during aggregation
96 Figure 5 4 Temperature Effect during the Fibrillization Process o f A§ 40/42 (a) Rate of aggregation per structure AFM morphological characterization of the A§ 40/42 fibril formation. (b) Percentage change of secondary structure content (c) Temperature effect on the change of parallel § sheet structures. (d) Temperature effect on the change of antiparallel § sheet structures From Fi gure 53(c), the evolution of secondary structures in the amide I region was calculated and represented in Figures 54(a) and (b). These graphs correspond to the change in integrated absorbance and its normalized values respectively. Figures 54 (c) and (d) compares the evolution of parallel and antiparallel § sheet structures at 25¡C and 37¡C; showing that at 37¡C the fibrillization takes
97 place rapidly (first 12 hours of incubation) when it reaches equilibrium. In comparison, the fibrillization process a t 25¡C, the stability of this process is reached at 40 hours of incubation. In addition Figure 55 shows the morphology of fibrils, which have similar appearance at 48 hours of incubation. Figure 55. Comparison between A§ 40/42 Fibrils Prepared at 25¡C and 37¡C. Images taken at 48hours of incubation with AFM at a scan size of 10x10 # m x y 4. 3 Chapter Remarks One important finding of this study is that preferentially, fibril formation is characterized by the increase of parallel § sheet structures, whil e if peptide species are in solution the preferential secondary structure is antiparallel § sheet configuration. The experiments and analysis in this chapter allowed the combinatorial study of two different A§ peptides isoforms A§ 1 40 A§ 1 42 and their equimolar mixture, A§ 40/42 The peptides were studied under 5 different set of conditions designed to control in different ways the aggregation of amyloid proteins. The conditions allowed for the formation of monomers in solution, monomers on a film, ol igomers, fibrils formed at 25¡C and fibrils formed at 37 ¡C The stability of
98 the aggregation process and the dominant secondary structures were assessed to provide key knowledge in the understanding of the effect of monoclonal antibodies (mAb) on A§ pept ides and their aggregation in later chapters. The results from this chapter have been summarized in a Table 4, which allows comparing the different cases from the entire study. Table 5 Dominant Structures a nd Stability Table A§ 1 40 A§ 1 42 A§ 40/42 HFIP Dominant Structures: Antiparallel sheet ( stable) HFIP Dominant Structures: Antiparallel sheet Helix (stable) HFIP Dominant Structures: Antiparallel sheet (stable) Film Dominant Structures: Parallel sheets Helix ( stable) Fil m Dominant Structures: Parallel sheets Antiparallel sheet Helix ( stable) Film Dominant Structures: Parallel sheets Helix ( stable) Mon omers 25¡C DMSO Dominant Structures: Antiparallel sheet ( stable) DMSO Dominant Structures: Antiparallel sheet ( stab le) DMSO Dominant Structures: Antiparallel sheet ( stable) Oligomers 25¡C Dominant at 60 min: Parallel sheets Antiparallel sheet (little stability) Dominant at 60 min: Helix Antiparallel sheet (little stability) Dominant at 60 min: Antiparall el sheet ( unstable )
99 Table 5 (Continued) A§ 1 40 A§ 1 42 A§ 40/42 Oligomers 25¡C Dominant during 48 hours: Parallel sheets (stable) Dominant during 48 hours: Helix Antiparallel sheet ( moderate stability) Dominant during 48 hours: Antiparallel sheet Parallel sheets (moderate stability) Dominant at 60 min: No perceived dominance (un stable) Dominant at 60 min: Parallel sheets Antiparallel sheet Helix (apparent stability) Dominant at 60 min: Antiparallel sheet (moderat e stability) Fibrils 25¡C Dominant during 48 hours: Antiparallel sheet Helix ( moderate stability) Reduced after 48 hours: Parallel § Sheet s Dominant during 48 hours: Parallel sheets (stable) Dominant during 48 hours: Parallel sheets ( moderate stabilit y) Fibrils 37¡C N/A Dominant during 48 hours: Parallel sheets ( moderate stability) Dominant during 48 hours: Parallel sheets Unordered ( moderate stability)
100 A simple assessment of A§ 1 40 and A§ 1 4 2 aggregation in oligomeric and fibril formation con ditions shows how the likelihood of stability of the process flips. Oligomers of A§ 1 40 reach stability much faster than oligomers of A§ 1 4 2 while the opposite is true for the fibrillar species where A§ 1 4 2 reaches stability considerably faster than fibri ls formed by A§ 1 4 0 peptide.
101 CHAPTER 5 MONOCLONAL ANTIBODIES MODIFY FIBRILLOGENESIS The main goal of this study was to elucidate and analyze the deviation from a typical fibrillization process caused by targeting A§ peptide monomers and oligomers in solution (before fibrillization occurs) with different anti A§ monoclonal antibodies (mAb). In addition to the two isoforms, the equimolar mixture of the A§ 1 40 and A§ 1 42 (A§ 40/42 ) was studied. The rationale of this relates to the idea of having antibodies with different specificities that would better target the peptide mixture. All the experiments were performed at 37¡C and at a substoichiometric molar ratio of 1 to 1000 (antibody to peptide). 5 .1. Summary In this chapter, the effects of antibodies were studied at a condition close physiological conditions in the brain. For this, temperature was kept constant at 37¡C, and solutions were kept at pH 7.4. A 1:1000 antibody to peptide molar ratio was chosen to approximate therapeutic cond itions. The antibodies were applied to monomers and oligomers in solution (with exception of the first study using 6E10 and 2H6 on preformed fibrils) at the initial stage of the A§ 40/42 fibrillization process.
102 It is worth mentioning, that the oligomeric s pecies of A§ peptide is recognized by different groups as the toxic specie from the aggregation process, due to its reactivity [15, 17 20, 87, 90 92] suggesting this species as a possible effective target for the a ntibody application. Table 6 describes the specificity for the monoclonal antibodies tested in this study. Briefly, 6E10 can bind to the N terminus of either A§ 1 40 or A§ 1 42 peptide, 2H6 binds to the C terminus of A§ 1 40 ; 4G8 binds to the mid domain (1 7 24) of either A§ 1 40 or A§ 1 42 ; and finally mIgG2b with not specificity to neither A§ 1 40 or A§ 1 42 peptides, that is used as a control antibody. Table 6 Specificity and Binding of Antibodies Tested Antibody Specificity Binding 6E10 n term 1 16 A § 1 x 2H6* c term 35 40 40 only 4G8 (Signet Dedham, MA) mid domain 17 24 A § 17 x mIgG2b 3 Control antibody does not bind A§ species A ntibodies provided by Rinat Pfizer All the antibodies tested were provided either by David Morgan's group (USF Healt h Byrd Alzheimer's Institute) or Rinat Pfizer (San Francisco, CA) directly. 3 Antibody raised against a pseudomonas aeruginosa antigen
103 5 2 Protocols a nd Methods ATR FTIR, AFM, and TEM analytical techniques were used in the same capacity as described in previous chapters. Moreover, some of the experiments with antibodies were also characterized using Western Blot. 5 2 .1. Antibody Peptide Solution An equimolar mixture of A§ 1 40 and A§ 1 42 (A§ 40/42 ) was first prepared as explained in section 4.1.4. In brief, A§ 1 40 and A§ 1 42 peptides were prepared separatel y. Each peptide's film was redissolved with DMSO and diluted to 100 # M with 0.01 M PBS. These two solutions were then combined in a 1 to 1 molar ratio stock solution. Amyloid peptides were incubated with each antibody from the beginning of the fibr il formation process. However, 6E10 and 2H6 antibodies were also tested against 5 days preformed fibrils. 188.8.131.52. Effects of Low Antibody Concentration on A§ 40/42 Fibrils Amyloid § peptide fibrils were prepared following the protocol described in 5.2.1 The A§ 40/42 solution at 100 # M was aliquoted in three portions; one aliquot was injected in the ATR FTIR flow cell, where the aggregation was monitored over time; the second portion was subdivided in different vials, each corresponding to different incub ation times, later analyzed by AFM and TEM. The third portion was incubated for later addition of antibody (at day 5) and analyzed by AFM and TEM at different time over 5 days after antibody addition. Hence, the
104 processes of fibril formation and antibody a ddition were verified with ATR FTIR spectroscopy, AFM and TEM. The antibody was added in a molar ratio of 1 to 1000, antibody to peptide, all the samples were incubated at 37¡C The control solution for this study was the equimolar mixture of A§ 40/42 pepti des incubated with no addition of antibody for a period of 10 days at 37¡C. 184.108.40.206. Targeting A§ 40/42 Monomers and Oligomers with Low Concentration of Different Monoclonal Antibodies As in section 220.127.116.11, each solution was prepared using a 1:1000 subst oichiometric molar ratio, antibody to amyloid peptide respectively. As described previously, the peptide's film was first redissolved with DMSO (0.2 mM) and diluted to 100 # M in 0.01 M PBS (pH 7.4), A§ 1 40 and A§ 1 42 peptides were prepared separately and then the solutions were combined in a 1 to 1 molar ratio. Immediately after combining the two peptide solutions the antibody was added to the peptide's mixture in a molar ratio 1:1000 of antibody to peptide. The solution antibody peptide was divided in two portions each incubated at 37¡C. The first portion of this solution was injected in the ATR FTIR flow cell accessory where it was monitored over 10 days of incubation (by spectroscopy). The other portion of the antibody peptide solution was subdivided for AFM, TEM, and Western Blot analysis. Samples were collected over a period of 10 days. The control for this study was a solution of A§ 40/42 peptide prepared with an antibody (mIgG2b) with not specificity to either peptide.
