Identification of cellular mechanisms that control α-synuclein aggregation using a genome-wide CRISPR-based functional genomics screen

The intercellular prion-like propagation of protein aggregation is an important mechanism in the progression of neurodegenerative diseases such as Parkinson’s disease and Multiple Systems Atrophy (MSA). However, our understanding of the cellular pathways that control the prion-like spread of these proteins, specifically the small neuronal protein α-synuclein, is incomplete. In order to comprehensively study these pathways and discover new mechanisms controlling the spread of α-synuclein aggregation we performed a genome-wide CRISPR interference (CRISPRi) screen in a mammalian cell-based model of α-synuclein aggregation that could be monitored by FRET (Fluorescence Resonance Energy Transfer). Our screen uncovered several cellular pathways, including components of the phosphatidylinositol pathway required for endosomal trafficking of α-synuclein aggregates. Specifically, we discovered that the knockdown and pharmacological inhibition of the kinase PIKfyve dramatically reduces aggregation. The inhibition of PIKfyve reduces α-synuclein transport from the early to late endosome, thereby limiting fibril escape from the lysosome, and reducing the amount of fibrils that can reach cytoplasmic α-synuclein to induce aggregation. These findings point to the endosomal transport of fibrils as a critical step in the propagation of α-synuclein aggregation and a potential therapeutic target in neurodegenerative diseases