Characterization of naturally occurring, pathogenic and benign Aβ multimers Why don't monkeys get Alzheimer's disease? Open Access

Rosen, Rebecca (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/pn89d6592?locale=en
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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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