MTOR SIGNALING IN ASTROCYTES AND NG2 GLIA REGULATES GLIAL HOMEOSTASIS AND CELLULAR RESPONSES TO STROKE

Stroke is a central nervous system pathology with limited therapeutic options, highlighting the need for new treatment strategies targeting cellular mechanisms of injury and recovery. We aim to assess how disturbed regulation of mTOR signaling in glia influences their responses and brain homeostasis following focal cerebral ischemia (FCI) in mice, modeled by middle cerebral artery occlusion. Spatial transcriptomics revealed increased expression of mTOR-related genes 3 days after ischemia in the penumbra, while 7 days post-injury the changes were observed in the ischemic core. Single-cell RNA sequencing further demonstrated that NG2 glia accumulate during the acute phase of ischemia at the expense of oligodendrogenesis, accompanied by elevated expression of mTOR-pathway-associated genes, which declined on day 14 after injury. To investigate cell-type-specific effects, genetic mouse models with inhibited or activated mTOR signaling were employed. Analysis of astrocytes isolated from conditional Aldh1l1-driven Rptor or Tsc1 knock-out mice revealed that mTOR activation (Tsc1^-/-) reduced expression of the Kir4.1 potassium channel and the ALPHA2 subunit of Na⁺/K⁺-ATPase, key components of astrocytic potassium buffering. These molecular changes were accompanied by region-specific alterations in astrocyte morphology and animal behavior, indicating that dysregulated astrocyte signaling can influence brain function at the systems level. Together, these results demonstrate that FCI alters mTOR signaling in glia and that modulation of this pathway profoundly affects glial function and brain homeostasis, underscoring mTOR as a critical regulator in the pathophysiology of stroke. Support: Czech Science Foundation (26-21470S), Czech Academy of Sciences (Strategy AV21, VP29), Grant Agency of Charles University (430825).