Large-scale bidirectional arrayed genetic screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation

In Parkinson’s disease and other synucleinopathies, α-synuclein (α-Syn) misfolds and forms Ser ¹²⁹ – phosphorylated aggregates (pSyn ¹²⁹ ). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn ¹²⁹ modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to α-Syn fibrils, pSyn ¹²⁹ signals were recorded by high-throughput fluorescent microscopy and aggregates were identified by image analysis. We found that pSyn ¹²⁹ was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn ¹²⁹ 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 pSyn ¹²⁹ across diverse α-Syn 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 α-Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.