N-Terminomic Changes of Neurons During Excitotoxicity Reveal Proteolytic Events Associated with Synaptic Dysfunctions and Inform Potential Targets for Neuroprotection

Excitotoxicity is a neuronal death process initiated by over-stimulation 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 perturbation of 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, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. In Brief Ameen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. Highlights Identification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. These pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses (A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKIIα and CaMKIIβ, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection One Sentence Summary Proteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.