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Physiological role of PrP in anti-oxidant defence 2

Since oxidative stress has been frequently implicated in neurodegeneration it was very interesting to investigate the influence of PrP expression in stimulated A74 on antioxidant enzymes activities and resistance to oxidative stress. PrPC expression in A74 cells increases significantly Cu/Zn-SOD, catalase, glutathione reductase activities and glutathione level cells. In addition stimulated cells were more resistant to oxidative stress caused by SIN-1(3-morpholinosydnonimine), a vasodilatatory drug widely used as a model for the continuous release of different free radicals. Increases in Cu/Zn SOD activity in stimulated cells is due to SOD like activity of PrP-Cu complexes in the outer side of the cell membrane. This is supported by the fact that no change Cu/Zn SOD protein levels still unchangeable in both stimulated and unstimulated cells. This antioxidant function observed in the outer of the cell membrane is very important, especially in neurons, to detoxify free radicals such as O2°-.

In contrast, stimulated cells showed decreased resistance to H2O2 toxicity this may be due to both the SOD like activity of murine PrP and to Fenton reaction via the increased amount of copper bound to the cell membrane. Stimulated cells show also high increase in glutathione level and glutathione reductase activity. Glutathione, a tripeptide consisting of glycine, cystein and glutamic acid moieties, is a major antioxidant and functions directly in the elimination of Reactive oxygen species (ROS). Glutathione acts as a cellular redox buffer and even modest variations in GSH concentrations can strongly modulate redox state.

We have also evaluated the antioxidant capacities of PrPC by PQ-induced oxidative stress in cultured A74 cells. PQ is cytotoxic, genotoxic, and mutagenic, mediated by its potential to generate the superoxide anion O2 in cultured cells. When exposed to PQ, survival of doxycycline-stimulated A74 cells was found to be significantly higher than that of unstimulated cells, supporting a protective role of PrPC against PQ-induced oxidative stress. Cellular membrane integrity of cells exposed to PQ was assessed in two ways:
- by assay of LDH released as a consequence of cell lysis and
- by the determination of MDA levels, which reflect lipid peroxidation.

Protective effect of PrPC also demonstrated against DNA damage induced by PQ cells exposition. PrPC stimulation in A74 cells increased CuZn-SOD and GPX activities significantly compared to unstimulated cells. Such antioxidant enzymes protect DNA against OH radicals, and the severity of PQ-induced DNA lesions as assessed by Comet assay in our system strongly supports the hypothesis that PrPC protects against PQ-induced DNA damage.

The protective effect of PrPC was also demonstrated in metal mediated cytotoxicity. Exposition of unstimulated and doxo-stimulated A74 cells to various copper ions concentration reveal that PrPC present a powerful protective effect against copper mediated cytotoxicity in A74 cells. However, expression of PrPC in A74 cells was unable to offer a protection against manganese mediated cytoxicity in these cells.

This finding was inexplicable since PrPC was also reported to possess a binding capacity to manganese ions. Studying the possible protective effect of PrPC toward high zinc concentration mediated cytotoxicity was then investigated. A surprisingly protective effect of the PrPC against high zinc mediated cytotoxicities was observed in PrPc expressed cells compared to the cells control. The protective effect of PrPC against high zinc cytotoxicities may explained by the fact that PrPC acts as an antiapoptotic protein in A74 cells. Indeed, accumulating evidences suggests that PrPC may serve has been reported that PrPC inhibits Bax-induced apoptosis in the primary culture of human neurons.

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