Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy

Hum Mol Genet. 2023 May 5;32(10):1683-1697. doi: 10.1093/hmg/ddad008.

Abstract

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain Diseases*
  • Mammals / metabolism
  • Membrane Fusion / physiology
  • N-Ethylmaleimide-Sensitive Proteins / chemistry
  • N-Ethylmaleimide-Sensitive Proteins / metabolism
  • Neurotransmitter Agents / metabolism
  • Rats
  • SNARE Proteins / chemistry
  • SNARE Proteins / metabolism
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism
  • Vesicular Transport Proteins* / metabolism

Substances

  • Vesicular Transport Proteins
  • SNARE Proteins
  • N-Ethylmaleimide-Sensitive Proteins
  • Neurotransmitter Agents
  • mTOR protein, rat
  • TOR Serine-Threonine Kinases
  • Nsf protein, rat