Amplification of amyloid-beta strains and their connection to tau in Alzheimer’s Disease Open Access
Li, Noel (Summer 2018)
Published
Abstract
The formation of amyloid-beta (Aβ) and tau aggregates are pathological hallmarks of Alzheimer’s disease (AD), yet their etiological roles in the disease are unknown despite decades of research. Different strains, or polymorphic structures, of Aβ have been resolved and it is thought that their conformations have an influence on their downstream toxicities. To determine how Aβ fibrils in AD and ND (non-demented healthy control) brains are different, we amplified them by seeding into synthetic Aβ. Disaggregation of purified Aβ peptides with NH4OH was essential for maximizing seeding efficiency. Brain homogenates were enriched for Aβ to reach the critical seed concentration, and we seeded them into Aβ40 and Aβ42 sequentially for two generations. The oligothiophene HS-68 spectrally distinguished quiescent, agitated and brain-seeded Aβ40, but not between AD- and ND-seeded Aβ40. Oligothiophene binding to seeded Aβ42 showed more differences, with pentamer formyl thiophene acetic acid (pFTAA) displaying different relative peak intensities at 425 nm in frontal lobe-seeded AD and ND brain. A 1% molar equivalent of assembled Aβ sufficiently lowered the critical assembly concentration of PHF6 (the nucleating core of tau) and catalyzed fibrillization. Both Aβ seeds and fibrils had similar cross-seeding rates, suggesting a combination of elongation and secondary nucleation mechanisms. PHF6 monomers cross-seeded by brain-seeded Aβ42 showed biphasic growth, which we attribute to cross-seeding in the initial ten hours and self-seeding taking over after a critical mass of PHF6 fibrils have formed. AD frontal lobe-seeded Aβ42 cross-seeded the formation of short PHF6 fibrils which self-seeded efficiently, while ND frontal lobe-seeded Aβ42 cross-seeded the formation of long, bundled PHF6 fibers which were ineffective seeds, analogous to observations in yeast prion strains. Our results suggest that structural differences in Aβ can be directly propagated to tau in AD, highlighting an attractive therapeutic opportunity at the Aβ-tau triggering point that needs to be explored, rather than current Aβ-focused approaches.
Table of Contents
Chapter 1. Introduction
Alzheimer’s Disease (AD)............................................................................................... 1
Molecular pathology ..................................................................................................... 3
1.1 Amyloid precursor protein (APP) ............................................................................... 3
1.2 Amyloid-beta (Aβ) production .................................................................................. 3
1.3 Structural heterogeneity in amyloid fibrils .................................................................. 7
1.4 Tau hyperphosphorylation and aggregation ................................................................ 9
Probes for detecting Aβ and tau ................................................................................... 11
1.5 Pittsburgh compound B (PiB) .................................................................................. 11
1.6 Luminescent conjugated oligothiophenes (LCOs) ....................................................... 14
1.7 Tau-specific ligands ............................................................................................... 16
Strategies for developing AD therapeutics ..................................................................... 17
1.8 β- and γ-secretase inhibitors .................................................................................. 17
1.9 Aggregation inhibitors and modulators ..................................................................... 17
1.10 Metal protein-attenuating compounds (MPACs) ....................................................... 18
1.11 Immune system activation.................................................................................... 18
1.12 Focused ultrasound (FUS) .................................................................................... 20
Conclusion ................................................................................................................ 20
References ................................................................................................................ 21
Chapter 2. Disaggregation of peptides into monomers
Introduction .............................................................................................................. 31
Results ..................................................................................................................... 34
2.1 Hexafluoroisopropanol (HFIP) ................................................................................. 34
2.2 Dimethyl sulfoxide (DMSO) .................................................................................... 41
2.3 Ammonium hydroxide ........................................................................................... 43
Conclusion ................................................................................................................ 48
Materials and Methods ................................................................................................ 49
References ................................................................................................................................ 51
Chapter 3. Pittsburgh Compound B fluorescence binding assay
Introduction .............................................................................................................. 53
Results ..................................................................................................................... 54
3.1 Factors influencing the fluorescence of PiB ............................................................... 54
3.2 Separation of bound and unbound PiB with spin filters ............................................... 63
3.3 Separation of bound and unbound PiB by centrifugation ............................................. 68
3.4 Separation of bound and unbound PiB with fiberglass filters ........................................ 71
Conclusion ................................................................................................................ 72
Materials and Methods ................................................................................................ 73
References ................................................................................................................ 75
Chapter 4. Seeding of brain extracts into synthetic Aβ
Introduction .............................................................................................................. 77
Results ..................................................................................................................... 78
4.1 Propagation of agitated Aβ fibrils under quiescent conditions ...................................... 78
4.2 Seeding of LSS (low-speed supernatant) brain extracts into synthetic Aβ ..................... 80
4.3 Stability of seeded Aβ fibrils to freeze-thawing .......................................................... 84
4.4 [3H]-PiB, [3H]-X-34 and pFTAA binding to brain-seeded fibrils .................................... 90
4.5 Enrichment of brain extracts for more specific amyloid seeding ................................... 92
4.6 Granddaughter Aβ40 fibrils seeded from enriched LSS brain extracts ........................... 97
4.7 Seeding of enriched LSS extracts into Aβ42 .............................................................. 98
4.8 Enrichment of brain homogenates and seeding into Aβ40 ......................................... 102
4.9 Seeding of enriched brain homogenates into Aβ42 ................................................... 116
4.10 Immunogold labeling of a high-affinity PiB-binding fraction ..................................... 123
4.11 Co-assembly of Aβ with nucleic acids and metal ions .............................................. 124
Conclusion ............................................................................................................... 129
Materials and Methods .............................................................................................. 131
References .............................................................................................................. 139
Chapter 5. Amyloid-β and Tau Interactions
Introduction ............................................................................................................ 144
Results ................................................................................................................... 145
5.1 Cross seeding of PHF6 by Aβ(16-22) E22Q and E22L variants .................................. 145
5.2 Co-assembly of Aβ40 and 42 with PHF6 ................................................................. 165
5.3 Cross-seeding of Aβ40 and 42 with PHF6 ............................................................... 168
Conclusion............................................................................................................... 176
Materials and Methods .............................................................................................. 177
References .............................................................................................................. 179
Chapter 6. Conclusion
Summary ................................................................................................................ 181
Outlook ................................................................................................................... 183
References .............................................................................................................. 185
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