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<p>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 <i>SLC39A9</i> and <i>C3orf58</i> genes, encoding respectively a Golgi-localized exporter of Zn<sup>2+</sup>, 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 <i>SLC39A9</i><sup>-/-</sup> and <i>C3orf58</i><sup>-/-</sup> cells indicated major defects in HS homeostasis. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in <i>C3orf58</i><sup>-/-</sup> cells. Interestingly, <i>C3orf58</i><sup>-/-</sup> 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.</p><p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月6日

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  • 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 Thomas,
  • Zhangjie Wang,
  • Wolfgang E. Reintsch,
  • Wen Luo,
  • Irina Shlaifer,
  • Fuming Zhang,
  • Ke Xia,
  • Zachary Steinhart,
  • Robert J. Linhardt,
  • Jean-François Trempe,
  • Jian Liu,
  • Thomas M. Durcan,
  • Stephane Angers,
  • Edward A. Fon

摘要

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.