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Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke.

ABSTRACT: In response to stroke-induced injury, astrocytes can be activated and form a scar. Inflammation is an essential component for glial scar formation. Previous study has shown that adjudin, a potential Sirt3 activator, could attenuate lipopolysaccharide (LPS)- and stroke-induced neuroinflammation. To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation, we used a transient middle cerebral artery occlusion (tMCAO) model with or without adjudin treatment in wild type (WT) and Sirt3 knockout (KO) mice and performed a wound healing experiment in vitro. Both our in vivo and in vitro results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression. In addition, adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice, which was blunted by Sirt3 deficiency. Furthermore, adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3. Both the suppression of Foxo3a and overexpression of N1ICD could alleviate the inhibitory effect of adjudin in vitro indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing experiment.

SUBMITTER: Yang X

PROVIDER: S-EPMC5744009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ischemic stroke has been reported to cause significant changes to memory, thinking, and behavior. Intriguingly, recently reported studies have indicated the association of Trimethylamine N-oxide (TMAO) with the acute phase of ischemic stroke. However, the comprehensive underlying mechanism remained unknown. The objective of the present study was to investigate the association between TMAO and recovery of neurological function after ischemic stroke. For this purpose, a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established and treated with TMAO or/and sh-ALK5, followed by the neurological function evaluation. Behaviors of rats were observed through staircase and cylinder tests. Moreover, the expression of Smurf2 and ALK5 was detected by immunohistochemistry while expression of GFAP, Neurocan, and Phosphacan in brain tissues was determined by immunofluorescence. Thereafter, gain- and loss-of-function assays in astrocytes, the proliferation, viability, and migration were evaluated by the EdU, CCK-8, and Transwell assays. Besides, Smurf2 mRNA expression was determined by the RT-qPCR, whereas, Smurf2, ALK5, GFAP, Neurocan, and Phosphacan expression was evaluated by the Western blotting. Finally, the interaction of Smurf2 with ALK5 and ALK5 ubiquitination was assessed by the co-immunoprecipitation. Notably, our results showed that TMAO promoted the proliferation of reactive astrocyte and formation of glial scar in MCAO/R rats. However, this effect was abolished by the Smurf2 overexpression or ALK5 silencing. We further found that TMAO upregulated the ALK5 expression by inhibiting the ubiquitination role of Smurf2. Overexpression of ALK5 reversed the inhibitory effect of Smurf2 on astrocyte proliferation, migration, and viability. Collectively, our work identifies the evolutionarily TMAO/Smurf2/ALK5 signaling as a major genetic factor in the control of reactive astrocyte proliferation and glial scar formation in ischemic stroke, thus laying a theoretical foundation for the identification of ischemic stroke.

Project description:Schwann cell (SC) transplantation is a promising approach for the treatment of spinal cord injury (SCI); however, SC grafts show a low migratory capacity within the astrocytic environment, which inevitably hampers their therapeutic efficacy. The purpose of this study was to explore mechanisms to modify the characteristics of SCs and astrocytes (ASs), as well as to adjust the SC-AS interface to break the SC-AS boundary, thus improving the benefits of SCI treatment. We observed that the expression levels of miR-124 in SCs and ASs were significantly lower than those in the normal spinal cord. Furthermore, overexpressing miR-124 in SCs (miR-124-SCs) significantly inhibited gene and protein expression levels of SC-specific markers, such as GFAP and Krox20. The expression of neurotrophic factors, Bdnf and Nt-3, was up-regulated in miR-124-SCs without affecting their proliferation. Further, the boundary assay showed an increased number of miR-124-SCs that had actively migrated and entered the astrocytic region to intermingle with ASs, compared with normal SCs. In addition, although Krox20 protein expression was down-regulated in miR-124-SCs, the luciferase assay showed that Krox20 is not a direct target of miR-124. RNA sequencing of miR-124-SCs revealed seven upregulated and eleven downregulated genes involved in cell migration and motility. Based on KEGG pathway and KOG functional analyses, changes in these genes corresponded to the activation of Hippo, FoxO, and TGF-beta signaling pathways, cytokine-cytokine receptor interactions, and the cell cycle. Finally, co-culturing of miR-124-SCs and ASs in a transwell system revealed that GFAP and p-STAT3 protein expression in ASs was significantly reduced. Collectively, these results show that overexpression of miR-124 in SCs promotes SC-AS integration in vitro and may attenuate the capacity of ASs to form glial scars. Thus, this study provides novel insights into modifying SCs by overexpressing miR-124 to improve their therapeutic potential in SCI.

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Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke.

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