Benoît Vanderperre, Amitha Muraleedharan, Marie‐France Dorion, Frédérique Larroquette, Esther del Cid‐Pellitero, Nishani Rajakulendran, Carol X.‐Q. Chen, Roxanne Larivière, Charlotte Michaud-Tardif, Thomas Goiran, Rony Chidiac, Damien Lipuma, Graham MacLeod, Rhalena A. Thomas, Zhangjie Wang, Wolfgang Reintsch, Wen Luo, Irina Shlaifer, Fuming Zhang, Ke Xia, Zachary Steinhart, Robert J. Linhardt, Jean‐François Trempe, Jian Liu, Thomas M. Durcan, Stéphane Angers, Edward A. Fon
Abstract Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-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 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. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn 2+ , and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-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 homeostasis. 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 α-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake.