StressMarq Biosciences, Inc.

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.

Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.