Background: Excitotoxicity, initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), is a major pathological process directing neuronal death in stroke and neurodegenerative diseases. The over-stimulated iGluRs allow massive influx of calcium ions into the affected neurons, leading to over-activation of neurotoxic enzymes such as the neurotoxic proteases calpains, protein kinases and phosphatases to perturb the structures, expression and phosphorylation of specific neuronal proteins. These perturbed proteins form signalling networks that direct neuronal death in excitotoxicity.
Methods: We used the quantitative “Terminal Amine Isotopic Labelling of Substrates” (TAILS) and phosphoproteomics methods to define the neurotoxic signalling networks governed by the perturbed proteins in cultured primary neurons. Specifically, we aim to identify the calpain substrates and the neuronal proteins of which the phosphorylation levels are perturbed in primary neurons undergoing excitotoxic cell death.
Results: We identified the cleavage sites in ~300 neuronal proteins proteolytically processed by proteases activated in excitotoxicity. Among them, the tyrosine kinase Src was cleaved by calpains at the unique domain to generate a neurotoxic truncated fragment (ΔNSrc), which acts as a mediator of excitotoxic neuronal death. We also demonstrated that blockade of cleavage of Src to form ΔNSrc could protect against excitotoxic neuronal death in vivo in a rat model of neurotoxicity. Additionally, we identified ~6,500 phosphosites in over 4,000 neuronal proteins exhibited dynamic changes in phosphorylation levels in excitotoxicity. Bioinformatic analysis predict over 20 protein kinases as the upstream regulatory kinases directly phosphorylating some of these phosphosites, suggesting that aberrant regulation of some of these kinases are key events directing neuronal death in excitotoxicity.
Conclusion: Results of our proteomic analyses form the conceptual framework for future investigation to define the molecular mechanism governing neuronal death in diseases.