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

Abstract

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

CRISPRi: a way to integrate iPSC-derived neuronal models

“…This approach is exemplified by a CRISPRi screen targeting both protein-coding genes and long non-coding RNAs to identify regulators of neural induction in iPSCs, as assessed by PAX6 staining [ 69 ]. In another example, a recent pooled genome-wide screen used FACS-based sorting to identify genes that modulate α-synuclein expression [ 71 ]. The primary CRISPRn screen used a melanoma cell line, and the secondary CRISPRi validation screen used iPSC-derived neurons, demonstrating that combining different cell models and gene perturbation methods can reveal cell-type independent modulators of α-synuclein expression [ 71 ].…”