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.