105 In addition to the control a bove, and to prove that our results correspond to the antibody peptide interaction rather than the single antibody and peptide contribution, a predicted model of secondary structures was calculated, this by mathematical adding the secondary structures calc ulated for A§ 40/42 peptide incubated without antibody and the secondary structures of antibodies 2H6 and 6E10 when incubated alone over the same period of time. This is A§ 40/42 + 2H6 and A§ 40/42 + 6E10. The A§ 40/42 2H6, and 6E10 controls were solutions pr epared and incubated as if they were combined in a 1:1000 antibody to peptide system. The antibodies used for these experiments (see Table 6) were received from the provider dissolved in PBS solution at variable concentrations (1 2 mg/ml), hence calculati ons of the appropriate volume of antibody were performed to assure the same molar ratio for all experiments. In order to avoid antibody contamination and unnecessary freeze unfreeze cycles, all the antibody stock solutions were aliquoted (in 5 portions) an d kept at 20¡C once received from the provider. Prior to use, 1 of the 5 vials containing the antibody was retrieved from the freezer and allowed to equilibrate to 25¡C. 5 2 .2. AFM and TEM Characterization AFM and TEM assisted to determine the state of aggregation of the peptides in contact with antibodies. In the initial state of aggregation, the peptide tends to spread fairly uniformly across the substrate where it is deposited. However, as the incubation time increases, and peptides aggregate in to fibrils, it becomes challenging for the nanoscale size tip of the AFM to depict
106 where fibrils are located on the substrate. At this point, it is better to combine TEM with AFM to determine the aggregation state and to examine the sample's surface dist ribution. Therefore, some results illustrating the morphology and/or topology of the samples will be shown with both techniques (TEM adapted to the AFM scale). 5 2 .3. ATR FTIR Analysis The background solution used for these studies was the solvent in wh ich the peptides/antibody systems were prepared, that was 2% DMSO to 98% 0.01M PBS (v/v). Therefore, all the collected spectra represent the changes of secondary structures of the antibody peptide mixture. 5 2 .4. SDS PAGE a nd Western Blot Analysis SDS PA GE and immunoblotting w as performed as described by Dahlgren et al. 2002  Briefly, individual aliquots of the stock solution collected at the testing times w ere centrifuged a t 14 000g for 10 min at 4¡C in order to remove large aggregates such as potential fibrils in the supernatant. Ice d cold Millipore water was used to dil ute the monomer and oligomer containing A 40/42 (or mAb A§ mixture) supernatant at 1:5 ratio and mixed with a buffer Note that there is no heating nor use of additional reducing agents. Th ese sample w ere then combined with a 12 % Bis Tris NuPage gel usin g MES as the running buffer (Invitrogen, US). See Blue pre stained molecular mass markers were run on each gel (Invitrogen, US). P eptides separated at 160 V for 1 h and subsequently
107 transferred to 0.22 mm PVDF membranes at 35 V for 1hr. The membranes were blocked for 1 h our at room temperature (RT) using 5 % non fat dry milk (International, Diagnostics Group, UK) in 20 mmol/LTris buffer, pH 7.6, with 137 mmol/L NaCl and 0.05 % (w/v) Tween 20. Subsequently, the membranes were incubated with a primary antibod y in blocking buffer overnight at 4¡C. The primary antibody 6E10 (a mouse monoclonal antibody directed against the human A amino acid residues 1 17) was used at a dilution of 1:1000. The membrane was then rinsed in Tris buffer, pH 7.6, 137 mmol/L NaCl and 0.05 % (w/v) Tween 20 three times and incubated with secondary antibody solution of HRP labeled rabbit antibodies against mouse IgG (diluted 1:2000) for 1 h at RT. Rinsing with 20 mmol/L Tris buffer, pH 7.6, with 137 mmol/L NaCl and 0.05 % (w/v) Tween 20 was applied again. Immunostained p eptides were visualized using chemiluminescence, in accordance with the manufacturer's recommendations (ECL, Amersham, Denmark). Western blots were scanned using a Fujif ilm Lass 300 Intelligent dark box scanner, and band density of each blot was quantified within linear range of detection using AlphaEase software This immunnochemical analysis was used to monitor the presence of monomers and oligomers during the aggregation process.
108 5 3. Results, Analysis and Discu ssions The outline for subsequent sections is as follows: 5.3.1. the effects of 6E10 and 2H6 antibodies on A§ 40/42 pre formed fibrils (at 5 days of incubation) are compared; 5.3.2. the effects 6E10 and 2H6 antibodies added at the beginning of the aggregat ion process (when only monomers and oligomers are present) are compared; and 5.3.3. the effects of 2H6, 6E10, and 4G8 antibodies are compared against mIgG2b, a non specific antibody. 5 3 .1. Effects of Antibodies on Species of A§ 40/42 Targeting the Peptide s C Terminus or N Terminus In this section, it is first shown the effects of 6E10 and 2H6 antibodies on fibrils pre incubated for 5 days, testing a molar ratio of 1:1000 (antibody to peptide). Then, these same antibodies were tested against an initial aggregation stage solution, were mainly oligomers and monomer were present. The rationale for comparing the effect of antibodies against fibrils or monomers+oligomers is to determine which species are more likely to be influenced by the addition of antibo dies. It is desired that if an antibody is going to be used in passive immunization, it should be able to break preformed fibrils or to stop or prevent their aggregation in the first place. The effect of the antibodies in this study has been measured depen ding on how effective they are to induce antiparallel beta sheet structures or to decrease parallel beta sheets structures (those structures were associated with monomeric and fibril species, respectively, in Chapter 4).
109 5.3.1 1. Effects of 6E10 and 2H6 on A§ 40/42 Pre Formed Fibrils As explained in previous sections, an equimolar mixture of A§ 40/42 peptides was used to form fibrils, and a substoichiometric molar ratio of 1:1000, antibody (either 6E10 or 2H6) to peptide respectively was used. Figures 56 (a) (c) show the spectral evolution for amide I and II bands with respect to incubation time for the control system (A§ 40/42 peptides mixture), the A§ 40/42 + 6E10 antibody and the A§ 40/42 + 2H6 antibody, respectively. These average spectra were obtained as de scribed in the Chapter 3 (Section 18.104.22.168 ). B riefly, continuous spectra corresponding to a selected time range were averaged into a single spectrum. As described above, at day 5, the antibody was added to a preformed fibril solution, indicated with an ar row ( +mAb) in the spectrum for the control (A§ 40/42 ) peptide solution ( Figure 56(a)). It is worth mentioning that the background spectrum for Figures 56(b) and (c) was obtained from the control system at day five of incubation (e.g., prior to adding the antibodies). Moreover, the Amide II band progression in Figure 55 shows a significant increase for the case when 6E10 antibody was added (Figure 56(b)). This means that 6E10 antibody promoted surface adsorption of the peptides to the surface. This same b and did not increase, and if anything, it shows a slight decrease when 2H6 antibody was added (Figure 56(c)). This implies that the preformed fibrils did not necessarily detach from the surface completely. Rather, it indicates that the fibrils attached to the surface were attacked by 2H6 antibody.
110 Figure 56 Influence of Low Concentration Antibodies Targeting A§ 40/42 Fibrils. Average spectra obtained at 37¡C, amide I and II regions are shown for (a) the control mixture of A§ 40/42 peptides, (b) 6E10 a ntibody added to A§ 40/42 peptide mixture (1:1000). This antibody targets the N terminus of both peptides, (c) 2H6 antibody added to A§ 40/42 peptide mixture (1:1000)., antibody with affinity to the C terminus of A§ 1 40 peptide only. The arrow shown in the spectrum for the control system at day 5 represents the point where antibodies were added to the peptide solution Figure 57 shows the most dominant secondary structures after the a mide I region was deconvoluted for the control and the two cases of the a ntibody to peptide systems. Only parallel and antiparallel beta sheet structures are shown because these two structures provide the most relevant information regarding
111 any dissolution effect on the preformed peptides fibrils Figure 57(a) shows that in the control system, parallel beta sheet structures increase continuously until they reach a plateau, which is when saturation has been reached after 48 h of incubation. Conversely, the progression for the peptide mixture +2H6 shows a peak with a maximum value at approximately 4 days of incubation in the parallel § sheet structures. Then, there is an abrupt decay of such structures, which implies that the antibody was able to render conformational changes tied to dissolution effects (Figure 57(a)). In the case of peptide mixture +6E10, the effect is better depicted in Figure 57(b), where the parallel beta sheet structures decreased to less than 5% of total structure content after day 3. Figure 57(d) shows the appearance of the fibrils found in the three samples after 10 days of incubation. It is observed that fibrils are still found in the case of 2H6 addition, but the morphology differs from the control system. That is, the fibril density is lower and fibril bundles are thinner than fibrils in the control pictu re (Top scan compare to bottom scan). In the case of 6E10, fibrils appear to be spread on the surface, but more abundant in comparison to the control system (+6E10 TEM Scan in Figure 57(d)).
