Investigating Structural Heterogeneities of Amyloid Aggregates with Spatially Resolved Infrared Spectroscopy

Abstract

The aggregation of proteins into fibrillar amyloids is central to the pathology of neurodegenerative disorders. However, the relationship between the structure of amyloid aggregates and disease progression remains incompletely understood, in part because amyloid systems are intrinsically heterogeneous: structurally distinct species coexist at every stage of aggregation, and conventional ensemble-averaged methods cannot always resolve them. This Account describes how infrared spectroscopy can be coupled to different imaging modalities to resolve this heterogeneity at multiple length scales: from single-aggregate level to amyloid species in diseased brain tissues.

Our findings demonstrate that (i) early stage tau fibrils exhibit pronounced structural polymorphism without morphological differences, converging to a single dominant conformation upon maturation that unexpectedly includes antiparallel β-sheet structure; (ii) Aβ42 oligomeric heterogeneity propagates through protofibrils into fibrils, where structurally distinct domains coexist along a single fibril, demonstrating that amyloid fibrils can deviate significantly from the canonical parallel cross-β architecture; (iii) Aβ(16–22) fibrils undergo a parallel-to-antiparallel β-sheet transition during maturation through disordered intermediates without morphological change, providing direct evidence of spontaneous structural reorganization in fibrillar species. Beyond single-protein systems, (iv) Aβ42 forms structurally distinct mixed polymorphs when aggregated with sequentially or structurally heterotypic seeds and when coaggregated with α-synuclein, establishing heterotypic interactions as an underappreciated source of aberrant aggregate structures; (v) coaggregation of Aβ with the islet amyloid polypeptide (IAPP) from the monomeric state produces a unique heterotypic polymorph in which both peptides adopt non-native structures, whereas cross-seeding with preformed IAPP fibrils yields predominantly homotypic Aβ aggregates, demonstrating that conformational flexibility at the monomeric stage is required for heterotypic fibril formation. These molecular-level findings are complemented by tissue-scale evidence: (vi) discrete frequency IR imaging reveals that amyloid plaques in AD brain exhibit chemical and structural heterogeneities not reflected in morphology, consistent with in vitro observations; and (vii) optical photothermal infrared (O-PTIR) spectroscopic imaging of vascular amyloid deposits in cerebral amyloid angiopathy (CAA) reveals a disease-stage-dependent increase in antiparallel β-sheet content that correlates specifically with colocalized lipids, recapitulated in vitro by coaggregating Aβ40 with lipid extracts. Collectively, these findings support the view that structural heterogeneity is not merely an artifact of in vitro conditions but a property that can persist in human brain tissues and illustrate how spatially resolved IR spectroscopy can complement cryogenic electron microscopy (cryo-EM) and solid-state NMR in the study of amyloid pathology.