Roles Of NF-κB/p53 Pathway In Autophagy And Apoptosis Mediated By Glutamate Receptors

Aim: To study the roles of Nuclear Factor-kappa B (NF-κB)/p53 pathway in autophagy and apoptosis mediated by glutamate receptors.Methods: The in vivo excitotoxic model was produced with stereotaxic administration of Quinolinic acid (QA) into unilateral striatum. The changes in the expression of p53 and its target genes involved in apoptosis and autophagy including PUMA, Bax, Bcl-2, damage-regulated autophagy modulator (DRAM), and other autophagic proteins including microtubule-associated protein 1 light chain 3 (LC3), and beclin1 were assessed with RT-PCR and Western blot analysis. The neuroprotective effects of the NF-κB inhibitor SN50 and the p53 inhibitor Pifithrin-alpha (PFT-α) were assessed with internucleosomal DNA fragmentation and Cresyl violet staining. Effects of SN50 or PFT-αon QA-induced increases in the level of p53, PUMA, Bax, decreases in the level of Bcl-2, and increases in the level of DRAM, LC3, Beclin1 were detected with RT-PCR and Western blot analysis. The neuroprotective effects of the Cathepsin L inhibitor Z-FY-DMK were assessed with Cresyl violet staining. Effects of Z-FY-DMK on QA-induced NF-κB activation were detected with immunohistochemical analysis. Effects of Z-FY-DMK on QA-induced IκBαdegradation and phosphorylation, changes in the levels of DRAM, Beclin1, P62 were detected with Western blot analysis. The in vitro excitotoxic model was produced with glutamate treatment of primary striatum neurons. The neuroprotective effects of SN50, PFT-αand Z-FY-DMK were assessed with LDH. Effects of SN50 and PFT-αon glutamate-induced decrease of mitochondrial transmembrane potential were assessed with immunofluorescence analysis. Effects of Z-FY-DMK on glutamate-induced changes of protein levels of Beclin1 and P62 were observed using Western blot analysis.Results: We have successfully established in vivo model of excitotoxicity by QA. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl-2. QA also upregulated DRAM, the ratio of LC3-II/LC3-I and the levels of Beclin1. QA- induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50 or PFT-α(p<0.01). SN50 and PFT-αreversed QA-induced increases in the expression of p53, PUMA, Bax, decrease in Bcl-2, and up-regulation of DRAM, the ratio of LC3-II/LC3-I and the levels Beclin1 in the striatum. QA-induced loss of striatal neurons were robustly inhibited by Z-FY-DMK (p<0.01). Z-FY-DMK reversed QA-induced activation of NF-κB. Z-FY-DMK inhibited QA-induced IκBαdegradation and phosphorylation, up-regulation of DRAM, Beclin1, and down-ragulation of P62. We have successfully established the in vitro excitotoxic model with glutamate in cultured primary striatal neurons. LDH tests showed that the damage of neurons induced by excitotoxicity was significantly depressed by the treatment with SN50, PFT-αand Z-FY-DMK. Immunofluorecsence analysis showed that glutamate-induced depolarization of mitochondrial membrane was attenuated by SN50 and PFT-α. Western blot analysis showed that glutamate-induced Beclin1 upragulation and P62 downregulation were attenuated by Z-FY-DMK.Conclusions: These results suggest that over-stimulation of glutamate receptors can induce NF-κB-dependent expression of p53. NF-κB/p53 pathway participates in excitotoxic neuronal death through both apoptotic and autophagic mechanisms. Cathepsin L inhibitor has neuroprotective effects by inhibiting glutamate receptors-induced IκBαdegradation and phosphorylation, NF-κB nuclear translocation and activation of autophagy.