Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp(0/0)) hippocampal neuronal cells in comparison to cellular prion protein (PrPC)-expressing (Prnp(+/+)) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrPC is associated with autophagy using Prnp(0/0) hippocampal neuronal cells under hydrogen peroxide (H2O2)-induced oxidative stress. We found that Prnp(0/0) cells were more susceptible to oxidative stress than Prnp(+/+) cells in a dose-and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 beta (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H2O2-treated Prnp(0/0) cells compared with H2O2-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H2O2-treated Prnp(0/0) cells, while the H2O2-treated Prnp(+/+) cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H2O2. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp(+/+) cells contributes to the prosurvival effect of autophagy against H2O2 cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrPC may impair autophagic flux via H2O2-induced oxidative stress.