Blocking SUR1-TRPM4 reduces neuronal loss in peri-infarct area via stimulating TGFα released by microglia

Abstract Background As a sequel of ischemic stroke, selective neuronal loss (SNL) mediated by activated microglia and consequential neuroinflammation affects the salvageable peri-infarct area (PIA) and hampers the functional recovery following reperfusion therapy. Recent evidence indicates that inhibition of sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) exerts a robust protective effect against inflammation. Hence, we hypothesized that blocking SUR1-TRPM4 channels reduced SNL after brain ischemia in the PIA. Methods Mice subjected to temporary middle cerebral artery occlusion (tMCAO) for 1 h and reperfusion for 24 h were adopted to mimic the pathophysiological changes of ischemic stroke. Gene expression, neuronal apoptosis, and protein content were tested by RNA-sequencing, TUNEL staining, Western blot, respectively. Results After tMCAO, abundant neuronal apoptosis appeared in the PIA, with remarkable up-regulation and co-localization of SUR1 and TRPM4. Blocking the SUR1-TRPM4 channel by glibenclamide (GLB, a SUR inhibitor) and Trpm4 gene deletion (Trpm4−/−) distinctly alleviated apoptotic neurons in the PIA. To explore the potential mechanism of blocking SUR1-TRPM4, we compared gene expression in brain tissues of Trpm4−/− and wild-type mice after tMCAO modeling using RNA-sequencing and identified 217 differentially expressed genes. Among them, the expression of Tgfα was significantly higher in Trpm4−/− mice compared with that in wild-type mice after tMCAO. GLB treatment significantly increased the expression of TGFα in microglia, as validated by Western Blot and immunofluorescence staining. Moreover, intracerebroventricular injection of recombinant TGFα significantly alleviated the neuronal loss in the PIA and improved neurological outcome after tMCAO. In vitro, we subjected BV-2 microglia cells to oxygen-glucose deprivation or lipopolysaccharide stimulation and found significant up-regulation and co-localization of SUR1 and TRPM4, while blocking SUR1-TRPM4 with GLB and 9-phenanthrol (9-Phe, a TRPM4 inhibitor) increased the expression and release of TGFα by activating the CaMKII/CREB pathway. BV-2 microglia derived conditioned medium after oxygen-glucose deprivation or lipopolysaccharide stimulation induced apoptosis of SH-SY5Y cells, which could be inhibited by applying GLB and 9-Phe on BV-2. Furthermore, direct application of recombinant TGFα alleviated neuronal apoptosis mediated by BV-2 microglia conditioned medium. Conclusions Collectively, our findings indicate that blocking SUR1-TRPM4 in microglia alleviates SNL, probably by up-regulating the expression and release of TGFα.