Investigation of the relationship between SIRT4 and the glutamate metabolism and its tumor suppressing potantial in glioblastoma primary tumor tissues

Objective: Accumulation of excess glutamate in the space between nerve cells is called excitotoxicity. Excitotoxicity plays a role in many brain diseases. Glutamate released into the interneuronal space is collected by GLT-1 (Glutamate Transporter 1) found in astrocytes. Some of the glutamate is converted to α-ketoglutarate by glutamate dehydrogenase (GDH) in astrocytes and enters the TCA cycle. As a second pathway, glutamate is converted to glutamine by glutamine synthetase (GS) in astrocytes. Glutamine completes this cycle by entering into the glutamatergic neuron. Material and Metedos: Glioblastoma Multiform (GBM) is the most common primary malignant brain tumor, accounting for 16% of all primary brain and central nervous system neoplasms. In glioblastoma, neurons are known to die by excitotoxicity. Glutamate Transporter 1 (GLT-1), also known as Excitatory Amino Acid Transporter 2 (EAAT2), is mostly responsible for glutamate uptake. Decreased activity of EAAT2 leads to accumulation of glutamate. This accumulation leads to glutamate excitotoxicity and possible necrosis of neurons, due to a large increase in intracellular Ca+2 and a decrease in cellular ATP levels. Results: Sirtuins are enzymes that posttranslationally modify proteins by deacetylation or ADP-ribosylation and are expressed in the nucleus, mitochondria, or cytoplasm. Sirtuin 4 (SIRT4) is a mitochondrial sirtuin that is also expressed in the brain. It has previously been shown that SIRT4 deletion in the mouse increases susceptibility to the excitotoxic agent, kainic acid, and reduces GLT-1-dependent glutamate uptake. In other studies, it has been shown that SIRT4 prevents excitotoxicity by modulating glutamate metabolism in cell lines. The expression of glutamate metabolism modulators in glioblastoma tumors will be a guide to understand the detection, prognosis, mechanism and progression of the disease. Conclusion: In this study, 84 primary tumor brain tissues (paraffinic sections) and 12 control brain tissues (paraffinic sections) were used. Total RNA was extracted from paraffin tissue sections using the RNA isolation kit by following the protocol provided. The RNA concentration and the quality were measured using the Nanodrop. Then, cDNAs were obtained using the cDNA synthesis kit by following the given protocol. The mRNA was transcribed from the obtained cDNA using primers specific to glutamate dehydrogenase (GDH), glutamine synthetase (GS) and Sirtuin 4 (SIRT4) by following the qPCR (quantitative PCR-quantitative polymer chain reaction) kit protocol. The GDH, GS and SIRT4 mRNA obtained was normalized to the beta actin mRNA. Then, Two-Tailed Unpaired, Student's t-test was used for the difference between the means of the two sample groups for the statistical analysis, One Way ANOVA test was used for cases with three or more groups.