112 Figure 57 Secondary Structure and Morphological Changes of A§ 40/42 Fibrils after Combined with 6E10 or 2H6 Antibodies. Analysis of the secondary structures performed from the spectra shown in Figure 56.The values of the control (labeled as A§ 40/42 ) peptides represent the change after the 5 days of incubation. The peptide mixture and the peptide mixture +antibody were further incubated for 10 days. (a) Comparison of the kinetics of parallel § sheet structures between the control and the peptide mixtures affected by 2H6 and 6E10 antibodies in a molar ratio 1:1000. (b ) Comparison of the parallel § sheets content for all three systems described above. (c) Comparison of the antiparallel § sheet structures content with respect to incubation time for the systems described above.(d) TEM scans of the control after a total o f 15 days of incubation at 37¡C (Top), peptide mixture+6E10 (Middle), and peptide mixture+2H6 (Bottom). Both systems with the antibodies added were incubated for 10 days at 37¡C
113 22.214.171.124 Effects of 6E10 and 2H6 on A§ 40/42 Monomers and Oligomers The stu dy on preformed fibrils on the previous section allows the statement that the effect of antibodies is limited on preformed fibrils. Continuing with this study, the effects taking place when antibodies are added at the initial stage of aggregation are prese nted. Figure 58 show the average spectra comparing the amide I and II regions of the control (A§ 40/42 ), and A§ 40/42 peptide treated with eider 6E10 or 2H6 antibodies. The main differences are in the amide I region for the peptide mixture+2H6 antibody, wher e most of the secondary structures undergo sudden transformations at day three of incubation (Figure 59a and (b)). This implies that the fibril formation is dramatically disrupted. On the other hand, the amide II region shows significant growth. This incre ase in the peptide adsorption is expected since peptide structures undergoing conformational changes may not be able to go back into solution because they have been corrupted (Figure 58c). This finding is verified by analyzing the deconvolution of the spec tra and the anatomic morphology of these spectra shown in Figures 59(a), (c) and (d), where the parallel beta sheet structures decrease, antiparallel beta sheet structures increase, and surface globular aggregates appear instead of fibrils. Note that even though antiparallel beta sheets content increased, peptide aggregation on the surface was relatively high. As for the case of having peptide mixture+6E10 antibodies, the Amide I and II regions follow the control system behavior (Figure 58(b) and Figure 59 (a) (c)). Therefore, this antibody was unable to prevent the
114 fibrillization process under these conditions and did not prevent peptide adsorption to the surface since Figure 59d shows that fibrils were still formed. Figure 58 Monitoring A§ 40/42 Fibril lization Process from the Beginning of the Incubation Process with N Terminus and C Terminus Antibodies. Spectra were collected at 37¡C. Amides I and II changes for (a) A§ 40/42 control system (b) 6E10 added to the beginning of the incubation process ofA§ 40 /42 peptides (1:1000 molar ratio). This antibody targets the N term of both A§ 40 and A§ 42 peptides. (c) 2H6 antibody incubated with A§ 40/42 peptides. 2H6 is affined to the C term of the A§ 1 40 peptide
115 Figure 59 Effects of 2H6 and 6E10 Antibodies on A§ 40/42 Monomers + Oligomers. (a) Compares the parallel § Sheet structures evolution for the control system and for when antibodies where added to the peptide mixture, (b) Compares the parallel § sheets content, (c) Compares the antiparallel § sheet conten t (d) AFM images representing the morphology of the fibrils immediately after antibody addition and after 10 days of incubation for the control system (Top), and for the aggregates formed with 6E10 (mid) and +2H6 (Bottom) addition In o rder to investig ate the changes in the initial species of the fibrillization process, that is, monomeric and oligomeric species, Western Blot analysis was also done to these samples. Results are shown in Figure 60 Figure 60( a ) shows the results from Western Blot analysis performed on samples at the same conditions as in Figure 5 8 That is, A§ 40/42 (control), A§ 40/42 + 6E10, and A§ 40/42 + 2H6. Figure 59(b) s hows the AlphaEase analysis of the soluble species
116 (monomers, low molecular weight (LMW) oligomers, and high molecula r weight (HMW) oligomers) Note that monome r s and oligomer s disappear from the solu tion after day 2 for all three cases This indicates that during the incubation process, fibrillization is forced and monomeric as well as oligomeric species undergo config urational changes into much higher molecular weight structures, which are transparent to/undetected by this technique (fibril aggregates). However, t he HMW oligomeric species abruptly increase s after day 3 for the case of A§ 40/42 peptide mixture treated wi th 2H6 antibody. This means that the antibody was able to reverse the production of fibril aggregates and induce the production of oligomers. Conversely t he control system and the samples treated with 6E10 antibody show that monomers and oligomers disappe ar and this antibody is not able to reverse this action These results obtained with Western Blot are in line with the data from ATR FTIR spectroscopy and AFM/TEM topological studies as depicted in Figures 58 and 59.
117 Figure 60 Effect o f 2H6 a nd 6E10 o n Soluble Species o f A§ 40/42 Peptide Equimolar Mixture d uring Fibrillogenesis. (a) Western Blot of the control system(A§ 40/42 ), A§ 40/42 + 6E10 antibody, and A§ 40/42 + 2H6 antibody. (b) AlphaEase analysis of soluble species for Monomers (Rig ht) l ow molecular weight (LMW) oligomers (Middle) and h igh molecular weight (HMW) oligomers (Left) 5.3.2 Proving Suitability of Results Before continuing with this study, it is necessary to prove that the results obtained correspond to the antibody pep tide interaction rather than to a pure contribution of antibody of peptide separately. For this, different control solutions were ran over 10 days and analyzed. The control solutions were, A§40/42 with no antibody addition, antibody 6E10, and antibody 2H6 In order to prove the results presented here, the secondary structures corresponding to peptide + 2H6, peptide + 6E10, and peptide +
118 average of 6E10 and 2H6 were mathematically added. The results show (Figure 61) that when comparing to the experimental data, the secondary structure kinetics is completely different from the predicted values. Thus depicting completely different behaviors. Figure 61. Compa rison of P redicted S econdary S tructures vs. E xperimental S tructures. (a) Predicted behavior for t he average values of antibodies control 6E10 and 2H6 when added to A§ 40/42 control. (b) Predicted behavior for the average values of antibodies control 6E10 when added to A§ 40/42 control. (c) Predicted behavior for the average values of antibodies control 2H6 when added to A§ 40/42 control. (d) Experimental values for 6E10: A§ 40/42 at 1:1000. Control. (e) Experimental values for 2H6: A§ 40/42 at 1:1000.
119 This study proves that when any antibody (that binds the peptide) is added to the peptide mixture, the r esulting spectra correspond to the interaction antibody peptide in solution, and not to the independent spectra. Hence, our results show a fair representation of the effect of antibodies on amyloid beta peptides during fibril formation or disruption. 5.3. 3 Effects of Antibodies on A§ 40/42 Monomers and Oligomers This study compares the effects of antibodies on an initial system mainly composed by monomers and oligomers in solution. The study compares three antibodies that bind either or both peptide agains t a non specific antibody. The results in Figure 62 show the existence and increase of a peak centered in the parallel § sheet frequency region (1630cm 1 ) for the cases of the control, peptide treated with 6E10 and peptide treated with 4G8, when in compa rison to the peptide treated with 2H6 this peak seems to decrease and with the shifting of peaks in other regions. Once Figures 62(a) (d) are deconvoluted and integrated, the comparison can have greater detail as shown in Figure 63(a) (c), proving that fr om all the antibodies only when the peptide is treated with antibody 2H6, there is a noticeable decrease in parallel § sheets and an increase in antiparallel § sheets structures after day 3 of incubation. In addition, Figure 63(d) compares the morphologic al appearance of the four systems under study, showing the formation of fibrils in the system of peptide treated with IgG2b, 6E10, and 4G8, when in the case of peptide treated with 2H6 amorphous aggregates are found rather than fibrils.
120 Figur e 62 Monitoring A§ 40/42 Fibrillization Process from the Beginning of the Incubation Process with N Terminus and C Terminus Antibodies. Spectra were collected at 37¡C. Amides I and II changes for (a) A§ 40/42 control system (b) 6E10 added to the beginning of the incubation process ofA§ 40/42 peptides (1:1000 molar ratio). This antibody targets the N term of both A§ 40 and A§ 42 peptides. (c) 2H6 antibody incubated with A§ 40/42 peptides. 2H6 is specific to the C term of the A§ 1 40 peptide. (d) 4G8 antibody in cubated with A§ 40/42 peptides. 4G8 has specificity to the mid domain of both A§ 1 40 and A§ 1 4 2 peptide.
121 Figure 63 Effects of 2H6, 6E10, and 4G8 Antibodies on A§ 40/42 Monomers+ Oligomers. (a) Compares the parallel § Sheet structures evolution for the control system and for when antibodies where added to the peptide mixture, (b) Compares the parallel § sheets content (c) Compares the antiparallel § sheet content (d) AFM images representing the morphology of the fibrils immediately after antibod y addition and after 10 days of incubation for the control system (Top), and for the aggregates formed with 6E10, 4G8, and 2H6 (Bottom) addition. This study proves that it is possible to discern and evaluate the effect of antibodies by analyzing the co ntent and evolution of secondary structures of the system, and that this results can be corroborated by the physical characterization of the sample using AFM and TEM.
122 5.4. Chapter Remarks The experiments in this chapter tested various monoclonal antib odies with different specificity against A§ peptides. A§ 1 4 0 and A§ 1 42 peptides were ratio in a molar mixture of 1 to 1, and for every case, the antibody to peptide molar ratio was 1 to 1000. 2H6 antibody targeting the C terminus of A§ 1 4 0 decreased the content of parallel sheets and increased the content of antiparallel sheets, these results are summarized in Table 6. The rise in the content of antiparallel sheets might indicate the presence of unstable A§ 40/42 oligomeric species (Chapter 4, Table 5). The effectiven ess of the evaluation methods was tested as a platform to evaluate the effects of antibodies used against amyloid peptides. In general, the use of antibodies to target Amyloid Beta during the fibrillization process seems to be more effective that targeting fully formed fibrils.
