Investigation of the protective effect of sirt4 (sirtuin4) against exitotoxicity in A cellular model

Glutamate has many neurological functions in the central nervous system and is found at high concentrations in the brain. At the nerve terminals, glutamate is densely present and has a much higher concentration outside the cell. By depolarization of the nerve cell membrane, glutamate is released from the vesicles of the presynaptic nerve endings into the intercellular space and the glutamate receptors in the postsynaptic neuron are stimulated. Stimulation is terminated by collecting glutamate from the intercellular space by glutamate transporters on glial cells. Increased levels of glutamate in the neuronal space are toxic, causing neuron damage and death. This is called glutamate-induced excitotoxicity. Excitotoxicity is among the causes of many neurological diseases such as trauma, cerebral ischemia, epilepsy, Parkinson's Disease, Alzheimer's Disease. Kainic acid is a glutamate analogue, a chemical model used to generate neurotoxicity. The homologues of Sir2, the first member of the silent information organizer (Sir) protein family, are called sirtuins. There are seven sirtuins (Sirt1-7) in mammals. Sirt4 is a mammalian sirtuin which is present in the mitochondria, showing deacetylase and ADP-ribosylase activities. Sirt4 regulates glutamine metabolism using GDH as a mediator. Therefore, Sirt4 is thought to regulate the response to increased glutamate levels during neuronal stimuli and excitotoxicity. In this study, N2A cells were treated with 10 µM, 50 µM, 200 µM and 1000 µM kainic acid concentrations. The viability of the cells was then assessed by the MTT test. There was a significant decrease in cell viability at 200 µM and 1000 µM kainic acid concentrations. In the following experiments, 1000 µM concentration of kainic acid was decided to be used. In N2A cells, Sirt4 expression was increased by transfection and the the enhanced expression was assesed by western blot method. N2A cell lines with or without Sirt4 overexpression were treated with kainic acid at a concentration of 1000 µM. After treatment, four different groups of N2A cells were obtained, control N2A, N2A treated with kainic acid, Sirt4 overexpressed, and kainic acid treated and Sirt4 overexpressed. The viability of these cells was measured by MTT test. Kainic acid treatment lead to a decrese in the cell viability. Increasing Sirt4 expression reduced the decrease in cell viability. In addition, glutamate release in these four different experimental groups was measured for 0, 12, 24, 48 hour periods. It was shown that in the experimental group with Sirt4 overexpression and kainic acid treatment, the cell death was less than that of the experimental group treated with kainic acid alone, therefore more glutamate was released. Thus, it has been shown that Sirt4 can reduce cell death by inhibiting excitotoxicity. Keywords: Excitotoxicity, glutamate, kainic acid, sirtuin, Sirt4.