Syndecan-4 in microglia mediates ischemic stroke-induced mitochondrial dysfunction and blood-brain barrier damage by interacting with Dishevelled

Abstract Ischemic stroke is a leading cause of disability and mortality, resulting in impaired mitochondrial function and disruption of the blood-brain barrier (BBB). Studies have demonstrated that Syndecan-4 (SDC4) influences BBB integrity and function, however, it remains unclear whether SDC4 influences mitochondrial function and BBB integrity following a stroke. We first obtained single-cell data from cortical tissue of transient middle cerebral artery occlusion (tMCAO) mice from public databases to identify changes in disease-associated cells and related molecular composition during disease progression. We then performed functional analyses to elucidate the functional characteristics of microglial subsets. Trajectory analysis was used to investigate cellular transition state signatures. Subsequently, we injected adeno-associated virus-shSDC4 (AAV-shSDC4) into the brain of C57BL/6J mice before tMCAO. Primary microglia were cultured and transfected with lentiviral-shSDC4 (LV-shSDC4) before oxygen-glucose deprivation/reoxygenation (OGD/R). Western blot, flow cytometry, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to investigate the expression, function, and mechanisms of SDC4. Molecular docking and molecular dynamics simulations were used to investigate binding. We identified 13 major cell populations, most of which underwent dynamic changes after MCAO. Microglia were the predominant cell population in all groups. Subsequent clustering analysis demonstrated that the relative abundances of repair- and anti-inflammatory as well as senescence-related microglial subpopulations were reduced following MCAO. Complementary trajectory inference modeling further uncovered a progressive upregulation of SDC4 expression in microglia throughout the course of disease progression. To validate these observations, we assessed SDC4 levels in experimental models of cerebral ischemia-reperfusion (I/R) injury and found elevated expression of this protein in both in vitro and in vivo settings. AAV-shSDC4 significantly improved I/R-induced motor impairment, restored mitochondrial morphology and function, increased occludin and claudin-5 expression, and protected BBB integrity. AAV-shSDC4 and LV-shSDC4 treatment enhanced Wnt/β-catenin signaling. XAV-939 reversed the protective effects of SDC4 interference. Mechanistically, SDC4 interacts with Dishevelled (Dvl) in microglia, physically sequestering Dvl and inhibiting Wnt/β-catenin signaling, ultimately leading to microglial mitochondrial dysfunction and associated BBB damage. The interaction between SDC4 and Dvl promotes mitochondrial dysfunction in microglia and is closely associated with the disruption of BBB integrity, thus offering a potential therapeutic strategy for the clinical treatment of cerebral ischemia.