Characterization of naturally occurring, pathogenic and benignAβ multimers Why don't monkeys get Alzheimer'sdisease? 公开
Rosen, Rebecca (2009)
Abstract
According to the amyloid cascade hypothesis of Alzheimer's disease (AD) pathogenesis, the aberrant multimerization of the β-amyloid peptide (Aβ) is a crucial early event in AD neurodegeneration and dementia. Aβ is a self-aggregating peptide that accumulates with age in the brains of humans, apes, and monkeys. However, the full spectrum of AD pathology and dementia has never been reported in a nonhuman primate. As our closest living relatives, nonhuman primates are thus unique models of nonpathologic Aβ accumulation. I hypothesized that toxic Aβ aggregates in AD brain are structurally distinct from Aβ multimers formed in nonhuman primate brains. To this end, I extensively characterized Aβ populations in postmortem cortical samples from AD patients, aged great apes (chimpanzees), Old World monkeys (rhesus macaques), and New World monkeys (squirrel monkeys). Using immunohistochemistry, ELISA, immunoprecipitation/MALDI-TOF MS, Western Blot and in vivo Aβ-seeding assays, I found that cortical Aβ populations are quantitatively and qualitatively similar in AD and aged nonhuman primates. The major cerebral Aβ isoforms (Aβ40 and Aβ42) accumulate at comparable levels and in similar ratios in AD and all nonhuman primate groups examined. Furthermore, post-translationally modified Aβ isoforms and low molecular weight Aβ multimers are strikingly similar in AD and aged nonhuman primate cortical homogenates. I then analyzed higher-order structural features of AD and nonhuman primate Aβ aggregates using Pittsburgh Compound B (PIB), a radioligand designed for in vivo PET imaging of β-amyloid. I confirmed that 3H-PIB binding correlates positively with levels of insoluble Aβ40 and Aβ42 in AD cortical homogenates. However, 3H-PIB binds with very low stoichiometry to Aβ in nonhuman primate cortical homogenates, even in cases with levels of Aβ equal to those in AD. These data suggest that, despite a common amino acid sequence, cerebral Aβ multimers are structurally distinct in AD and aged nonhuman primates, and that high-affinity PIB binding may be selective for a pathogenic molecular conformation of Aβ in AD brain. The identification of structural differences between naturally occurring, pathogenic and benign Aβ multimers could yield important clues to the uniquely human susceptibility to AD, and thereby reveal new molecular targets for AD therapeutics.
Table of Contents
Chapter 1. Introduction and Background...1
The Evolution of Human Aging and Alzheimer's Disease...1 Alzheimer's disease: History...9 Alzheimer's disease: Diagnosis...12 The Aβ Amyloid Cascade Hypothesis of Alzheimer's Disease Pathogenesis...18 Alzheimer's Disease: Therapeutics...23 Toxic Aβ Multimers in AD Brain...26 Animal Models of Cerebral Aβ Accumulation...29 Cerebral Aβ Deposition in Nonhuman Primates...32 The Evolution of ApoE and Alzheimer's Disease...36 Toxic and Benign Aβ "Strains"...38 Conclusions and Hypothesis...41
Chapter 2. Is Alzheimer's a Human Specific Disease?...43
Introduction...43 Materials and Methods...47
Subjects...47 Collection and preparation of cerebral tissue samples...48 Antibodies and reagents...49 Histochemistry...51 Quantitative mapping of tau and Aβ lesions...53 Electron microscopy...54 Aβ ELISA...55 DNA extraction and MAPT genotyping...56
Results...70
Tau histopathology...70 Aβ histopathology...71 Aβ ELISA...72 MAPT genotyping...72
Discussion...73
Chapter 3. Characterization of Aβ Peptide Populations in Aged Human and Nonhuman Primate Brain...86
Introduction...86 Methods...92
Subjects...92 Tissue collection and preparation...93 Antibodies...94 Immunohistochemistry...95 Quantitative Aβ ELISA...96 Aβ Immunoprecipitation/MALDI-TOF MS...96 Western blot...98 Statistical analysis...99
Results...123
Aβ Immunohistochemistry...123 Quantification of Aβ40 and Aβ42...124 MALDI-TOF MS...126 Aβ Immunoblot...128
Discussion...129
Aβ Immunohistochemistry...130 Quantification of Aβ isoforms...131 Mass spectrometric detection of modified Aβ isoforms...132 Multimeric Aβ in AD and nonhuman primate brain...133
Chapter 4. PIB Binding in Aged Primate Brain: Enrichment of High-Affinity Sites In Humans with Alzheimer's Disease...136
Introduction...136 Methods...139
Subjects...139 Preparation of tissue samples...140 ELISA quantification of Aβ40 and Aβ42...140 3H-PIB binding assay...141 Immunohistochemistry...142 3H-PIB autoradiography...143 Statistical analysis...144
Results...155
Aβ and tau pathology in human and nonhuman primate brain...155 Quantification of Aβ40 and Aβ42...155 In vitro 3H-PIB binding...156 3H-PIB autoradiography...158
Discussion...158
Chapter 5. Deficient High-Affinity Binding of Pittsburgh Compound B in a Case of Alzheimer's Disease...163
Introduction...163 Materials and Methods...165
Subject...165 Comparison subjects...166 Tissue preparation...166 DNA sequencing for the β-amyloid precursor protein (APP), presenilin 1 (PSEN-1) and presenilin 2 (PSEN-2)...167 Immunohistochemistry...168 Electron microscopy...169 3H-PIB binding assay...169 3H-PIB autoradiography...171 ELISA quantification of Aβ40 and Aβ42...171 Western blot...172 Immunoprecipitation/MALDI-TOF MS...172
Results...190
APP, PSEN1, and PSEN2 gene sequencing...190 Histopathology...190 3H-PIB binding and Aβ analysis...191 Aβ MALDI-TOF mass spectrometry...193
Discussion...193
Chapter 6. In vivo Characterization of Naturally Occurring, Pathogenic and Benign Aβ Multimers...203
Introduction...203 Materials and Methods...206
Subjects...206 Tissue preparation...207 Stereotaxic mouse surgeries...208 Antibodies...209 Immunohistochemistry...210 Aβ ELISA...211 Autoradiography...212
Results...225
Aβ deposition in AD and aged nonhuman primate brain...225 In vivo seeding with AD and aged squirrel monkey cortical extract...226 Seeding of high-affinity PIB binding sites in transgenic mouse brain...227
Discussion...228
Chapter 7. Discussion...233
Adventures in Tissue Collection...235 Alzheimer's Pathology in a Chimpanzee?...236 Aβ in Aged Nonhuman Primate Brain...239 Aβ is Structurally Distinct in AD and Nonhuman Primate Brain...241 Can PIB Distinguish Strains of Aβ in AD Brain?...243 In vivo Seeding with Aβ Multimers from AD and Monkey Brain...244 Significance and Future Directions...246
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