Kinomic analysis of excitotoxicity in primary neuronal cultures

Glutamate excitotoxicity plays a critical role in neurodegeneration by triggering NMDA receptorhyperactivation, leading to elevated synaptic calcium levels and subsequent neuronal cell death. Tobetter understand how glutamateaffects neurons in neurological diseases, we conducted acomprehensive analysis of molecular changes at the transcriptome and kinome levels.Our research used primary cortical cultures from rat embryos to study glutamate excitotoxicity. Non-neuronal cells,like astrocytes, typically enhance neuron tolerance to glutamate excitotoxicity. We useda neuron-rich cultured system and observed that the effects of glutamate on neurons are concentration-dependent. Intermediate doses of glutamate had significant neurotoxic effects, while high and lowdoses resulted in less cell mortality, aligning with previous findings related to calcium influx.Glutamate is known to inhibit protein synthesis in neurons and leads to a rise in cytosolic calcium, a keystep in synapticplasticity and delayed neuronal cell death. Kinome profiling indicated activation of PKAand PKG phosphorylation, essential for synaptic plasticity-related gene expression. Rat neuron cells (5 x 104cells per mL, 3 mLtotal volume)were plated on a 6-well poly-D-lysine coated plate. Incubated the plate at 37°C and 5% CO2 for 8 dayswith half of the growth medium (1.5 mL) replaced every other day. Prior to treatment, plates werescanned in the IncuCyte S3 (25 scans perwell, 20 X magnification). After scanning, the growth mediumwas aspirated and replaced with a glutamate-based medium (2 mL, 250 μM final concentration) or freshgrowth medium and scanned in IncuCyte S3 at the same specification for 4 h