123 Table 7 Summary of Observations for the Antibodies Tested in this Chapter Antibody Specificity Observable Effect 6E10 A§ 1 40 and A§ 1 42 n terminus (1 16) No effect to preformed fibrils No effect on secondary structure con tent No effect on fibrillization process 2H6 A§ 1/40 c terminus (35 40) Decrease Parallel sheet content (both on preformed and in fibrillization) Increase antiparallel sheet content (only in fibrillization) Limited Effect on preformed fibers Stopped fi brillization only allowing oligomers 4G8 A§ 1/40 and A§ 1/42 Mid domain (17 24) No effect on secondary structure content Stopped fibrillization only allowing oligomers mIgG2b Non specific No effect on secondary structure content No effect on fibrillization process
124 CHAPTER 6 MIMICKING T HE NEURON MEMBRANE SURFACE 6 .1. Lipid Bilayer Membranes a nd t heir Relevance t o A§ Plaque Formation Lipid bilayer membranes are an important element of living cell s, supported by a network of structural proteins ca lled the cytoskeleton. An important characteristic of lipid bilayer membranes is their natural fluidity, which allows free movement of lipid mole cules on their own monolayer. This fluidity shows differences throughout the cell membrane, and varies dependin g on the composition of the principal molecules forming the lipid bilayer: phospholipids, cholesterol, and glycolipids The biophysical characteristics of the lipid membrane also allow certain proteins to embed and anchor or penetrate through them. The Alz heimer's disease A hallmark supposes the formation of extracellular A plaques ; these plaques are formed on the cell membrane by binding to membrane molecules and by partial insertion in to the cell membrane. It is evident that altering the homeostasis of membrane components such as cholesterol, leads to accelerated plaque formation that might be related to the anchoring or the release of A  Besides the biological implications, the
125 biophysical role of higher cell membrane contents of cholesterol and other intramembran e components is yet not well understood. However, recent studies suggest that cholesterol and membrane rigidity might hinder A penetration resulting in amyloid accumulation on cell membranes  This chapter presents two methods for building in vitro lipid bilayers with the potential to be used as a platform for studying biophysical p henomena related to A plaque formati on on lipid bilayer membranes. Two techniques for creating lipid bilayers were explored: Langmuir Blodgett deposition technique and self assembled bilayer membranes from lipid vesicles. In both cases, the lipid bilayer membranes were deposited on a thin film of polyethylene glycol (PEG), which acts as a cushion layer to the lipid bilayers and serves to mimic membrane fluidity  The composition o f model in vitro membranes can be made to mimic different conditions important to the understanding of A plaque formation by reproducing in a monolithic way local characteristics of membrane domains. One of the challenges encountered in reproducing cell m embrane characteristics in vitro lipid bilayer is reproducing membrane fluidity; this can be overcome by using a cushion layer below the in vitro lipid membrane. 6.2. Soft Support Layer U sing Polyethylene Glycol Polyethylene glycol (PEG) is an amphiphi lic oligomer used to build the soft supported layer. PEG is a linear or branched neutral polyether that is soluble in water and most organic solvents. The following is t he chemical formula for
126 P EG : HO (CH2CH2O)n CH2CHOH. PEG is utilized in this study becau se its physicochemical properties allow its use as a cushion substrate or coating polymer, and it has been a part of previous studies by our group  When in aqueous solution, PEG rejects other polymers and proteins, and can form two phase systems with other polymers. This prope rty is based on its molecular conformation in aqueous solution, whereby PEG exposes uncharged hydrophilic groups and shows very high surface mobility (steric exclusion). PEG can also be used to coat surfaces with very little chemistry modification to thes e surfaces. When surfaces are coated with PEG, the y become hydrophilic and protein rejecting. This protein rejecting characteristic of PEG makes it of value as a cushion layer under a lipid bilayer, as it would reduce the probability of proteins adhering t o the substrate underneat h the lipid bilayer membrane. Additionally when chemically attached to a substrate, PEG maintains its biological and biocompatible properties. For all these reasons, PEG is an attractive substrate for using it as a support or cush ion layer to increase in vitro fluidity of lipid bilayer membranes. 6.2.1. Construction o f t he Soft Support Layer U sing PEG Following a protocol developed by Alcantar et al. in 2000  a polished glass surface (rectangular hemacytometer cover glass, 12 519 10, Fisher Sci ) was activated by submerging it in a 10 % w /v sodium hydroxide (NaOH, 1310 73 2, Acros Organics) solution and sonicating (Model FS30, Fisher Sci.) it for 5
127 minutes. During the surface activation of polished glass, silanol groups (Si OH) are produced at the solid air interface (Fig ure 64 ). The glass was retrieved from the NaOH solution and cleaned by rinsing it multiple times with Millipore water. U ltrapure nitrogen was then applied to remove any water traces until the s urface was completely dry. The a ctivated glass surface was then submerged in the polyethylene glycol (PEG, 400 Da, P3265 500G, Sigma) for one hour at 100¡C T his resulted in the PEG being grafted to the surface of the activated polished glass (Figure 64 .b) T his process occurs through a reaction between the end alcohol groups of the PEG molecules with the Si OH molecules on the activated glass (Figure s 64 .a and 64.b). (a) (b) Figure 64 PEG Cushion Layer Construc tion. ( a ) During the surface activation of polished glass, silanol groups (Si OH) are produced at the solid air interface (b) Schematic of the PEG s urface grafting reaction, where silanol groups on the surface of the activated polished glas s lose the hydrogen and forms water with the end hydroxyl group of the PEG molecules This result in the PEG being grafted to the surface of the activated polished glass
128 The PEG grafted glass was retrieved from the PEG solution while still hot and immed iately rinsed avoiding direct contact with the water flow F inally, the surface was dried by gently exposing it to a flow of ultrapure nitrogen. 6 .3. Lipid Bilayer Construction Techniques 6 .3.1. Langmuir Blodgett Deposition Technique This technique is used to deposit multiple organized and co mpact layers, one monolayer at a time. Any organic or inorganic molecule of amphiphilic nature can be deposited [98, 99] Typically, a bed of water is used as the initial surface for deposition (subphase) in which the molecules to be deposited are gently placed F or this a suitable solvent with lower density than the subphase (and with high vapor pressure) is used to dissolve the molecules T his solvent is easily evaporable leaving a monolayer of the molecule of interest on the water's surface. Once a monolayer of the molecule is formed on the subphase, the deposition takes place by slowly pulling out a previous ly submerged substrate, where the monolayers will be depos ited in an upward followed by a downward direction T hese movements are perpendicul ar to the subphase surface ( Figure 65 .a).
129 Thi s technique is extremely simple and allows deposition of a single layer, bilayers or multiple layers one at a time. Depending on the orientation of the amphiphilic molecule layers with respect to the substrate and to each other, the deposition can take one of three for ms: X type, Y type, or Z type ( Figure 65 .b). Figure 65. Langmuir Blodgett Deposition Technique. ( a ) Schematic of the deposition meth odology. (b) Types of deposition based on the order of layers The Langmuir Blodgett (LB) trough (Figure 66 ) requires an environment completely free of contamination to avoid particles clinging to the surface. These particles can contaminate and perturb the deposition quality. The trough is located inside a laminar flow cabinet to avoid potential contamination. However, the airflow is turned off during lipid deposition in order to protect the trough from the environment. When necessary, Millipore water a nd sometimes organic
130 solvents (for instance: chloroform) are used to clean the trough. A common indicator of cleanliness is the ab sence of air bubble formation. T he presence of bubbles with the addition of water indicates potential contamination. Hence, b ubble s are eliminated by suction. Figure 66 Langmuir Blodgett Trough. The principal components of the trough are highlighted in this photo The LB trough is connected to a computer system, and is run by software that allows the control of parameters such as surface area, superficial tension, and substrate deepness necessary for the actual deposition. The area, deposition pressure, and lipid concentration used for the depositions are chosen based on calibration experiments that are run beforehand and are a function of the lipid's nature.
131 126.96.36.199. Layer b y Layer Lipid Membrane Construction Lipid bilayers composed of 1,2 Distearoyl sn Glycero 3 P hosphoethanolamine (DSPE, 850715X, Avanti Polar Lipids Inc.) were deposited onto the grafted PEG layer usin g the Langmuir Blodgett deposition technique (LB trough, Model 611D, Nima Technology) as explained earlier in this chapter. The polished glass surfaces containing the soft supported PEG layer were kept inside of a laminar flow cabinet (Class 100) to minimi ze contamination. The initial uncompressed surface area was fixed at 300 cm 2 The lipids were compressed using a LB barrier speed of 25 cm 2 /min. The parameters used for this experiment, such as area, deposition pressure, and lipid concentration, were devel oped in a previous study  A volume of 100 # L of DS PE w as dissolved in chloroform (HPLC grade, 67 66 3, Acros Chemicals) at a concentration of 15 # g/ # L. This solution was gently poured on the air/water interface using a gas tight Hamilton syringe ( allowing 5 minutes for the solvent to evaporate ) During the process of deposition, the surface pressure was kept constant at 30 mN/m. The process of deposition of the first lipid monolayer took place by dipping the activated polished glass with the PEG layer at a velocity of 2 cm/min; the second lip id layer is deposited when the dipping motion is reversed, rem oving the glass from the trough and placed in a fluid reservoir for transportation (Figure 67 ).
132 Figure 67 Schematic o f Lipid Bilayer Construction. ( a ) Schematic of first lipid monolayer d eposited on the cushioned layer (PEG) ( b ) Representation of the soft supported lipid bilayer Hydrophilic and hydrophobic domains are shown c A double layer of lipid is deposited on top of the PEG layer using Langmuir B lodgett deposition technique 6. 3.1 2. Soft Supported Lipid Bilayer Characterization: Surface Topography Atomic Force Microscopy (AFM, MFP 3D, Asylum Research, Santa Barbara, CA) was used to physically characterize the surface topography/topology of the soft supported lipid bilayer membr anes. Tapping mode was used to scan the surfaces, which were at all times submerged in deionized water to preserve the i ntegrity of the lipid bilayer. The cantilevers used had typical spring constant < 1 N/m. Scan sizes were 10x10 # m. (Figure 68 ).
133 Figu re 68 Characterization of Soft Supported Lipid Bilayer Membrane. (a) RMSR change at different steps of construction. (b) AFM images (0.5x0.5 # m x y scan size) at the different steps of the lipid bilayer assembly The lipid bilayer membranes wer e kept in contact with saturated lipid solution at all times to avoid air exposition, which could consequentl y cause damage to the samples. While LB is a simple and very reliable technique that allows deposition of homogenous monolayers, there are some lim itations to this technique. First, the substrate geometry has to be open and flat, second, the size of the substrate cannot be greater than 4 cm x 4 cm and third, the transfer from the LB trough (where the deposition takes place) to a container is diffi cult because any expo sure of the bilayer to the air would destroy it This makes it difficult to stor e and unsuitable for certain characterization techniques (like closed ATR FTIR flow cell). Therefore, other methods to construct lipid bilayers must be c onsidered, as is the case of vesicle based self assemble d lipid bilayers.
