Id1/NR2B Receptor Pathway Regulates Rat Cochlear Sensory Epithelial Cell Survival after Radiation

Survival of cochlear sensory epithelial cells may be regulated by inhibitor of differentiation-1 (Id1) and the N-methyl-D-aspartic acid (NMDA) receptor. However, it is unclear whether Id1 and the NMDA receptor are involved in the radiation-mediated survival of rat cochlear sensory epithelial cells. Here, we show that the percentage of apoptotic cells increased, the percentage of cells in the S phase decreased, Id1 mRNA and protein expression decreased and the NMDA receptor subtype 2B (NR2B) mRNA and protein level increased in OC1 cells after radiation. Cells infected with the Id1 gene exhibited higher Id1 mRNA and protein levels and lower NR2B mRNA and protein levels than the control cells. In contrast, after transfection of the Id1 siRNA into OC1 cells, Id1 mRNA and protein expression decreased and NR2B mRNA and protein expression increased relative to that of the control group. Additionally, treatment with ifenprodil for 24 h before radiation reduced apoptosis and increased the percentage of cells in the S phase. Our results suggest that Id1 and NR2B might regulate the survival of OC1 cells following radiation.

Characterization of Glutamatergic Phenotypes in Hybrid Septal Neuroblastoma (SN56) cells

Septal Neuroblastoma (SN 56) cells are hybrid cells made through cell fusions between quiescent medial septum neurons (cholinergic) and tumoral neuroblastoma cells. Cholinergic cells synthesize and release the neurotransmitter acetylcholine. Preliminary studies in our laboratory revealed that SN 56 neurons also express the vesicular glutamate transporter type 1 (VGluT1), a protein that is normally produced by glutamatergic neurons. This discovery prompted us to hypothesize that SN 56 neurons may also co-express a glutamatergic phenotype which is important because glutamatergic neurons have been associated to the pathogenesis of neurological disorders such as Alzheimer’s disease. To assess whether SN 56 neurons express in fact both phenotypes, we conducted experiments in differentiated and no differentiated SN 56 cell, to confirm the expression of glutamatergic phenotype, by qPCR, western blotting and Immunocytochemistry assay. The cells are cultured in an incubator gassed with 5% CO2 at 37°C. After differentiation for 3-5 days with cAMP and retinoic acid, SN 56 cells were prepared for qPCR, western blotting and immunocytochemistry. Cells were separated by each experiment, primary antibodies or primers against NMDA glutamate receptor subunit NR2B, VG luT1 and vesicular cholinergic transport (ChAT) how positive control were used to confirm our hypothesis,. Expression of these markers will indicate a glutamatergic phenotype. After secondary detection with appropriate fluorescently-labeled antibodies we confirmed that differentiated SN 56 neurons express glutamate NR2B receptor subtype and the VGluT1 transporter in both post-synaptic and presynaptic structures respectively. Hence, these findings support our hypothesis that SN 56 neurons can co-express both cholinergic and glutamatergic phenotype.