N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection

S. Sadia Ameen, Nane Griem-Krey, Antoine Dufour, M. Iqbal Hossain, Ashfaqul Hoque, Sharelle Sturgeon, Harshal Nandurkar, Dominik F. Draxler, Robert L. Medcalf, Mohd Aizuddin Kamaruddin, Isabelle S. Lucet, Michael G. Leeming, Dazhi Liu, Amardeep Dhillon, Jet Phey Lim, Faiza Basheer, Hong Jian Zhu, Laita Bokhari, Carli L. Roulston, Prasad N. ParadkarOded Kleifeld, Andrew N. Clarkson, Petrine Wellendorph, Giuseppe D. Ciccotosto, Nicholas A. Williamson, Ching-Seng Ang, Heung-Chin Cheng

Research output: Contribution to journalArticleResearchpeer-review


Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Original languageEnglish
Article number100543
Number of pages28
JournalMolecular & Cellular Proteomics
Issue number5
Publication statusPublished - 1 May 2023


  • calpains
  • CaM kinase IIa
  • CaM kinase IIb
  • CRMP2
  • excitotoxicity
  • neuronal death
  • neuroprotection
  • proteolytic processing
  • Src
  • synaptic damage

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