p53 Mediates autophagy activation and mitochondria dysfunction in kainic acid-induced excitotoxicity in primary striatal neurons

The present study sought to investigate if p53 mediates autophagy activation and mitochondria dysfunction in primary striatal neurons in kainic acid (KA)-induced excitotoxicity. The excitotoxic model of primary striatal neurons was established with KA. The levels of p53, microtubule-associated protein 1 light chain 3 (LC3), Beclin1, and p62 were examined by Western blot and immunostaining. Autophagy activation was also determined with electron microscope. To evaluate the contribution of p53 to autophagy activation and mitochondria dysfunction in KA-induced excitotoxicity, the protein levels of LC3, Beclin1, and p62, the mitochondrial transmembrane potential and the mitochondrial Reactive oxygen species (ROS) after pretreatment with the p53 inhibitor pifithrin-alpha (PFT-α) and the autophagy inhibitor 3-methyladenine (3-MA) were analyzed. Excitotoxic neuronal injury was induced after KA treatment as demonstrated by increases in lactate dehydrogenase (LDH) leakage and was significantly inhibited by PFT-α. Western blot and immunostaining showed that the induction of p53 protein occurred in the cytosol and the nucleus. Increases in autophagic proteins LC3 and Beclin1 were observed, whereas the protein levels of p62 decreased after KA treatment. Electron microscope analysis showed increased autophagosomes in the cytoplasm. The changes in LC3, Beclin1, and p62 levels were blocked by PFT-α, PFT-μ, 3-MA, and E64d but not Z-DEVD-FMK. JC-1 staining showed the depolarization of mitochondrial membrane potential after excitotoxic insult. Mito-tracker and RedoxSensor Red CC-1 staining showed an increased production of mitochondrial ROS after excitotoxic insult. These effects were significantly suppressed after pretreatment with PFT-α and 3-MA. This study suggests that p53 mediates KA-induced autophagy activation and mitochondrial dysfunction in striatal neurons.

▶p53 activates autophagy in primary striatal neurons. ▶KA-induced autophagy activation is associated with mitochondrial depolarization. ▶KA-induced autophagy activation is associated with free radical production. ▶KA-induced autophagy activation may initiate mitochondria apoptotic pathway. ▶This study reveals a novel p53 downstream mechanism contributing to excitotoxicity.