A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Abstract

Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. Understanding how these aggregates propagate from cell to cell in a prion-like fashion thus holds great therapeutic promises. Here, we focused on understanding the cellular processes involved in the entry and accumulation of pathological α-syn aggregates. We used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout (KO) screening to identify genes that regulate the accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. We show that all confirmed hits affect heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including of α-syn and tau. Intriguingly, KO ofSLC39A9andC3orf58genes, encoding respectively a Golgi-localized exporter of Zn2+, and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs uptake but not of tau oligomers, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains indicated major defects in HS maturation inSLC39A9andC3orf58KO cells, explaining the cell surface binding deficit. Our findings now clearly establish these two genes as HSPG-modulating factors. Interestingly,C3orf58KO human iPSC-derived microglia exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data establish HSPGs as major receptors for α-syn PFFs binding on the cell surface and identifies new players in α-syn PFFs cell surface binding and uptake.