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bioRxiv · 10.1101/2023.03.03.530975

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity

Upadhyay, A.·Chhangani, D.·Rao, N. R.·Kofler, J.·Vassar, R.·Rincon-Limas, D. E.·Savas, J. N.
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Abstract

BackgroundThe accumulation of amyloid beta (A{beta}) peptides in fibrils is prerequisite for Alzheimers disease (AD). Our understanding of the proteins that promote A{beta} fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. MethodsTo investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of A{beta} immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic A{beta}42 flies, and A{beta}42 seeded cultured neurons. ResultsAmyloid fibrils are primarily composed by A{beta}42 and unexpectedly harbor A{beta}38 but generally lack A{beta}40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 17 new amyloid-associated proteins. Notably, we confirmed 126 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic A{beta}42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Eight RNAi lines suppressed and 11 enhanced A{beta}42 toxicity. ConclusionsThese discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

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Upadhyay, A., Chhangani, D., Rao, N. R., Kofler, J., Vassar, R., Rincon-Limas, D. E., Savas, J. N.. 2023-03-03. Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity. https://doi.org/10.1101/2023.03.03.530975

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