Investigation of the regulation of the GLT-1 (Glutamate transporter 1) degradation pathway by SİRT4 (Sirtuin 4) in glia and gliablastoma cells

Objective: In this study, we aimed to elucidate the regulation of Glutamate Transporter-1 (GLT-1) degradation pathway in glia and gliablastoma cells by SIRT4. It was aimed to show the change in GLT-1 protein expression levels, change in PKC mRNA levels and protein expression, change in phosphoNedd4-2 levels, SIRT4-PKC interaction, and ubiquitination and acetylation of GLT-1 posttranslational modifications in this pathway. Material and Methods: Firstly, SIRT4 silenced groups were obtained by using SIRT4 shRNA lentiviral particles in IHA and U87 cells. PKC mRNA levels in four different cell groups were determined by qPCR and protein expression levels were determined by Western blotting. The change in phosphorylated Nedd4-2 levels between cell groups was demonstrated using Western Blot method. SIRT4 protein obtained from cells was immunoprecipitated using PKC antibody and PKC proteins were immunoprecipitated using SIRT4 antibody and SIRT4-PKC interaction was demonstrated by this method. In the immunoprecipitation method using GLT-1 proteins obtained from cell groups, ubiquitination and acetylation levels were determined. Results: After SIRT4 shRNA in the cells was silenced with the lentiviral particle, four groups were obtained: IHA control cells, SIRT4 silenced IHA cells, U87 control cells and SIRT4 silenced U87 cells. SIRT4 expression levels were examined in these four cell groups and it was shown that the silencing process was successfully performed. It was found that GLT-1 dimer and trimer expression decreased, monomer expression did not change and GLT-1 ubiquitination increased in glioblastoma cells after SIRT4 silencing. In glia cells, GLT-1 monomer, dimer, trimer and ubiquitination levels did not change after SIRT4 silencing. Nedd4-2 phosphorylation and PKC expression did not change in glioblastoma cells but increased in glia cells. SIRT4 was also found to deacetylate PKC. In the light of all these findings, it was concluded that the regulation of GLT-1 in glia and glioblastoma cells is carried out by different mechanisms. Conclusion: In this study, we investigated the regulation of GLT-1 through SIRT4 in glia and glioblastoma cells and obtained various results. According to these results, SIRT4 silencing in both glia and gliablastoma cells affects different mechanisms within the cell. SIRT4 silencing in glia cells has no effect on GLT-1 expression. However, changes in GLT-1 regulation after SIRT4 silencing in gliablastoma cells suggest that this pathway may be effective for excitotoxicity.