Strain-Distinct α-Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal...

The co-occurrence of α-synuclein (αSyn) and Tau in synucleinopathies and tauopathies suggests a complex interplay between these proteins. Their cross-seeding enhances fibrillization, leading to the formation of diverse amyloid-specific structures enriched with β-sheets, which may influence their biological functions. However, existing tools cannot differentiate structural polymorphs directly in cells, as conventional microscopic approaches have limitations in providing structural insights into aggregates. As a result, a structurally relevant characterization of amyloids in their native cellular environment has not yet been achieved. In this study, we characterize the structural rearrangements of newly formed αSyn inclusions cross-seeded by different αSyn and Tau preformed fibrils (PFFs) directly in cells, using a correlative approach that combines submicron optical photothermal infrared (O-PTIR) microspectroscopy and confocal microscopy. We found that hybrid PFFs synthesized from αSyn, and two Tau isoforms (Tau3R and Tau4R) exhibit variations in αSyn and Tau composition. Specifically, structural polymorphs composed of αSyn and Tau3R exhibit the highest β-sheet content and most potent seeding potency, leading to enhanced phosphorylation within cellular inclusions. Importantly, we demonstrate that cellular inclusions inherit structural motifs from their donor seeds and exhibit distinct spatial and structural evolution. By providing subcellular-resolution structural imaging of amyloid proteins, our study uncovers divergent mechanisms of αSyn aggregation induced by αSyn/Tau PFFs in both mixed and hybrid formats.