Investigation of the regulation of the GLT-1 (glutamate transporter 1) degradation pathway by SIRT 4 (sirtuin 4) in excitotoxic neuroblastoma cells induced by kainic acid

Objective: In this study, we aimed to investigate how SIRT4 regulates the expression and degradation of GLT-1 in neuroblastoma cells and to clarify its effect on transcription factors in the GLT-1 promoter. In addition, it was aimed to observe how this regulation changes especially in the case of kainic acid-induced excitotoxicity. Material and Methods: In SH-SY5Y human neuroblastoma cell line, lentiviral particles containing SIRT4 shRNA were used to silence the SIRT4 gene and a stable SH-SY5Y cell line with silenced SIRT4 gene was obtained. Western blotting was used to demonstrate SIRT4 silencing. A single cell line that met the appropriate conditions was selected and continued to be propagated for further experiments. Control SH-SY5Y cells and SIRT4 gene silenced SH-SY5Y cells were treated with kainic acid (1 mM) and excitotoxicity model was established. A total of four experimental groups were formed. These are; control group, kainic acid-induced excitotoxicity model, SIRT4 silenced group and kainic acid-induced excitotoxicity model and SIRT4 silenced group. GLT-1 expressions in the four groups were analyzed using Western Blot method. GLT-1 mRNA levels in the four groups were determined using qPCR method. The interaction of GLT-1 promoter and transcription factors (CREB and REST) was demonstrated by CHIP (Chromatin Immunoprecipitation) method in four groups. Results: In order to determine the GLT-1 expression level in the control group, Kainic acid-excitotoxicity induced group, SIRT4 silenced group and Kainic acid-excitotoxicity induced and SIRT4 silenced groups, protein isolation was performed from each group and Western Blot method was applied. The results showed that GLT-1 formed oligomeric structures in neuroblastoma cells. A statistically significant decrease in total GLT-1 expression was observed between the two SIRT4 gene silenced groups to induce excitotoxicity. In addition, silencing the SIRT4 gene between the two excitotoxicity-induced groups was found to statistically significantly reduce total GLT-1 expression. GLT-1 mRNA levels in the four experimental groups were then determined using qPCR method. According to the results obtained, it was observed that GLT-1 mRNA levels did not show a statistically significant difference between the four groups. Conclusion: Our results suggest that SIRT4 silencing and excitotoxicity in neuroblastoma cells affect GLT-1 expression at the protein level rather than the mRNA level.