In-Vitro Cell-Model Investigation of Alpha-Synuclein Aggregate Morphology Using Spectroscopic Imaging

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.