Roles And Mechanisms Of P53 In An Autophagy Pathway Mediated By KA Receptors

Aim: To study the molecular mechanisms of p53 in an autophagy pathway mediated by kainic acid (KA) receptors in primary striatum neurons.Methods: The excitotoxicity model of primary striatum neurons was established by kainic acid. The injury of neurons induced by KA was detected with LDH test and immunofluorescence. The activation of autophagy in neurons after excitotoxicity was observed using MDC staining. Protein levels of LC3, Beclin-1, and P62 were determined by Western Blot analysis and immunefluorescence. The neuroprotective effects of the autophagy inhibitor 3-methyladenine (3-MA) and the apoptosis related protein p53 inhibitor Pifithrin-alpha (PFT-α) was assessed with LDH and immunofluorescence. The autophagy related protein LC3, Beclin-1, and P62 as well as the apoptotic protein p53, Bcl-2 were assessed with Western Blot analysis. In order to evaluate the relationship between the autophagy and mitochondria in the excitotoxicity model, we assessed the mitochondrial transmembrane potential, the level of calcium ions and the generation of ROS after the pre-treatment with 3-MA and PFT-αby immunofluorescence analysis.Results: We have successfully established the excitotoxicity model of primary striatum neurons by kainic acid. Immunofluorecsence and LDH test showed that neurons were severly injured after excitotoxicity on the time dependence and the dose dependence. MDC staining revealed that autophagy was activated after excitotoxicity. Western blot analysis showed that the expression of protein LC3, p53 and Beclin-1, increased and the expression of protein P62 and Bcl-2 decreased. From LDH test and Immunofluorecsence, we observed that the damage of neurons induced by excitotoxicity was significantly depressed after the pre-treatment with 3-MA and PFT-α. Immunofluorecsence analysis showed that the mitochondrial membrane depolarization, elevated intracellular Ca2+ levels, stimulated ROS production after excitotoxicity, but these were significantly depressed after the pre-treatment with 3-MA and PFT-α. Conclusions: We have successfully established the excitotoxicity model of primary striatum neurons. Autophagy was activated by kainic acid and the excitotoxicity was significantly depressed after the pre-treatment with p53 inhibitor PFT-α. From this study, we learned that the excitotoxicity induced the mitochondrial membrane depolarization, elevated intracellular Ca2+ levels, stimulated ROS production and these were inhibited after the pre-treatment with 3-MA and PFT-α.