134 6.3.2. Vesicle Based Self Assembled Lipid Bilayers Vesicle self assembly technique provides an attractive alternative to overcome the limitations of the LB lipid deposition techniqu e, such as size and geometry of the starting substrate and difficult handling. The process consists o f the spontaneous fusion of small unilamellar vesicles  onto a surface substrate. Vesicles are made of lipid aggregations when they are forced to associate in cores as micelles for instance (unilamel l ar) at controlled media conditions. A general methodology is to dissolve the lipids in a solvent, i.e. chloroform, and then the lipid in suspension is placed in a rounded flask T he solvent is left to evaporate overnight and vacuum for 4 hours before further preparation. Once the film is completely dry, water is used to hydrate the film by heating and gently agitating it; once the transition temperature of the lipid is overcome the lipid will completely be dissolved. The last step in the preparation of vesicles is to slowly cool down the solution allowing the lipids to organize, resulting in vesicles formation The deposition by self assembly technique, is acco mplished by the interaction between the prepared vesicles and the treated surface substrate. This interaction burst the vesicles and drives a self assembled lipid bilayer formation. 188.8.131.52. Self A ssembled Lipid Bilayers from Vesicles As a second stage o f this study, the formation of lipid bilayer membranes by self assembly technique was performed.
135 Lipid bilayers were prepared with the same lipid used in the Section 6 .3 .1.1. : 1,2 Distearoyl sn Glycero 3 Phosphoethanolamine (DSPE). Following the principle s explained in the previous section, for this study the vesicles were prepared by using 2 mg of lipids dissolved in CHCl3. The solvent was evaporated from the lipid solution using an ultrapure stream of nitrogen. The lipids were then hydrated for 20 minute s (in a concentration of 2 mg/ml in Millipore water) at a temperature above their highest transition temperature (100¡C). In the next step, the solution was sonicated 5 times for periods of 2 minutes (to form small unilamellar vesicles evenly distributed i n size)  Finally, the vesicle solution wa s used to submerge the PEG grafted glass slides The temperature was kept in the 90¡C range, which was gradually lowered to allow vesicles to diffuse onto the PEG cushion layer. 184.108.40.206. Self A ssembled Soft Supported Lipid Bilayer Characte rization In the preparation of self assembled membranes using vesicles, the first step into characterization is the analysis of the vesicles size. Using the dynamic light scattering (DLS) technique and instrument (Zetasizer Nano S, Malvern, PA). The second characterizati on step is the study of the surface topography of the bilayer, and as explained for membranes deposited with LB, this surface characterization is to be done with atomic force microscopy. However, because the technique to provide the lipid bilayers using v esicles is still under investigation, only the characterization of the vesicles size was performed.
136 The results showed high instability in the formation of self assembled lipid bilayers The sizes of the vesicles obtained, were highly variably in dimensi ons (as shown in T able 8 ), and they were between 485 nm and 2348 nm. Table 8 Dynamic Light Scattering R esults giving the sizes of the multilamellar vesicles f ormed Peak Particle's D iameter (nm) 1 2347.5 401.9 2 484.8 189.3 The pres ence of large vesicles is an inconvenien ce in the formation of the lipid bilayer with this method (the optimum size should b e between 40 nm to 100 nm). The lipid used in this study (DSPE), has a long hydrophobic tail, which determine s first the creation o f rather stables and large vesicles S econd causes a high transition temperature (100 ¡ C) This high transition temperature is similar to the one required for PEG surface grafting, which could be altering this last process. T hese reasons might explain the adverse results obtained for this particular lipid In o rder to overcome the inconvenience in this technique further testing should be accomplished using short hydrophobic tail lipids with low molecular weights, such as the following : 1,2 Dipalmitoyl sn Glycero 3 Phosphocholine (DPPC), Sphingomyelin (SM. Ceramide 1 Phosphocholine. Brain, Porcine), and 1,2 Distearoyl sn Glycero 3 Phosphocholine (DSPC); all with MW between 730
137 790. Additionally, c holesterol (Ovine wool, MW 387) can also be used in combinat ion with these lipids to attain the same effect. 6.4. Chapter Remarks In this study, a fluid cell membrane in vitro model was constructed to present a brain like environment approach  with the purpose of providing a platform for future A aggregation studies. In the first stage of this proposed project, lipid (DSPE) bilayers were formed on polyethylene gl ycol cushion support (PEG) using the Langmuir Blodgett (LB) deposition technique [104, 105] The PEG layer was successfully used as a stabilizer for the bilayers. I t was observed that the PEG cushion layer offer ed high mobility to the lip id bilayer membrane (Figure 68). Also, the PEG layer was used to support self assemble d lipid bilayers formed from small unilamellar vesicles an alternative technique to prepared lipid bilayers Two important characteristics make this model a potential go od platform for in vitro A aggregation studies: First, the high fluidity provided by the cushion PEG layer underneath the lipid bilayer  give s the system fluidity that better represents that of cell membranes in mammalian cells Second, protein exclusion characteristic of PEG also prevents proteins such as A from breaching the lipid bilayer and adsorbing directly on the substrate; this property makes this platform also ideal for studies involving antibody peptide interactions by confining these to t he surface of the lipid bilayer.
138 CHAPTER 7 CONCLUSI ONS Alzheimer disease (AD) represents the main leading cause of dementia typically among individuals over the age of 65. As life expectancy increases (76 year in 2006) also does the number of AD cases increases. As of today the number of AD cases worldw ide is approximately 26 million of individual affected by the disease; and this number is estimated to increase to 106 million of cases by the year 2050. AD is known to affect the cognitive function, alter the social behavior and body motor skills, among other affections related to the nervous system Nowadays, t he causes of Alzheimer disease and onset mechanisms are yet unclear. There are still pieces missing in the puzzle to fully understand the disease and discern effective therapies to avoid and eradic ate AD From the pathology of the disease, are known three hallmarks commonly found in patients with AD, the extracellular formatio n of amyloid beta (A§) plaques, the presence of intracellular neurofibrillary tangles, and finally the neuronal death. The hypothesis that gathers these hallmarks (amyloid hypothesis) to explain the possible mechanism states that imbalance on the catabolic process of A§ peptide leads to its accumulation forming neuronal
139 plaques that trigger the formation of intracellular neuro fibrlillary tangles and finally terminate the neurons [51, 88, 89] This work was studies and charact erizes the mechanisms involved in the process of A§ plaque formation and its possible clearance by using an immunotherapeutic approach. Two isoforms of the amyloid beta peptide were investigated separately and combined in a n equimolar mixture. These were, A§ 1 40 and A§ 1 42 peptides. These two A§ isoforms are the main component of the neuritic A§ deposits (A§ plaques) found in the brain of patients with AD In order to understand the process of aggregation initial attention was rendered to the study and characterization of the different species involved in the aggregation process. When many species might be found during the process of aggregation, for the effects of this study the species were classified as monomeric oligomeric, and fibrillar. It is rele vant to mention that a myloid beta owes its name to th e characteristic of its native secondary structure composition, which is mainly the § structure type. § structures or more commonly known as § sheet structures are structures composed by lateral connecti ng § strand structures through 5 or more hydrogen bonds. § strand s are extended parts of the peptide formed by 5 to 10 amino acids. The § sheet structures can be either parallel or antiparallel, depending on the orientation of the strands forming the § s heet structures.
140 U nderstand ing these molecular configurations was important to interpret the results obtained with ATR FTIR spectroscopy; since this technique allows monitoring the secondary structures change by detecting the interactions occurring thro ugh hydrogen bonding within and between the peptide molecules. There are different points of view and disagreements on which is the most energetically stable § strands configuration (parallel or antiparallel). However, the results in this work showed that the stability of these structural configurations highly depends on the peptide isoform under study. For instance, it was observed that when A§ 1 40 fibril were forming the dominant structural configuration (in percentage) present along the process was the antiparallel type, in addition to helix structures while parallel structures were reduced considerably. In contrast, in the case of A§ 1 42 peptide fibrillization, the structural configuration of the parallel type seems to dominate the aggregation. When oligomeric species are formed the configuration of the strand structures also varied in accordance with the peptide, as for A§ 1 40 oligomeric species, the dominating arrangement was the parallel type, flipping to antiparallel when the peptides are set to form fibrils, and as for the A§ 1 42 oligomeric species the antiparallel arrangement seems to dominate (and a helix) flipping to parallel configuration or rearrangement type. Results in this study also suggested that the combination of the two peptide isofo rms shows competition between the secondary structures making the fibrils morphologically different (thicker) from the peptides alone.
141 This work included the addition of monoclonal antibodies (mAb) with specificity to A§ with the purpose to modify or rever se the fibrillization process. All experiments were carried out at close to physiological conditions (37 ¡ C and pH 7.4). The antibodies used were chosen for their particular specificity to A§ peptides and some for their potential use in therapeutic setti ngs. These antibodies were 6E10 ( binds the N terminus of both peptide isoforms ) 2H6 ( preferentially binds to the C terminus of A§ 1 40 ) 4G8 ( binds the mid domain either isoform ) and a control antibody mIgG2b that has no specificity for either A§ peptide Preformed fibrils targeted with antibodies 6E10 and 2H6 suffer secondary structural change but not in a degree that allow fibril dissolution. The initial state of aggregation resulted a convenient phase for the addition of antibodies. This is evidenced by experiments in which the target point for antibody addition where monomeric and oligomeric species. This resulted in antibody binding and altering the fibrillization process (Chapter 6). This phenomenon was observed when in two cases under equal incuba tion conditions but varying the species targeted by the antibody. Preformed fibrils in solution were not easily dissolved, but targeting monomer + oligomers in solution (at the initial fibrillization phase) resulted in an evident effect of the antibody ca using the disruption of the fibrillization process. This study points to the structural configuration of the § strands forming the § sheet structures as the key to understand and explain at the molecular
142 level the effects caused by antibodies to the diffe rent species of the peptides isoforms studied in this work. The combination of the analytical techniques (ATR FTIR, AFM, TEM) allowed the testing and evaluation of the peptide characteristics during aggregation and allowed to explain the effects of ant ibodies observed when targeting the different species of the A§ peptides. Finally, it was possible to prepare a soft substrate mimicking the cell membrane's surface (Chapter 7), laying the groundwork for future experiments. A polymeric film (PEG) grafted to SiO2 served as a cushion layer for the deposition of a double layer of lipids. Langmuir Blodgett resulted as the preferential deposition technique due to the control on monolayer uniformity before and during the lipids deposition. In the future, this m embrane could be optimized by inserting other cell membrane molecules; such as cholesterol, other lipids, sugar groups, or proteins. This interface could potentially serve to test the surface role on the process of plaque formation as function of rigidity for instance varying the amount of cholesterol inserted in the bilayer membrane.
