Extraction of\nIn-Cell β‑Amyloid Fibrillar\nAggregates for Studying Molecular-Level Structural Propagations Using\nSolid-State NMR Spectroscopy

Molecular-level structural polymorphisms of β-amyloid\n(Aβ)\nfibrils have recently been recognized as pathologically significant.\nHigh-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy\nhas been utilized to study these structural polymorphisms, particularly\nin ex-vivo fibrils seeded from amyloid extracts of post-mortem brain\ntissues of Alzheimer’s disease (AD) patients. One unaddressed\nquestion in current ex-vivo seeding protocol is whether fibrillation\nfrom exogenous monomeric Aβ peptides, added to the extracted\nseeds, can be quantitatively suppressed. Addressing this issue is\ncritical because uncontrolled fibrillation could introduce biased\nmolecular structural polymorphisms in the resulting fibrils. Here,\nwe present a workflow to optimize the key parameters of ex-vivo seeding\nprotocols, focusing on the quantification of amyloid extraction and\nthe selection of exogenous monomeric Aβ concentrations to minimize\nnonseeded fibrillation. We validate this workflow using three structurally\ndifferent 40-residue Aβ (Aβ<sub>40</sub>) fibrillar seeds,\ndemonstrating their ability to propagate their structural features\nto exogenous wild-type Aβ<sub>40</sub>.