C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Hum Mol Genet. 2014 Jul 1;23(13):3579-95. doi: 10.1093/hmg/ddu068. Epub 2014 Feb 18.

Abstract

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Amyotrophic Lateral Sclerosis / genetics
  • Amyotrophic Lateral Sclerosis / metabolism*
  • Animals
  • Biological Transport
  • C9orf72 Protein
  • Endosomes / metabolism*
  • Frontotemporal Dementia / genetics
  • Frontotemporal Dementia / metabolism*
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / genetics
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism
  • Humans
  • Mass Spectrometry
  • Mice
  • Proteins / genetics
  • Proteins / metabolism
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • C9orf72 Protein
  • C9orf72 protein, human
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Proteins
  • UBQLN2 protein, mouse
  • hnRNP A2
  • rab7 protein
  • rab11 protein
  • rab GTP-Binding Proteins

Supplementary concepts

  • Amyotrophic lateral sclerosis 1