Loss of MUNC18-1 leads to retrograde transport defects in neurons

J Neurochem. 2021 May;157(3):450-466. doi: 10.1111/jnc.15256. Epub 2020 Dec 12.

Abstract

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport / genetics*
  • Cell Death
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Cholera Toxin / metabolism
  • Endoplasmic Reticulum / metabolism
  • Golgi Apparatus / pathology
  • Golgi Apparatus / ultrastructure
  • Immunohistochemistry
  • Intracellular Membranes / metabolism
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Metabolic Networks and Pathways / genetics
  • Mice
  • Mice, Knockout
  • Munc18 Proteins / deficiency*
  • Munc18 Proteins / genetics*
  • Neurodegenerative Diseases / genetics
  • Neurodegenerative Diseases / metabolism
  • Neurodegenerative Diseases / pathology
  • Neurons / pathology
  • Neurons / ultrastructure
  • SNARE Proteins / deficiency
  • SNARE Proteins / genetics

Substances

  • Membrane Glycoproteins
  • Munc18 Proteins
  • SNARE Proteins
  • Stxbp1 protein, mouse
  • Tgoln2 protein, mouse
  • Cholera Toxin

Associated data

  • RefSeq/15256
  • RefSeq/RRID:AB_398141
  • RefSeq/RRID:AB_449893
  • RefSeq/RRID:AB_1524486
  • RefSeq/RRID:AB_310445
  • RefSeq/RRID:AB_2077527
  • RefSeq/RRID:AB_2721247
  • RefSeq/RRID:AB_371415
  • RefSeq/RRID:AB_2096814