RHBDF2 governs microglial neuroinflammation during cerebral ischemia-reperfusion injury and is positively regulated by the m6A reader YTHDF1.

BACKGROUND

Neuroinflammation mediated by microglia activation is the key pathological mechanisms for cerebral ischemia-reperfusion injury (CIRI). This study investigated the role and underlying molecular mechanism of Rhomboid 5 homolog 2 (RHBDF2) in neuroinflammation during CIRI.

METHODS

The in vivo middle cerebral artery occlusion and reperfusion (MCAO/R) mouse model and in vitro HMC3 microglia subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established to mimic CIRI. Real-time PCR, western blot, immunohistochemistry, immunofluorescence, flow cytometry, and co-immunoprecipitation assays were used to confirm RHBDF2 expression and explore the molecular mechanism of microglia-specific RHBDF2 knockdown in CIRI. Methylated RNA immunoprecipitation was used to detect the m6A methylation level of RHBDF2 mRNA both in vivo and in vitro. RNA sequencing analysis was performed in OGD/R-treated HMC3 cells with or without RHBDF2 knockdown.

RESULTS

Our finding showed that RHBDF2 expression increased in both in vivo and in vitro CIRI models. Microglial-specific RHBDF2 knockdown reduced brain injury in MCAO/R mice, as evidenced by the reduction in the cerebral infarct volume and amelioration of the neurological deficits. Furthermore, we demonstrated that RHBDF2 knockdown alleviated neuroinflammation by inhibiting microglial M1 polarization and promoting microglial M2 polarization in MCAO/R mouse ischemic penumbra. Mechanistically, RHBDF2 interacted with STING and promoted the activation of the STING-TBK1-IRF3/p65 signaling pathway. Rescue experiments confirmed that RHBDF2 knockdown suppressed inflammation via the inhibition of STING-TBK1 signaling pathway. In addition, the m6A methylation level of RHBDF2 mRNA was significantly increased in the MCAO/R mouse brain tissues and OGD/R-treated HMC3 cells. YTHDF1 recognized the m6A sites of RHBDF2 and promote its expression in an m6A manner. Through RNA-seq, the possible downstream effectors of RHBDF2 in CIRI was predicted.

CONCLUSIONS

Microglial-specific RHBDF2 knockdown inhibits neuroinflammation in CIRI via STING-TBK1 signaling pathway, and is positively regulated by the m6A reader YTHDF1. This suggests RHBDF2 as a potential therapeutic target in ischemic stroke.