In vitro investigation of caspase 3-dependent and caspase-independent apopitotic process in cortex and hypocampal neurons in cell death triggered with sistein-S-sulphate

There are studies in literature that cysteine-S-Sulfate (SSC) toxicity may be the cause of severe neuropathology in sulfite oxysdase enzyme deficiency or molybdenum cofactor deficiency. The neurotoxic effect of SSC has been demonstrated, but the apoptotic process has not been elucidated. In this study, it was aimed to investigate the possible caspase-dependent and independent apoptotic mechanisms that SSC can trigger in the secondary hippocampal (HT-22) and primary cortical neuron (PKN) lines. For this purpose, cytotoxicity experiments were performed to determine the LD50 and the safe doses of apoptotic inducing factor (AIF), calpain and cytochrome c inhibitors in HT-22 and PKN arrays, and to determine the effect of inhibitors on suppressing the toxicity of SSC. Triggering time of apoptosis was determined via time-dependent reduced glutathione (GSH) quantification analysis by adding SSC to neuron lines. AIF, cytochrome c, calpain and caspase levels were measured colorimetrically. The LD50 value of SSC was found to be 150 µM for HT-22 and 155 µM for PKN. Safe doses of Kalpain 1, AIF and cytochrome c inhibitors were found to be 10µM, 10nM, 20µm respectively for PKN cell line, 10µM, 10nm, 10µM for HT-22 cell line. In time-dependent GSH measurements, it was observed that the amount of GSH increased in PKN sequences with SSC added over a 2-8-hour time period and decreased over an 8-16-hour time period. It was found that the amount of GSH increased in the groups with SSC added at the second and eighth hours in the HT-22 cell line compared to the control group. It was determined that the amount of calpain, AIF, cytochrome C and caspase 3 in the PKN cell line and the amounts of AIF and calpain in the HT-22 sequences were higher than those of the control groups. It was found that AIF, cytochrome c and calpain inhibitors reduced the toxic effect of SSC. In this thesis, it was demonstrated that SSC-induced apoptosis was induced by a caspase-independent pathway by increasing AIF, calpain activity in the HT-22 cell line. On the other hand, in PKN cell line, it was shown that SSC effect induced apoptosis by increasing AIF, cytochrome c and calpain and caspase 3 activities, by caspase dependent (cytochrome c, caspase 3 activity) and independent (AIF, calpain activity) pathways. It was concluded that apoptosis inhibitors can be used in the development of treatment methods against SSC-induced neurotoxicity.