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Background/Aims: Autophagy is essential for maintaining cellular homeostasis and the survival of terminally differentiated cells as neurons. In this study, we aim to investigate whether mitofusin 2, a mitochondrial fusion protein, mediates autophagy in cerebral ischemia/reperfusion (I/R) injury. Methods: Primary cultured neurons were treated with oxygen-glucose deprivation/reperfusion to mimic cerebral I/R injury in vitro. Autophagosomes were visualized upon TEM. Autophagy-markers were then detected to monitor autophagy by western-blot and real-time PCR, and the autophagic flux was tracked with a mRFP-GFP-LC3 construct by fluorescence as well as autophagy inhibitors and agonists. The up- and downregulation of Mfn2 were through transfecting a lentivirusexpression vector respectively. And neuronal injury was detected by cell counting kit and TUNEL assay. Results: Results showed I/R increased autophagosome formation and inhibited autolysosome degradation. Furthermore, use of autophagy related agents demonstrated that I/R injury was caused by insufficient autophagy and aggravated by impaired autophagic degradation. The results also indicated that mitofusin 2 could ameliorate I/R injury through increasing autophagosome formation and promoting the fusion of autophagosomes and lysosomes. In contrast, downregulation of mitofusin 2 aggravated the I/R injury by inhibiting autophagosome formation and the fusion of autophagosomes and lysosomes. Additionly, mitofusin 2 overexpression did not lead to autolysosome accumulation induced by I/R. Conclusions: In summary, this study explicitly demonstrated that mitofusin 2 could ameliorate I/R injury mainly through promoting autophagy, which represented a potential novel strategy for neuroprotection against cerebral I/R damage.

Neuronal oxidative stress is involved in diverse neurological disorders. Homer1a, as an important member of the Homer family and localized at the postsynaptic density, is known to protect cells against oxidative injury. However, the exact neuroprotective mechanism of Homer1a has not been fully elucidated. Here, we found that Homer1a promoted cell viability and reduced H2O2-induced LDH release. The overexpression of Homer1a enhanced autophagy after H2O2 treatment, which was confirmed by increased expression of LC3II, Beclin-1, and greater autophagosome formation. In addition, we demonstrated that activating autophagy improved cell survival and reduced H2O2-induced oxidative stress and mitochondrial damage. Moreover, the autophagy inhibitor 3-MA partially prevented the protective effects of Homer1a against oxidative challenge. We also found that the upregulation of Homer1a after H2O2 treatment increased the phosphorylation of AMPK. Furthermore, the AMPK inhibitor compound C inhibited Homer1a-induced autophagy and abolished Homer1a-mediated neuroprotection. All the above data suggests that Homer1a confers protection against H2O2-induced oxidative damage via AMPK-dependent autophagy.

Glutamate induced excitotoxicity is common in diverse neurological disorders. RNF146 as an E3 ubiquitin ligase protects neurons against excitotoxicity via interfering with Poly (ADP-ribose) (PAR) polymer-induced cell death (parthanatos). However, the neuroprotective role of RNF146 has not been fully understood. We aimed to investigate the role of RNF146 in modulating autophagy in HT22 cells under glutamate excitotoxicity injury. Here we found that induction of RNF146 decreased the cellular damage and excitotoxicity induced by glutamate. RNF146 also suppressed the excessive autophagy, which is detrimental to HT22 cells survival, induced by glutamate or rapamycin treatment. In addition, we find that Wnt/β-catenin was a negative regulation factor for autophagy in glutamate excitotoxicity. Over-expression of RNF146 promoted Wnt/β-catenin signaling, which was related to destabilization of β-catenin destruction complex. These results indicated that RNF146 acted as a neuroprotective agent against glutamate-induced excitatory damage, and this neuroprotection might be at least partly dependent on the inhibition of excessive autophagy by regulating Wnt/β-catenin signaling.

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