Abstract
Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death. Our data, available in ProteomeXchange with identifier PXD008353, identified over 100 changed proteins exhibiting significant alterations in abundance and/or phosphorylation levels at different time points (5–240 min) in neurons after glutamate overstimulation. Bioinformatic analyses predicted that many of them are components of signalling networks directing defective neuronal morphology and functions. Among them, the well-known neuronal survival regulators including mitogen-activated protein kinases Erk1/2, glycogen synthase kinase 3 (GSK3) and microtubule-associated protein (Tau), were selected for validation by biochemical approaches, which confirmed the findings of the proteomic analysis. Bioinformatic analysis predicted Protein Kinase B (Akt), c-Jun kinase (JNK), cyclin-dependent protein kinase 5 (Cdk5), MAP kinase kinase (MEK), Casein kinase 2 (CK2), Rho-activated protein kinase (Rock) and Serum/glucocorticoid-regulated kinase 1 (SGK1) as the potential upstream kinases phosphorylating some of the changed proteins. Further biochemical investigation confirmed the predictions of sustained changes of the activation states of neuronal Akt and CK2 in excitotoxicity. Thus, future investigation to define the signalling pathways directing the dynamic alterations in abundance and phosphorylation of the identified changed neuronal proteins will help elucidate the molecular mechanism of neuronal death in excitotoxicity.
Highlights
Excitotoxicity is a prominent pathological process directing neuronal death
Since these events occur at the initial phase of the cytotoxic signalling pathway, their identification is best conducted with glutamate-treated neurons showing no signs of cellular damage but would subsequently progress to excitotoxic cell death
With no signs of reduced viability from 15 to 60 min of glutamate treatment (Fig. 1a), we selected time points from 5 to 60 min to investigate how the abundance and phosphorylation of neuronal proteins are perturbed at the early stage of excitotoxicity (Fig. 1b)
Summary
Excitotoxicity is a prominent pathological process directing neuronal death. It is initiated by aberrant stimulation of neurons by the excitatory neurotransmitter glutamate,(iGluRs) including N-methyl-D-aspartate (NMDA) receptor, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA) receptor and kainate receptor[1]. Phosphoproteomic analysis revealed dynamic changes in phosphorylation levels of multiple neuronal proteins induced by glutamate overstimulation
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