Microglial DNA G-quadruplex accumulation is associated with altered autophagy-related responses and aggravated ischemic brain injury.

Ischemic stroke creates a hypoxic and nutrient-deprived microenvironment that rapidly activates microglia, a central driver of post-ischemic inflammation and a key determinant of secondary tissue damage and neurological recovery. G-quadruplex (G4) structures are non-canonical nucleic acid conformations that can reshape stress responses in diverse settings, yet whether microglial DNA G4 relates to ischemic pathology remains unknown. Here, using a transient middle cerebral artery occlusion (tMCAO) mouse model and an oxygen-glucose deprivation/reoxygenation (OGD/R) primary microglia model, we examined how DNA G4 dynamics relate to microglial stress responses and post-ischemic outcomes. We found that cerebral ischemia induced a dynamic accumulation of microglial DNA G4 signal, accompanied by transient changes in autophagy-related markers. Pharmacological stabilization of G4 with pyridostatin (Pds) was associated with increased mTOR phosphorylation under ischemic stress, increased inhibitory ULK1 phosphorylation, and alterations in autophagy-related proteins, including a reduced LC3-II/LC3-I ratio, p62 accumulation, and downregulation of Beclin-1. In parallel, Pds treatment was associated with increased overall cellular stress under ischemic conditions. Under the prophylactic in vivo paradigm, Pds-treated mice showed larger infarct burden and worse neurological deficits. Importantly, the mTOR inhibitor rapamycin partially reversed Pds-associated autophagy-related changes and partially improved tissue and functional outcomes, although it did not fully normalize the broader stress-associated alterations. Collectively, our findings suggest that a pre-existing G4-stabilized state is associated with altered mTOR-ULK1/autophagy-related responses and aggravated ischemic outcomes, highlighting the G4-mTOR-autophagy-related axis as a potential contributor to post-ischemic microglial stress responses.