Effect of C3orf58 or SLC39A9 knock-out on gene expression in human RPE-1 cells

Synucleinopathies are characterized by the accumulation and propagation of a-synuclein (a-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. In this study, we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of a-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in a-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including a-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn2+, and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of a-syn PFFs, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9 -/- and C3orf58 -/- cells indicated major defects in HS maturation. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in C3orf58 -/- cells. Interestingly, C3orf58 -/- human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize a-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate a-syn PFFs cell surface binding and uptake. Overall design: Total RNA sequencing of wild type RPE-1 cells and monoclonal knock-out lines for each gene SLC39A9 or C3orf58. Triplicate sample were analyzed for each genotype (WT, C3orf58 -/-, SLC39A9 -/-). RNA transcripts were calculated and differential gene expression was analyzed to access the effect of each gene on transcription.