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E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion.

ABSTRACT: Cerebral ischemia/reperfusion (I/R) causes brain damage accompanied by ubiquitin accumulation and impairment of proteasome activity. In this study, we report that E2-25K, an E2-conjugating enzyme, is SUMOylated during oxidative stress and regulates cerebral I/R-induced damage. Knockdown of E2-25K expression protects against oxygen/glucose deprivation and reoxygenation (OGD/R)-induced neuronal cell death, whereas ectopic expression of E2-25K stimulates it. Compared with the control mice, cerebral infarction lesions and behavioral/neurological disorders are ameliorated in E2-25K knockout mice during middle cerebral artery occlusion and reperfusion. In particular, E2-25K is SUMOylated at Lys14 under oxidative stress, OGD/R and I/R to prompt cell death. Further, E2-25K downregulates the proteasome subunit S5a to impair proteasome complex and thus restrain proteasome activity under oxidative stress. This proteasome inhibitory activity of E2-25K is dependent on its SUMOylation. These results suggest that E2-25K has a crucial role in oxidative stress and cerebral I/R-induced damage through inhibiting proteasome via its SUMOylation.

SUBMITTER: Jeong EI

PROVIDER: S-EPMC5261013 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion.

Jeong Eun Il EI Chung Hae Won HW Lee Won Jea WJ Kim Seo-Hyun SH Kim Hyunjoo H Choi Seon-Guk SG Jung Yong-Keun YK

Cell death & disease 20161229 12

Cerebral ischemia/reperfusion (I/R) causes brain damage accompanied by ubiquitin accumulation and impairment of proteasome activity. In this study, we report that E2-25K, an E2-conjugating enzyme, is SUMOylated during oxidative stress and regulates cerebral I/R-induced damage. Knockdown of E2-25K expression protects against oxygen/glucose deprivation and reoxygenation (OGD/R)-induced neuronal cell death, whereas ectopic expression of E2-25K stimulates it. Compared with the control mice, cerebral ...[more]

PMID: 28032866

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Project description:Rationale: Recent studies indicate that microglial activation and the resulting inflammatory response could be potential targets of adjuvant therapy for ischemic stroke. Many studies have emphasized a well-established function of Annexin-A1 (ANXA1) in the immune system, including the regulation of microglial activation. Nevertheless, few therapeutic interventions targeting ANXA1 in microglia for ischemic stroke have been conducted. In the present study, Tat-NTS, a small peptide developed to prevent ANXA1 from entering the nucleus, was utilized. We discovered the underlying mechanism that Tat-NTS peptide targets microglial ANXA1 to protect against ischemic brain injury. Methods: Preclinical studies of ischemic stroke were performed using an oxygen-glucose deprivation and reperfusion (OGD/R) cell model in vitro and the middle cerebral artery occlusion (MCAO) animal model of ischemic stroke in vivo. Confocal imaging and 3D reconstruction analyses for detecting the protein expression and subcellular localization of microglia in vivo. Co-immunoprecipitation (Co-IP), immunoblotting, ELISA, quantitative real-time PCR (qRT-PCR), Luciferase reporter assay for determining the precise molecular mechanism. Measurement on the cytotoxicity of Tat-NTS peptide for microglia was assessed by CCK-8 and LDH assay. TUNEL staining was used to detect the microglia conditioned medium-mediated neuronal apoptosis. Adeno-associated viruses (AAVs) were injected into the cerebral cortex, striatum and hippocampal CA1 region of adult male Cx3cr1-Cre mice, to further verify the neurofunctional outcome and mechanism of Tat-NTS peptide by TTC staining, the modified Neurological Severity Score (mNSS) test, the open field test (OFT), the novel object recognition task (NORT), the Morris water maze (MWM) test, the long-term potentiation (LTP) and the Transmission electron microscopy (TEM). Results: It was observed that administration of Tat-NTS led to a shift of subcellular localization of ANXA1 in microglia from the nucleus to the cytoplasm in response to ischemic injury. Notably, this shift was accompanied by an increase in ANXA1 SUMOylation in microglia and a transformation of microglia towards an anti-inflammatory phenotype. We confirmed that Tat-NTS-induced ANXA1 SUMOylation in microglia mediated IKKα degradation via NBR1-dependent selective autophagy, then blocking the activation of the NF-κB pathway. As a result, the expression and release of the pro-inflammatory factors IL-1β and TNF-α were reduced in both in vitro and in vivo experiments. Furthermore, we found that Tat-NTS peptide's protective effect on microglia relieved ischemic neuron apoptosis. Finally, we demonstrated that Tat-NTS peptide administration, through induction of ANXA1 SUMOylation in microglia, reduced infarct volume, improved neurological function and facilitated behavioral recovery in MCAO mice. Conclusions: Our study provides evidence for a novel mechanism of Tat-NTS peptide in regulating microglial ANXA1 function and its substantial neuroprotective effect on neurons with ischemic injuries. These findings suggest that Tat-NTS peptides have a high potential for clinical application and may be a promising therapeutic candidate for treating cerebral ischemia.

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E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion.

Publications

E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion.

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