Novel regulators of heparan sulfate proteoglycans modulate cellular uptake of α-synuclein fibrils

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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 Zn2+, 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.
Funding Information
  • Michael J. Fox Foundation for Parkinson's Research (021129)
  • Gouvernement du Canada | Canadian Institutes of Health Research (MFE-152571, Canada Research Chair (Tier 1) in Parkinson’s disease)
  • Canada First Research Excellence Fund, Healthy Brain, Healthy Lives awarded to McGill University
  • Proteo

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