(47b) Novel Technologies for the Detection of Alpha-Synuclein Misfolded Toxic Species

Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 50 years of age. The hallmarks of PD pathogenesis are loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions. These inclusions, called Lewy bodies, are mainly composed of the pre-synaptic protein alpha-synuclein and ubiquitin, suggesting a direct correlation between misfolding and abnormal accumulation of alpha-synuclein and impairment of the proteasome ubiquitin system (UPS). Alpha-synuclein is an intrinsically unfolded protein characterized by high conformational plasticity. Depending on the environment, it can misfold and oligomerize, giving rise to the formation of soluble prefibrillar intermediates, and precipitate into insoluble fibrillar aggregates, as those found in Lewy bodies. Recent evidence suggests that the soluble intermediate species exert cellular toxicity, while the insoluble aggregates serve as a protective mechanism by confining aberrant misfolded proteins in a localized space and preventing deleterious interactions with other cellular components. Enhancement of cellular degradation of soluble, UPS-accessible intermediates has been proven to alleviate cellular toxicity and is suggested as an approach for the development of efficient therapeutic strategies. This approach, however, is currently limited by the lack of technologies to reliably monitor the formation of alpha-synuclein distinct misfolding intermediates within the cellular environment . We recently developed two cell-based assays that allow distinguishing i) soluble vs. insoluble asyn species, based on adapting the previously reported split Green Fluorescent Protein (GFP) assay, and ii) monomeric vs. oligomeric soluble asyn intermediates thought GFP Proximity Imaging (GFP PRIM). The significance of these novel and enabling technologies is underscored by their amenability to high-throughput screening of chemical and genetic libraries for the isolation of small molecules and endogenous proteins that influence the accumulation of toxic asyn species with potential therapeutic applications for PD.