Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons

Alpha-synuclein (alpha Syn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering alpha Syn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous alpha Syn using sequential CRISPR-knockout and -interference screens in an alpha Syn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover alpha Syn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous alpha Syn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic alpha Syn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous alpha Syn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous alpha Syn, identifies mechanisms mediating the degradation of nonacetylated alpha Syn, and illustrates potential therapeutic pathways for decreasing alpha Syn levels in synucleinopathies.