143 CHAPTER 8 FUTURE DIRECTIONS The studies presented in this work leave a door open for new possible paths to investigate, as described below. Measurement of surface energy of A§ peptide species, in particular oligomers and protofibrils, which are the intermediate species in the process of fibrillization, this can be performed with sur face force apparatus (SFA) The effects observed in the ATR FTIR spectroscopic analy sis can be used to investigate synthetic analogs to antibodies that act on A§ peptides. ATR FTIR information can be utilized to detect secondary structure fingerprints in molecule screening processes. Other amylogenic processes such as the hyperphosphoryla tion of Tau protein (other hallmark of AD) can be studied using the methods presented in this dissertation. Complementary techniques could be used to study binding and rate of aggregation and dissolution to complement the ATR FTIR such as quartz crystal mi crobalance (QCM).
144 It is suitable to introduce polarized light to the ATR FTIR spectrometer to produce more complete information in regards to the structural orientation of the molecules. Lipid bilayer membrane construction by self assembled vesicles can be improved by introducing a mixture of lipids with different tail lengths, which might allow a controlled system at lower temperatures more suitable for the preparation of these membranes.
145 LIST OF REFERENCES  Alzhe imer's Association, 2008 Alzheimer's disease facts and figures, Alzheimer's & Dementia 4 (2008) 110 133.  Alzheimer's Association, 2009 Alzheimer's Disease Facts and Figures, Alzheimer's & Dementia 5 (2009).  R. Katzman, The Prevalence and Malignancy of Alzheimer Disease: A Major Killer, Alzheimer's and Dementia 4 (2008) 378 380.  P.C. Wong D.L. Price L. Bertram R.E. Tanzi Alzheimer's Disease: Genetics, Pathogenesis, Models, and Experimental Therapeutics in: L.P. Guarente L. Partridge D.C. W allace (Eds.), Molecular Biology of Aging Cold Spring Harbor Monograph 2007 pp. 371 408.  Alzheimer's Association, 2009 Alzheimer's Disease Facts and Figures, Alzheimer's & Dementia 5 (2009) 80.  J.R. Harris, F. Fahrendholz, Alzheimer's Disease: C ellular and Molecular Aspects of Amyloid §, Springer 2005.  J.R. Cirrito, P.C. May, M.A. O'Dell, J.W. Taylor, M. Parsadanian, J.W. Cramer, J.E. Audia, J.S. Nissen, K.R. Bales, S.M. Paul, R.B. DeMattos, D.M. Holtzman, In vivo assessment of brain intersti tial fluid with microdialysis reveals plaque associated changes in amyloid beta metabolism and half life., The Journal of Neuroscience 23 (2003) 8844 8853.  I.H. Cheng, J.J. Palop, L.A. Esposito, N. Bien Ly, F. Yan, L. Mucke, Aggressive amyloidosis in m ice expressing human amyloid peptides with the Arctic mutation, Nature Medicine 10 (2004) 1190 1192.
146  J. Naslund, V. Haroutunian, R. Mohs, K.L. Davis, P. Davies, P. Greengard, J.D. Buxbaum, Correlation Between Elevated Levels ofAmyloid § Peptide in th e Brain and Cognitive Decline, Journal of American Medical Association 283 (2000) 1571 1577.  Harvard Medical School, How the Brain Works, AARP, 2007.  B. Cordell, § Amyloid Formation as a Potential Therapeutic Target for Alzheimer's Disease, Annu. Rev. Pharmacol. Toxicol. 34 (1994) 69 89.  D.J. Selkoe, M.B. Podlisny, Deciphering the Genetic Basis of Alzheimer's Disease, Annu. Rev. Genomics Hum. Genet. 3 (2002) 67 99.  N. Suzuki, C.T. T., X.D. Cai, A. Odaka, O.J. L, C. Eckman, T.E. Golde, S. G. Younkin, An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants, Science 264 (1994) 1336 1340.  G. Bitan, M.D. Kirkitadze, A. Lomakin, S.S. Vollers, G.B. Benedek, D.B. Teplow, A myloid § Protein (A§) Assembly: A §40 and A §42 Oligomerize Through Distinct Pathways, PNAS 100 (2003) 330 335.  I. Klyubin, D.M. Walsh, C.A. Lemere, W.K. Cullen, G.M. Shankar, V. Betts, E.T. Spooner, L. Jiang, R. Anwyl, D.J. Selkoe, M.J. Rowan, Amylo id protein immunotherapy neutralizes A oligomers that disrupt synaptic plasticity in vivo Nature Medicine 11 (2005) 556 561.  G. Mamikonyan, M. Necula, M. Mkrtichyan, A. Ghochikyan, I. Petrushina, N. Movsesyan, E. Mina, A. Kiyatkin C.G. Glabe, D. H. Cribbs, M.G. Agadjanyan, Anti A§ 1 11 Antibody Binds to Different § Amyloid Species, Inhibits Fibril Formation, and Disaggregates Preformed Fibrils but Not the Most Toxic Oligomers, Journal of Biological Chemistry 282 (2007) 22376 22386.  R. Kayed, E. Head, J.L. Thompson, T.M. McIntire, S.C. Milton, C.W. Cotman, C.G. Glabe, Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis, Science 300 (2003) 486 489.
147  J.P. Cleary, D.M. Walsh, J.J. Hofmeister, G.M. Shankar, M.A. Kuskowski, D.J. Selkoe, K.H. Ashe, Natural oligomers of the amyloid § protein specifically disrupt cognitive function, Nature Neuroscience 8 (2005) 79 84.  K.N. Dahlgren A.M. Manelli, W.B. Stine Jr., L.K. Baker, G.A. Krafft, M. Jo LaDu, Oligomer ic and fibrillar species of amyloid beta peptides differentially affect neuronal viability, J. Biol. Chem. 277 (2002) 32046 32053.  C.G. Glabe, Structural Classification of Toxic Amyloid Oligomers, Journal of Biological Chemistry 283 (2008) 29639 29643  H.V.A. Olsson, N. Andreasen, G. De Meyer, A. Wallin, B. Holmberg, L. Rosengren, E. Vanmechelen, K. Blennow, Simultaneous Measurement of B amyloid (1 42), Total Tau, and Phosphorylated Tau (Thr181) in Cerebrospinal Fluid by xMap Technology, Clinical Chemistry 51 (2005) 336 345.  J.H. Growdon, S. Corkin, E. Ritter Walker, R.J. Wurtman, Aging and Alzheimer's disease : sensory systems neuronal growth, and neuronal metabolism, New York Academy of Sciences, New York, 1991.  S. Saddichha, V. Pandey, Alzheimer's and Non Alzheimer's Dementia: A Critical Review of Pharmacological and Nonpharmacological Strategies, American Journal of Alzheimer's Disease and Other Dementias 23 (2008) 150.  National Institute of Aging, Alzheimer's Disease Medications Fact Sheet, in: U.S.D.o.H.a.H. Services (Ed.), Alzheimer's Disease Education & Referral Center, 2008, p. 4.  D. Steinberg, Companies Halt First Alzheimer Vaccine Trial Investigators are looking into what Inflamed patients' brains?, The Scientist 16 (20 02) 22 23.  K. Senior, Dosing in phase II trial of Alzheimer's vaccine suspended, Lancet Neurol. 1 (2002) 3.  D. Schenk, Amyloid § immunotherapy for Alzheimer's disease: the end of the beginning., Nature Reviews Neuroscience 3 (2002) 824 828.
148  K. Aterman, A historical note on the iodine sulphuric acid reaction of amyloid Histochemistry and Cell Biology 49 (1976) 1432 1119X.  J.D. Sipe, A.S. Cohen, Review: History of Amyloid Fibril, Journal of Structural Biology 130 (2000) 88 98.  Jean D. Sipe, The pleated sheet conformation and protein folding: A brief history, in: J.D. Sipe (Ed.), Amyloid Proteins, vol. 1, Wiley VCH, 2005, pp. 49 61.  T.P. Pankaj D. Mehta, Sangita P. Mehta, Eugene A. Sersen, Paul S. Aisen, Henryk M. Wisniewski, Pla sma and cerebrospinal fluid levels of amyloid b proteins 1 40 and 1 42 in Alzheimer Disease, Archives of Neurology 57 (2000) 100 105.  M. Gralle, M.G. Botelho, F.S. Wouters, Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers, Journal of Biological Chemistry 284 (2009) 15016 15025.  T.C. Saido, A Metabolism and Alzheimer's Disease 2003.  S.A. Gravina, L. Ho, C.B. Eckman, K.E. Long, L. Otvos Jr., L.H. Younkin, N. Suzuki, S.G. Younkin, Amyloid § Protein (A§) in Alzheimer's Disease Brain, The Journal of Biological Chemistry 270 (1995) 7013 7016.  R. Garrett, C.M. Grisham, Biochemistry, Saunders College Publishing, Michigan, 1995.  R.H.a.F. Fahrendholz, Alzheimer's Disease Cellular and Molec ular Aspects of Amyloid b, Subcellular Biochemistry 2005.  W.B. Stine, K.N. Dahlgren, G.A. Krafft, M.J. LaDu, In vitro characterization of conditions for amyloid beta peptide oligomerization and fibrillogenesis, Journal of Biological Chemistry 278 (200 3) 11612 11622.  J.D. Harper, S.S. Wong, C.M. Lieber, P.T. Lansbury, Assembly of A§ Amyloid Protofibrils: An in Vitro Model for a Possible Early Event in Alzheimer's Disease, Biochemistry 38 (1999) 8972 8980.  J.M. Berg, J.L. Tymoczko, L. Stryer, B iochemistry, Fifth Edition ed., W. H. Freeman, New York, 2001.
