Examination of excitotoxicity status and GLT-1 levels in MPP+ (1-methyl-4-phenylpyridinium)-treated mouse neuroblastoma (N2A) cells

Parkinson's disease is defined as a dysfunction resulting from the accumulation of intraneuronal aggregates of α-synuclein, expressed as lewy neurites and lewy bodies, with loss of dopamine-producing cells in the substantia nigra and thus degeneration of dopaminergic neurons. Excitotoxicity is one of the common molecular mechanisms of neurodegeneration. Glutamate excitotoxicity occurs by triggering excessive release of glutamate from presynaptic nerve terminals and astrocytes into the intracellular domain and by overexcitation of glutamate receptors. Glutamate carriers prevent excess activation of glutamate receptors by accumulating excess glutamate. In particular, GLT-1 (Glutamate Transporter-1) or EAAT2 (Excitatory Amino Acid Transporter2) is responsible for 95% of total glutamate absorption in the brain. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is an important chemical model for Parkinson's disease and a commonly used effective toxin. It interferes with mitochondrial function by inhibiting Complex 1 in the electron transport chain. In this study, MPT+ (1-methyl-4-phenylpyridinium), the active metabolite of MPTP, was used to model excitotoxicity in neuron and glia cells and the effect of MPP+ on GLT-1 expression was investigated. After N2A (mouse neuroblastoma cells) and glia (IHA-immortalized human astrocytes-immortalized human astrocytes) cells were treated with MPP+, the viability of cells, glutamate release and changes in GLT-1's mRNA and protein levels were investigated. After MPP+ treatment, total protein was isolated from IHA cells and GLT-1 protein expression was examined by western blot. It was observed that MPP+ treated IHA cells showed a significant increase in GLT-1 expression when compared to control. Total RNA was isolated from MPP+ treated and untreated (control) N2A cells, total RNA was isolated and cDNA was produced. GLT-1 mRNA level was measured by Real Time PCR (Real-time Polymerase Chain Reaction). Consistent with the results of IHA cells, a significant increase in GLT-1 mRNA level was observed in MPP+ treated N2A cells compared to control cells. In the end, glutamate release was measured by glutamate assay. As a result, it was observed that GLT-1 expression increased as a rescue mechanism in response to the excitotoxicity induced by MPP+. Therefore, it was observed that glutamate release decreased in the first 12 hours. However, glutamate release continued to increase as the effect of neurotoxicity continued. It was thought that the decrease in glutamate secretion may be due to cell death. It will be important to investigate the relevant molecular pathways with the aim of increasing GLT-1 expression regularly, and to develop treatment against Parkinson's disease by reducing excitotoxicity. Keywords: Excitotoxicity, GLT-1, glutamate, lewy body, MPP+, Parkinson