Microglial RIOK3 promotes post-ischemic neuroinflammation by facilitating YBX1 phosphorylation and nuclear accumulation.

Abstract

Excessive activation of microglia exacerbates secondary brain injury after ischemic stroke, yet the upstream mechanisms governing this response remain incompletely understood. This study aimed to investigate the role of RIO kinase 3 (RIOK3) in microglia-mediated neuroinflammation after ischemic stroke and to explore the underlying molecular mechanisms. RIOK3 expression was examined in mice subjected to transient middle cerebral artery occlusion and in primary microglia exposed to lipopolysaccharide or oxygen-glucose deprivation/reoxygenation. Microglia-targeted RIOK3 knockdown was achieved using a Cre-dependent adeno-associated virus-shRNA strategy in Tmem119-CreERT2 mice. RIOK3 was markedly upregulated in microglia after cerebral ischemia and in primary microglia following LPS or OGD/R stimulation. Microglia-targeted RIOK3 knockdown reduced infarct volume, improved early neurological and sensorimotor outcomes, preserved microglial process complexity, and reduced post-ischemic inflammation. In vitro, RIOK3 knockdown reduced microglial inflammatory responses and microglia-mediated neurotoxicity. Transcriptomic and biochemical analyses further showed that RIOK3 knockdown suppressed NF-κB-related transcriptional programs and decreased the phosphorylation of IκBα and p65. Mechanistically, immunoprecipitation assays identified Y-box-binding protein 1 (YBX1) as a RIOK3-interacting protein and mapped this interaction to the C-terminal kinase domain-containing region of RIOK3. RIOK3 enhanced YBX1 Ser102 phosphorylation and nuclear accumulation, whereas YBX1 knockdown attenuated NF-κB activation and the pro-inflammatory effects of RIOK3 overexpression. These findings identify microglial RIOK3 as an important driver of post-ischemic neuroinflammation and highlight the RIOK3-YBX1 axis as a potential therapeutic target for ischemic stroke.