Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be as-sociated to clinical subtypes of alpha-synucleinopathies like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to a caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in-vitro method to explore various alpha-synuclein (αsyn) aggregates morphotypes. We performed a spectroscopic investigation of the HEK293 cell model transfected with human wildtype-αsyn and A53T-αsyn variants using the amyloid fibril specific heptameric luminescent oligomeric thio-phene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In-vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant Kd < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The en-suing intracellular aggregates were stained with h-FTAA followed by evaluation of spectral features and fluorescence lifetime of intracellular syn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell-culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in-vitro and in-vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early sub-type-specific diagnosis is of utmost importance for prognosis and treatment response. The po-tential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtypes specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation specific features of various protein pathologies.

Disease-specific alpha-synuclein (αsyn) strains have been linked to different synucleinopathies. Current αsyn biomarkers are limited to binary detection of pathogenic αsyn in peripheral tissue biopsies or fluids, limiting differential diagnosis. Hence, there is an urgent need for methods that allow strain-specific detection and characterization of αsyn strain architecture. Notably, luminescent conjugated oligothiophenes (LCOs) have been successfully used to detect distinct protein strain conformers in prion diseases and Alzheimer’s disease, highlighting their utility in differentiating disease-specific amyloid structures. Species-dependent differences in αsyn structure are increasingly recognized as one of the critical aspects that shape how fibrils form, propagate and interact with molecular LCO probes. Here, we evaluate the potential of the LCO h-FTAA to differentiate species-specific αsyn strains and conduct a translational investigation using peripheral cardiac tissue of a gut-first synucleinopathy rodent model. Our in vitro data demonstrate strain-specific probe–fibril interactions, reflecting a differential strain architecture and cellular micro-environment. While h-FTAA binds with comparable efficiency to mouse (mo-) and human (hu-) pre-formed fibrils (PFFs), h-FTAA exhibits markedly lower quantum yield when bound to moPFFs versus huPFFs. Spectral imaging revealed h-FTAA-moPFF binding produces blue-shifted maxima (505–550 nm), contrasting with the red-shifted maxima (545–580 nm) of huPFFs. Fluorescence lifetime imaging microscopy confirmed h-FTAA’s intrinsic sensitivity to species-dependent variations through distinct temporal fluorescence signatures (moPFFs: ~0.60–1.5 ns vs. huPFFs: ~0.65–1.0 ns). Our translational investigation showed h-FTAA binding to peripheral cardiac pathology exhibits comparable red-shifted emission, but distinct fluorescence lifetimes of h-FTAA-bound aggregates in moPFF-injected (~1.0–1.4 ns) versus huPFF-injected (~0.69–0.8 ns) rats. Interestingly, we observed distinct blue-shifted emission profiles in a few selected regions of the heart of moPFF-injected rodents, further characterized by extra-long fluorescence decay shifts (~1.5–1.9 ns), reflecting differences in both aggregate conformation and maturity in moPFF-induced compared with huPFF-induced rats. Taken together, our findings underscore the potential of LCO ligands, like h-FTAA, to enable more precise disease staging and diagnosis through peripheral biopsies, complementing existing αsyn biomarker methods.