Arrayed CRISPR activation and knockout screens of ~4,700 mitochondria/trafficking genes identified OXR1 activation as promoting Ser129-phosphorylated α-synuclein aggregation via mitochondrial dysfunction and EMC4 ablation as reducing aggregation by enhancing ER-driven autophagic/lysosomal…

Provides actionable, validated targets—EMC4 for enhancing ER-autophagy to broadly lower α-syn aggregation and OXR1/mitochondrial pathways as modulators of polymorph-specific aggregation—making these high-priority leads for Parkinson’s therapeutic discovery and target development.

In Parkinson's disease and other synucleinopathies, αSynuclein (αSyn) misfolds and forms Ser129-phosphorylated aggregates (pSyn129) with the factors controlling this process largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondria, trafficking, and motility functions in HEK293 cells. After exposure of cells to αSyn fibrils, pSyn129 signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn129 across diverse αSyn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in αSyn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.