Development and Characterization of a Novel α-Synuclein-PEST H4 Cell Line for Enhanced Drug Screening in α-Synucleinopathies

Alpha-Synuclein (α-Syn) is a presynaptic neuronal protein implicated in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, primarily through its aggregation into insoluble fibrils. The extended α-Syn half-life necessitates treatment durations that are incompatible with efficient high-throughput drug screening, can risk compound stability or cause cellular toxicity. To address this, we inserted a PEST sequence, a motif known to promote rapid protein degradation, at the C-terminus of the <i>SNCA</i> gene using CRISPR/Cas9 to create a novel cell line with reduced α-Syn half-life. This modification accelerates α-Syn turnover, providing a robust model for studying α-Syn dynamics and offering a platform that is applicable to other long-lived proteins. Our results demonstrate a six-fold reduction in α-Syn half-life, enabling the rapid detection of changes in protein levels and facilitating the identification of molecules that modulate α-Syn production and degradation pathways. Using inhibitors of the proteasome, transcription, and translation further validated the model's utility in examining various mechanisms that impact protein levels. This novel cell line represents a significant advancement for studying α-Syn dynamics and offers promising avenues to develop therapeutics for α-synucleinopathies. Future research should focus on validating this model in diverse experimental settings and exploring its potential in high-throughput screening applications.