Regulation of the Glutamate Transporter 1 (GLT-1) promoterin Sirtuin 4 (SIRT4) silenced glial cells

Objective: Glutamate Transporter 1 (GLT-1) is a member of the excitatory amino acid transporter (EAAT) protein family that collects excess glutamate present in the synaptic cleft. SIRT4 (Sirtuin 4) is a mitochondrial sirtuin expressed in glial cells in the brain and can deacetylate or ADP-ribosylate various proteins. SIRT4 protects against excitotoxicity by regulating GLT-1 protein expression. Therefore, the study aimed to determine whether SIRT4 is effective in the transcriptional regulation of the GLT-1 gene. Material and Methods: First, IHA cells with the SIRT4 gene silenced were obtained by using lentiviral particles containing the shRNA gene designed to silence SIRT4 in the IHA (Immortalized Human Astrocytes) cell line. GLT-1 mRNA levels were determined using the qPCR method. To show that SIRT4 was silenced, SIRT4 protein expression was measured using the Western Blot method. The interaction of GLT-1 promoter and transcription factors (CREB, REST and YY1) was demonstrated in both experimental groups by the CHIP (Chromatın Immunoprecipitation) method. Results: After SIRT4 was silenced in IHA cells with lentiviral particles containing the shRNA gene designed to silence SIRT4, two experimental groups were obtained: IHA control and SIRT4 silenced IHA cells. By isolating protein from control and SIRT4 silenced IHA cells, SIRT4 protein levels were examined by western blot and it was shown that the silencing of the SIRT4 gene was successfully carried out. Subsequently, total RNA was isolated from both cell groups, cDNA was made and qPCR analysis was performed to determine the GLT-1 mRNA level. As a result of the analysis, it was revealed that there was no statistically significant difference between GLT-1 mRNA levels in both groups. CHIP experiments have shown that CREB, REST and YY1 transcription factors are located in the GLT-1 promoter. In CHIP analysis, DNA regions to which transcription factors bind are amplified by qPCR. In the SIRT4 silenced cell group, CREB, REST and YY1 transcription factors bound to the GLT-1 promoter. Silencing SIRT4 did not make a significant difference in the CHIP analysis. Conclusion: In this study, research was conducted considering that SIRT4 regulates GLT-1 at the transcriptional level and various results were obtained. In IHA cells, silencing the SIRT4 gene did not alter the GLT-1 mRNA level. Silencing the SIRT4 gene had no effect on the binding of CREB, REST and YY1 transcription factors to the GLT-1 promoter.