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Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.

ABSTRACT: The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and β-Catenin signaling cascade, but only Erk inhibitors reversed RSPO3-induced neuroprotection against OGD/R. In mouse embryonic fibroblasts (MEFs) and neuronal cells, RSPO3-induced LGR4-Gab1-Gαi1/3 association was required for Erk activation, and either silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. In mice, middle cerebral artery occlusion (MCAO) increased RSPO3 expression and Erk activation in ischemic penumbra brain tissues. Endothelial knockdown or knockout of RSPO3 inhibited Erk activation in the ischemic penumbra brain tissues and increased MCAO-induced cerebral ischemic injury in mice. Conversely, endothelial overexpression of RSPO3 ameliorated MCAO-induced cerebral ischemic injury. We conclude that RSPO3 activates Gαi1/3-Erk signaling to protect neuronal cells from ischemia/reperfusion injury.

SUBMITTER: Liu TT

PROVIDER: S-EPMC10560285 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.

Liu Ting-Tao TT Shi Xin X Hu Hong-Wei HW Chen Ju-Ping JP Jiang Qin Q Zhen Yun-Fang YF Cao Cong C Liu Xue-Wu XW Liu Jian-Gang JG

Cell death & disease 20231007 10

The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and β-Catenin signaling cascade, but onl ...[more]

PMID: 37805583

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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.

Dataset Information

Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.

Publications

Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.

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