mTOR/S6 Kinase Pathway Contributes to Astrocyte Survival during Ischemia

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2^−/−) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, “in vivo” effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.Astrocytes are the most abundant cells in the central nervous system. Their functions are crucial for central nervous system homeostasis, because they provide trophic, metabolic, and antioxidant support to neurons. In addition, astrocytes show the ability to modulate synaptic activity and are responsible for preserving neuronal integrity in conditions of disease and injury. In this regard, recent evidence indicates that they are protective for neurons during cerebral ischemia (1). As there is a growing consensus that astrocyte dysfunction may compromise the ability of neurons to survive, the need for studies that clarify the molecular mechanisms involved in the astrocytic response to ischemia is plainly justified.Among the intracellular pathways that integrate signals from nutrients and oxygen, the mammalian target of rapamycin (mTOR)² kinase plays an evolutionary conserved role in the regulation of cell growth, proliferation, survival, and metabolism (2). mTOR exists in the cell in at least two distinct complexes with different partners, mTORC1 and mTORC2. The activity of mTORC1 is exquisitely sensitive to the energy status of the cell and is blocked by the macrolide antibiotic rapamycin. Glucose and oxygen deprivation inhibits mTORC1 activity, respectively, through the regulation of AMP-activated kinase and REDD1/REDD2 proteins (3–5). These factors favor the action of the tuberous sclerosis proteins TSC1 and TSC2, which suppress mTORC1 by forming a complex with GTPase-activating protein (GAP) activity for the small GTPase Rheb (6).In turn, mTORC1 phosphorylates at least three distinct classes of substrates, the eIF4E-binding proteins (4EBP-1 to -3), the ribosomal protein S6 kinases (S6K1 and S6K2) and the serum- and glucocorticoid-inducible kinase 1. However, the pathophysiological role of the mTOR pathway during hypoxia-induced brain damage and the involvement of the distinct mTOR effectors remain to be established.The anabolic actions of the mTOR pathway may in part depend on the regulation of protein synthesis. mTOR associates with the translation initiation factor eIF3 (7). In turn the mTORC1 substrates 4EBPs interact with the cap-binding protein eIF4E (8), while S6Ks phosphorylate the ribosomal protein rpS6 and eIF4B (9, 10). However loss of function mouse mutants of 4EBPs and S6Ks failed to uncover a role of these effectors in global protein synthesis during resting conditions, while instead suggesting an involvement in energy homeostasis and mitochondrial function (11–13). Therefore, mTOR plays critical anabolic and energetic functions still poorly understood, raising the appealing possibility that hypoxia-induced down-regulation of the mTOR pathway could be linked to brain damage. In this regard, S6K, besides stimulating phosphorylation of the ribosomal proteins rpS6 and eIF4B, has been shown to inactivate the anti-apoptotic factor BAD and the insulin receptor substrate IRS (13, 14), demonstrating additional targets that are not directly involved in protein synthesis and may be relevant for the physiological action of the pathway. Moreover, S6K activity is decreased in in vivo paradigms of global and focal brain ischemia (15–17); whereas insulin-activated cardioprotection during ischemia/reoxygenation-induced injury is linked to S6K activation (18).Here we show that oxygen and glucose deprivation (OGD) decreases S6K1 mRNA levels in astrocyte cultures, leading to a reduction of S6K1 protein and activity. S6K loss of function leads to increased astrocyte death during ischemia, impairment of protein synthesis recovery, unbalance between mitochondrial pro- and anti-apoptotic factors and rise in ROS levels. Finally we reveal an effect of S6K suppression on mouse mortality and infarct volume following permanent middle cerebral artery occlusion. Our data indicate a novel role of S6K1 promoting astrocyte survival, protein synthesis, and brain protection in conditions of ischemic stress.