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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 direct

Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association.

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.