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

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Parkinson's disease and synucleinopathies involve alpha-synuclein (αSyn) misfolding and Ser129 phosphorylation (pSyn129).
• The specific factors regulating αSyn phosphorylation remain largely unknown.

Purpose of the Study:

• To discover novel modulators of pSyn129 formation using large-scale genetic screening.
• To elucidate the roles of identified genes in αSyn aggregation and cellular processes.

Main Methods:

• Employed arrayed CRISPR-mediated gene activation and ablation in HEK293 cells targeting mitochondrial, trafficking, and motility genes.
• Utilized high-throughput fluorescence microscopy and image analysis to quantify pSyn129 levels after αSyn fibril exposure.
• Validated findings in human induced pluripotent stem cell (iPSC)-derived neurons.

Main Results:

• OXR1 activation increased pSyn129 by affecting mitochondrial function (ATP levels, membrane potential).
• EMC4 ablation reduced pSyn129 by enhancing ER-associated autophagy and lysosomal clearance.
• OXR1 modulation was specific to certain αSyn polymorphs (MSA-derived), while EMC4 ablation had broader effects.
• Confirmed OXR1 and EMC4 roles in primary neurons, affecting somatic and neuritic αSyn aggregates.

Conclusions:

• Uncovered OXR1 and EMC4 as previously unrecognized key regulators of αSyn aggregation.
• These findings provide new mechanistic insights into synucleinopathies and potential therapeutic targets.