Abstract Body: Introduction: Post-stroke neuroinflammation is a hallmark of ischemic stroke. This study investigates the role of IGSF6 in microglia during ischemic stroke and its contribution to neuroinflammatory responses and brain injury following stroke.

Methods: IGSF6 expression was examined in murine microglia under various inflammatory stimuli. Using a middle cerebral artery occlusion (MCAO) model, we evaluated the impact of microglial Igsf6 knockdown (Igsf6-KD) on neuroinflammation and ischemic outcomes. Pro-inflammatory cytokine levels were measured using qPCR and ELISA in both Igsf6-KD and wild-type (WT) mice, and functional outcomes were assessed through neurological tests. Subcellular localization and cytokine trafficking studies were conducted using immunofluorescence, electron microscopy, and protein interaction assays.

Results: IGSF6 expression was significantly upregulated in microglia following MCAO. Knockdown of Igsf6 in microglia resulted in reduced secretion of pro-inflammatory cytokines (TNF, IL-6, CCL2, CCL5) at day 1 post-stroke, despite unchanged mRNA levels. Morphological analysis of microglia from Igsf6-KD mice revealed a less reactive phenotype. Additionally, Igsf6-KD mice exhibited a reduction in infarct volume and improved functional outcomes, including enhanced performance on rotarod, grip strength, and foot-fault tests.

Mechanistically, IGSF6 was shown to localize to the trans-Golgi network (TGN) and mediate cytokine trafficking. Knockdown of Igsf6 disrupted this process, reducing the secretion of key cytokines without affecting their transcription or translation.

Conclusions: Microglial IGSF6 exacerbates post-stroke neuroinflammation by promoting the secretion of pro-inflammatory cytokines. Its knockdown attenuates neuroinflammatory responses, reduces ischemic brain injury, and improves functional recovery. These findings highlight the detrimental role of IGSF6 in stroke pathology and suggest that targeting IGSF6 may offer therapeutic benefits for mitigating neuroinflammation and improving outcomes after ischemic stroke.