α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent β-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs.

Aggregated Tau protein is associated with over 20 neurological disorders including Alzheimer’s disease. Previous work has shown that Tau’s sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length Tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of Tau aggregation. Two structures of this segment determined by the cryo EM method MicroED explain its dominant influence on Tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit Tau aggregation but also inhibit the ability of exogenous full-length Tau fibrils to seed intracellular Tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of Tau that may account for a subset of prion-like strains of Tau.

Alzheimer’s disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

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