149  C.A. Borrebaeck, Antibody Engineering, Oxford University Press US 1995.  D.R.B.a.G. C.Howard, Basics Methods in Antibody Production and Charaterization, CRC Press LLC, Boca Raton, FL, 2001.  B. Solomon, R. Koppel, E. Hanan, T. Katzav, Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta amyloid peptide, PNAS 93 (1996) 452 455.  B. Solomon, R. Koppel, D. Frankel, E. Hanan Aharon, Disaggregation of Al zheimer § amyloid by site directed mAb, Proceedings of the National Academy of Sciences of the United States of America 94 (1997) 4109 4112.  C. Hock, U. Konietzko, J. Streffer, J. Tracy, A. Signorell, B. Mller Tillmanns, U. Lemke, K. Henke, E. Moritz E. Garcia, Antibodies against § amyloid slow cognitive decline in Alzheimer's disease, Neuron 38 (2003) 547 554.  D. Morgan, B.D. Gitter, Evidence supporting a role for anti Ab antibodies in the treatment of Alzheimer's disease, Neurobiology of Aging 25 (2004) 605 608.  D. Morgan, D.M. Diamond, P.E. Gottschall, K.E. Ugen, C. Dickey, J. Hardy, K. Duff, P. Jantzen, G. DiCarlo, D. Wilcock, K. Connor, J. Hatcher, C. Hope, M. Gordon, G.W. Arendash, A§ peptide vaccination prevents memory loss in an anim al model of Alzheimer's disease, Nature 408 (2000) 982 985.  D. Morgan, B.D. Gitter, Evidence supporting a role for anti A§ antibodies in the treatment of Alzheimer s disease, Neurobiology of Aging 25 (2004) 605 608.  D.M. Wilcock, S.K. Munireddy, A. Rosenthal, K.E. Ugen, M.N. Gordon, D. Morgan, Microglial activation facilitates A § plaque removal following intracranial anti A § antibody administration, Neurobiolog y of Disease 15 (2004) 11 20.  D.M. Wilcock, A. Rojiani, A. Rosenthal, G. Levkowitz, S. Subbarao, J. Alamed, D. Wilson, N. Wilson, M.J. Freeman, M.N. Gordon, D. Morgan, Passive Amyloid Immunotherapy Clears Amyloid and Transiently Activates Microglia in a Transgenic Mouse Model of Amyloid Deposition, The Journal of Neuroscience 24 (2004) 6144 6151.
150  N.C. Carty, D.M. Wilcock, A. Rosenthal, J. Grimm, J. Pons, V. Ronan, P.E. Gottschall, M.N. Gordon, D. Morgan, Intracranial administration of deglycosylat ed C terminal specific anti A antibody efficiently clears amyloid plaques without activating microglia in amyloid depositing transgenic mice, Journal of Neuroinflammation 3:11 (2006) 1 11.  D. Morgan, Immunotherapy for Alzheimer's disease Journal of A lzheimer's Disease 9 (2006) 425 432.  D.M. Wilcock, J. Alamed, P.E. Gottschall, J. Grimm, A. Rosenthal, J. Pons, V. Ronan, K. Symmonds, M.N. Gordon, D. Morgan, Deglycosylated Anti Amyloid § Antibodies Eliminate Cognitive Deficits and Reduce Parenchymal Amyloid with Minimal Vascular Consequences in Aged Amyloid Precursor Protein Transgenic Mice, The Journal of Neuroscience 26 (2006) 5340 5346.  D.M. Wilcock, P.T. Jantzen, Q. Li, D. Morgan, M.N. Gordon, Amyloid § vaccination, but not nitro nonsteroida l anti inflammatory drug treatment, increases vascular amyloid and microhemorrhage while both reduce parenchymal amyloid, Neuroscience 144 (2007) 950 960.  D. Morgan, The rationale for an immunological approach to Alzheimer's therapeutics, Pharmacologi cal Mechanisms in Alzheimer's Therapeutics, Springer, 2008, pp. 141 148.  T.N. Bhat, G.A. Bentley, G. Boulot, M.I. Greene, D. Tello, W. Dall' Acqua, H. Souchon, F.P. Schwarz, R.A. Mariuzza, R.J. Poljak, Bound water molecules and conformational stabiliz ation help mediate an antigen antibody association, Proceedings of the National Academy of Sciences of the United States of America 91 (1994) 1089 1093.  G.A.B. T. Narayana Bhat, Ginette Boulot, Mark I. Greene, Diana Tello, William Dall Acqua, Helene S ouchon, Frederick P. Schwarz, Roy A. Mariuzza, Bound water molecules and conformational stabilization help mediate an antigen antibody association, Proceedings of the National Academy of Sciences of the United States of America 91 (1994) 1089 1093.  D. L. Ed Harlow, Using Antibodies, Cold Spring Harbor Laboratory Press 1998.  S.E. Amelinckx, D.E. van Dyck, J.E. van Landuyt, G.v.T. (Editor), Electron Microscopy: Principles and Fundamentals, Wiley 2008.
151  V.J. Morris, A.R. Kirby, A.P. Gunning, Atomi c force microscopy for biologists, Imperial College Press 1999.  I. Horcas, R. Fernndez, J.M. Gmez Rodrguez, J. Colchero, J. Gmez Herrero, A.M. Baro, WSXM: A software for scanning probe microscopy and a tool for nanotechnology, Revier of Scientific Instruments 78 (2007) 013705 013701 to 013705 013708.  A. Baumketner, S.L. Bernstein, T. Wyttenbach, G. Bitan, D.B. Teplow, M.T. Bowers, J. E. Shea, Amyloid b protein monomer structure: A computational and experimental study, Protein Science 15 (2006) 420 428.  M. Jackson, H.H. Mantsch, The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Struture, Critical Reviews in Biochemistry and Molecular Biology 30 (1995) 95 120.  E. Goormaghtigh, V. Cabiaux, J.M. Ruysschaert, Determin ation of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds., Sub cellular Biochemistry 23 (1994) 329 362.  T. Wiyasawa, T. Shimanouchi, S. i. Mizushima, Normal Vibrations of N Methylac etamide, Journal of Chemical Physiscs 29 (1958) 611 616.  W. Moore, S. Krimm, Transition Dipole Coupling in Amide I Modes of ss Polypeptides, Proceedings of the National Academy of Sciences 72 (1975) 4933 4935.  D.M. Byler, H. Susi, Examination of the secondary structure of proteins by deconvolved FTIR spectra Biopolymers 25 (1986) 469 487.  J.T. Pelton, L.R. McLean, Review: Spectroscopy Methods for Analysis of Protein Secondary Structure, Analytical Biochemistry 277 (2000) 167 176.  M.A. Wo uters P.M.G. Curmi, An analysis of side chain interactions and pair correlations within antiparallel beta sheets: the differences between backbone hydrogen bonded and non hydrogen bonded residue pairs., Proteins 22 (1995) 119 131.
152  M. Jackson, H.H. Mantsch, Protein secondary structure from FT IR spectroscopy: correlation with dihedral angles from three dimensional Ramachandran plots Canadian Journal of Chemistry 69 (1991) 1639 1642.  Y. Abe, S. Krimm, Normal vibrations of crystalline polyglycine I, Biopolymers 11 (1972) 1817 1839.  S. Krimm, Y. Abe, Intermolecular Interaction Effects in the Amide I Vibrations of § Polypeptides, Proceedings of the National Academy of Sciences of the United States of America 69 (1972) 2788 2792.  J. Jimenez, Systematic study of amyloid beta peptide conformations: Implications for Alzheimer's disease, Department of Chemical Engineering, University of South Florida, Tampa, 2005, p. 103.  Y.N. Chirgadze, N.A. Nevskaya, Infrared spectra and resonance interact ion of amide I vibration of the antiparallel chain pleated sheet, Biopolymers 15 (1976) 607 625.  N. Demirdoven, C.M. Cheatum, H.S. Chung, M. Khalil, J. Knoester, A. Tokmakoff, Two dimensional infrared spectroscopy of antiparallel § sheet secondary str ucture, Journal American Chemical Society 126 (2004) 7981 7990.  C. Warthon, Infrared Spectroscopy of Enzyme Reaction Intermediates, Natural Products Report 17 (2000) 447 453.  A. Barth, Infrared Spectroscopy, in: V.N. Uversky, E.A. Permyakov (Eds. ), Methods in Protein Structure and Stability Analysis: Vibrational Spectroscopy, Nova Publisher, New York, 2007, pp. 69 151.  I.H.a.M.Y. Abraham Fisher, Progress in Alzheimer's and Parkinson's Diseases, New York, 1998.  G. Tiana F. Simona R.A. B roglia G. Colombo Thermodynamics of § amyloid fibril formation The Journal of Chemical Physics 120 ( 2005 ) 8307 8317  H.A. Lim, N.A. Kolchanov, Computer analysis of genetic macromolecules: structure, function, and evolution, World Scientific 1994.
