Large‐scale bidirectional arrayed genetic screens identify <scp> <i>OXR1</i> </scp> and <scp> <i>EMC4</i> </scp> as modifiers of <scp>αSynuclein</scp> aggregation

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.