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

In synucleinopathies, including Parkinson’s disease, α-synuclein (α-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn). Mitochondrial and lysosomal dysfunction, along with impairments in protein and organelle trafficking, exacerbate α-Syn pathology through poorly defined molecular mechanisms. We used CRISPR-mediated gene activation and ablation to systematically interrogate mitochondrial, trafficking and motility-related genes to identify pSyn regulators in HEK293 cells exposed to α-Syn fibrils. We found that activation of the mitochondrial protein OXR1 increased pSyn by impairing ATP synthesis and altering the mitochondrial membrane potential, whereas ablation of the endoplasmic reticulum (ER)-associated protein EMC4 reduced pSyn by enhancing ER-driven autophagic flux and lysosomal clearance. Assays with distinct strains of α-Syn fibrils revealed that OXR1 was preferentially synergistic with multiple system atrophy (MSA)-associated fibrils, whereas EMC4 broadly reduced pSyn across diverse α-Syn polymorphs. These findings were validated in human iPSC-derived cortical and dopaminergic neurons, with OXR1 preferentially driving somatic aggregation and EMC4 depleting both somatic and neuritic aggregates. This work identifies OXR1 and EMC4 as organelle-specific regulators of α-Syn proteostasis and underscores the involvement of mitochondrial and ER-lysosomal pathways in synucleinopathies.