153 [ 80] C. Hock, U. Konietzko, A. Papassotiropoulos, A. Wollmer, J. Streffer, R.C. von Rotz, G. Dvaey, E. Moritz, R.M. Neitsch, Generation of antibodies specific for beta amyloid by vaccination of patients with Alzheimer's disease, Nature medicine 8 (2002) 127 0.  J.P. Jimenez, Systematic Study of Amyloid Beta Peptide Conformations: Implications for Alzheimer's Disease, Chemical Engineering Deptartment vol. M.S. University of South Florida Tampa, 2005, p. 91.  R. Riek, S. Hornemann, G. Wider, M. Billet er, R. Glockshuber, K. Wuthrich, NMR Structure of the Mouse Prion Protein Domain PrP(121 231), Nature 382 (1996) 180 182.  J.D. Sipe, A.S. Cohen, Review: History of the Amyloid Fibril, Journal of Structural Biology 130 (2000) 88 98.  J.D. Sipe, The § pleated sheet conformation and protein folding: A brief history, in: J.D. Sipe (Ed.), Amyloid Proteins: The Beta Sheet Conformation and Disease, vol. 1, Wiley VCH, 2005, pp. 49 61.  H. James D., L.J. Peter T., MODELS OF AMYLOID SEEDING IN ALZHEIMER' S DISEASE AND SCRAPIE:Mechanistic Truths and Physiological Consequences of the Time Dependent Solubility of Amyloid Proteins, Annual Review of Biochemistry 66 (1997) 385 407.  H. Shao, S. c. Jao, K. Ma, M.G. Zagorski, Solution structures of micelle bou nd amyloid § (1 40) and § (1 42) peptides of Alzheimer's disease, Journal of Molecular Biology 285 (1999) 755 773.  G. Bitan, M.D. Kirkitadze, A. Lomakin, S.S. Vollers, G.B. Benedek, D.B. Teplow, Amyloid protein (A ) assembly: A 40 and A 42 oligomeri ze through distinct pathways, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 330 335.  D.I. Dominguez, B.D. Strooper, Novel therapeutic strategies provide the real test for the amyloid hypothesis of Alzheimer 's disease, Trends in Pharmacological Sciences 23 (2002) 324 330.  J. Hardy, D.J. Selkoe, The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics, Science 297 (2002) 353 356.
154  M. Zhu, S. Han, F. Zhou, S.A. C arter, A.L. Fink, Annular Oligomeric Amyloid Intermediates Observed by in Situ Atomic Force Microscopy, J Biol. Chem. 279 (2004) 24452 24459.  A.J. Modler, K. Gast, G. Lutsch, G. Damaschun, Assembly of Amyloid Protofibrils via Critical Oligomers A Nove l Pathway of Amyloid Formation, Journal of Molecular Biology 325 (2003) 135 148.  G. Bitan, M.D. Kirkitadze, A. Lomakin, S.S. Vollers, G.B. Benedek, D.B. Teplow, Elucidation of primary structure elements controlling early amyloid beta protein oligomeri zation., The Journal of Biological Chemistry 278 (2003) 34882 34889.  M. Simons, P. Keller, J. Dichgans, J. Schulz, Cholesterol and Alzheimer's disease Is there a link?, Neurology 57 (2001) 1089 1093.  S. Dante, T. Hau§, N. Dencher, Cholesterol inh ibits the insertion of the Alzheimer's peptide A§ (25 35) in lipid bilayers, European Biophysics Journal 35 (2006) 523 531.  J. Jimenez, A.J. Heim, W.G. Matthews, N. Alcantar, Construction and Characterization of Soft Supported Lipid Bilayer Membranes for Biosensors Application, Proceedings of the 28th IEEE. EMBS Annual International Conference. New York City, USA Aug 30 Sept 3 (2006) 4119 4122.  N.A. Alcantar, Plasma Assisted Synthesis and Characterization of Biocompatible Surface coatings, Chemica l Engineering, University of California Santa Barbara, Santa Barbara, 2000, p. 237.  N. Alcantar, J. Israelachvili, J. Boles, Forces and ionic transport between mica surfaces: implications for pressure solution, Geochimica Et Cosmochimica Acta 67 (2003 ) 1289 1304.  K.B. Blodgett, Films Built by Depositing Successive Monomolecular Layers on a Solid Surface, Journal of the American Chemical Society 57 (1935) 1007 1022.  K.B. Blodgett, Monomolecular Films of Fatty Acids on Glass Journal of the Amer ican Chemical Society 56 (1934) 495.
155  L. Aho, J. Jolkkonen, I. Alafuzoff, § Amyloid Aggregation in Human Brains With Cerebrovascular Lesions, Stroke 37 (2006) 2940 2945.  A.D. Bangham, Membrane Models with Phospholipids, Prog. Biophys. Mol. Biol. 18 (1968) 29.  J.M. J. Y. Wong, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith, Polymer Cushioned Bilayers. I. A Structural Study of Various Preparation Methods Using Neutron Reflectometry, Biophysical Journal 77 (1999) 1445 1457.  J imenez Jeffy, Systematic Study of Amyloid Beta Peptide Conformations: Implications for Alzheimer's Disease, Department of Chemical Engineering, University of South Florida, Tampa, 2005, p. 103.  J. Jimenez, A.J. Heim II, G. Matthews, N. Alcantar, Cons truction and Characterization of Soft Supported Lipid Bilayer Membranes for Biosensors Application Proceedings of the 28th IEEE. EMBS Annual International Conference. New York City, USA Aug 30 Sept 3 (2006) 4119 4122.  N.A. Alcantar, E.S. Aydil, J.N. Israelachvili, Polyethylene glycol coated biocompatible surfaces, Journal of Biomedical Materials Research 51 (2000) 343 351.
156 APPENDI C ES
15 7 Appendix A. Isoelectric Points of A§ Peptides Figure 69 Titration Curve of A§ 1 40 Indicating its Isoelectric Point. Obtained from http://biophysics.cs.vt.edu/H++/ (Virginia Tech) using the Protein Data Bank for the peptide sequence. Figure 70 Titrat ion Curve of A§ 1 42 Indicating its Isoelectric Point. Obtained from http://biophysics.cs.vt.edu/H++/ (Virginia Tech) using the Protein Data Bank for the peptide sequence.
158 Appendix B. ATR Crystals Table 9 Limitations and General Specifications of ATR Crystals Material n1 LWL [cm 1 ] dp [ # m] S water [g/100g] pH Range Hardness [Kg/mm 2 ] AMTIR 2.5 625 1.46 Insoluble 1 9 170 Diamond/ZnSe 2.4 525 1.66 Insoluble 1 14 5,700 Diamond/KRS 5 2.4 250 1.66 Insoluble 1 14 5,700 Germanium 4.0 780 0.65 Insoluble 1 14 550 KRS 5 2.4 250 1.73 0.05 5 8 40 Silicon 3.4 1500 0.84 Insoluble 1 12 1150 Silicon/Znse 3.4 525 0.84 Insoluble 1 12 1150 ZnS 2.2 850 2.35 Insoluble 5 9 240 ZnSe 2.4 525 1.66 Insoluble 5 9 120 n1 = re fractive index of ATR crystal, LWL = long wave length cut off, dp = depth of penetration at 1000 cm 1 S water = Solubility in Water
ABOUT THE AUTHOR Jeffy Pilar Jimnez received her Bachelors degree in Chemical Engineering from Los Andes University in Mrida-Venezuela in 2001 and her Masters degree in Chemical Engineering from the University of South Florida (USF) in 2005. In 2005 she started her Ph.D. in Chemical Engineering program at USF, in 2006 she had a summer internship exper ience at the R&D department for Dow Chemical. In every step of her career she has been moved by the passion for research. Jeffy has been exchanging and leaving scientific, mentoring, and cultural experiences in the group of Norma Alcantar (Ph.D.) since 2003, also guided by David Morgan (Ph.D.). Jeffys research focused on elucidating mechanisms responsible for A aggregation and dissolution by the mean of monoclonal antibodies, studies that have been conducted in-vitro at the Nanosurface-Chemistry and Green Materials Chemistry Laboratory. She successfully defended her doctoral dissertation in April 20 10 at the University of South Florida.
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Effects of monoclonal anti-abeta antibodies on the amyloid beta peptide fibrillogenesis and their involvement in the clearance of alzheimer's disease plaques
h [electronic resource] /
by Jeffy Jimenez.
[Tampa, Fla] :
b University of South Florida,
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Dissertation (Ph.D.)--University of South Florida, 2010.
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ABSTRACT: Alzheimer's disease (AD) is the most common cause of senile dementia worldwide. AD is a neurodegenerative disorder characterized by the loss of memory and language skill, collapse of the cognitive function, and distortion of social behavior. As of today, the onset mechanisms of AD and cure are unknown; however, three hallmarks are commonly encountered: extra and intracellular accumulation of amyloid beta (Abeta) peptide plaques, formation of intracellular neurofibrillary tangles, and inevitable neuronal death. Hypothetically, a possible scenario provoking or involved in the onset of AD is a cascade effect that starts with an imbalance in the production and clearance of Abeta peptide that consequently leads to its accumulation, formation of tau protein tangles and neuronal death. This work studied and characterized the mechanisms governing Abeta peptide aggregation and the effects of using anti-Abeta monoclonal antibodies to modify this process. These mechanisms play an important role in the formation of AD plaques and are critical in the search for therapies involving Abeta peptide plaque clearance. Yet, antibody-based therapies for plaque clearance are not well understood, adding to the existing concerns about side effects in humans, hence there is a necessity of knowledge in this matter. In this work different N-terminus, C-terminus, and Mid-domain antibodies were used against Abeta peptide species (monomers, oligomers, and fibrils) to probe peptide aggregates modification and disruption. Additionally, construction of a soft supported lipid bilayer membrane was proposed to study the adhesion mechanisms of Abeta peptide and interactions with antibodies, mimicking the neuronal cell surface. The main characterization techniques used in this work were: atomic force microscopy (AFM) and transmission electron microscopy that allowed the physical exploration and visualization of the different processes of aggregation in terms of adhesion, size evolution, and distribution of the peptide; and attenuated total reflectance Fourier spectroscopy (ATR/FTIR) which allowed monitoring the change of secondary structures for the peptide during the processes studied. It is endeavored that this work will help to elucidate the effects attributed to the molecular interactions between Abeta peptide species and antibodies to target Abeta plaque's clearance in the brain of AD patients. Ultimately, this study provides novel information critical for the formulation of effective therapies to prevent and treat AD with less collateral effects. It also represents a contribution to the basic scientific knowledge regarding peptide-antibody interactions with application to other diseases related to protein misfolding.
Advisor: Norma Alcantar, Ph.D.
x Chemical Engineering
t USF Electronic Theses and Dissertations.