Alternatively, astrocytes were electroporated (2 106astrocytes with 2 g of DNA or shRNA) before seeding using an astrocyte Nucleofector kit (Lonza, Switzerland). be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active Ioversol AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring Ioversol of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may type part of the brain defense mechanism against oxidative stress injury. Keywords: Akt PKB, astrocyte, FOXO, insulin resistance, insulin-like growth factor (IGF), neuroprotection, oxidative stress, p38 MAPK, phosphatase and tensin homolog (PTEN) == Introduction == IGF-I2is a pleiotropic growth factor with important prosurvival effects in neurons (1). One of the main downstream targets of IGF-I is the Ser/Thr kinase AKT (2), which mediates cell survival and proliferation (3). Upon its activation, the IGF-I receptor recruits and phosphorylates IRS docking proteins (4), which allows translocation of phosphatidylinositol 3-kinase (PI3K) to the plasma membrane where it catalyzes the formation of the lipid second messenger phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (5). AKT is recruited to the membrane by interaction with these messengers so that it can be fully activated by PDK1 and mTORC2 kinases (3, 6). This pathway is switched off (to prevent uncontrolled proliferation) through activation of the lipid and protein phosphatase PTEN, which catalyzes PIP3dephosphorylation (4). Cell metabolism generates potentially harmful reactive oxygen species (ROS), which at moderate levels can act as second messengers (7). However , chronic and/or sudden increases of ROS (uncontainable by detoxification through cellular defenses) generates oxidative stress, a pathological cellular condition that can interfere with redox-sensitive signaling pathways (7). Neurons are particularly vulnerable to oxidative stress because of their low ROS detoxifying capacity (8). We previously described that oxidative stress interferes with the IGF-I/PI3K/AKT pathway Ioversol by redox activation of p38 kinase to induce neuronal death (9). IGF-I signaling impairment by oxidative stress has been recently confirmed by others in neurons (10, 11) and others cell types (12). However , a substantial body of evidence in vertebrates and invertebrates also indicates that IGF-I signaling may reduce cell Ioversol defenses to oxidative stress (1315) that, in turn, would affect neuron survival. These results question the neuroprotective role of IGF-I in response to brain oxidative insults. However , IGF-I has shown antioxidative activity in the majority of cell types present in the mammalian brain (1618) and clinical benefits in brain pathologies associated with oxidative stress Ioversol (17, 19, 20). Furthermore, our group has recently showed that IGF-I cooperates with other trophic signals produced by astrocytes, essential contributors to neuronal homeostasis (21), to protect neurons against oxidative insults (16). Collectively, these results suggest that the antioxidative functions of IGF-I could be cell- and context-dependent and could play a neuroprotective role during brain oxidative insults (16). In the present work, we describe two molecular adaptations present in astrocytes that preserve the activation of AKT by IGF-I during oxidative stress. These adaptations include 1) insensitivity of the IGF-I/PI3K/AKT pathway to modulation by the kinase p38 and 2) phosphorylation of PTEN by IGF-I, which leads to its cytosolic translocation. Armoring of AKT activation by IGF-I in astrocytes contributes to normalize ROS levels and to prevent cell death during oxidative stress. Of note, the neuroprotective role of astrocytes is also enhanced SK by these adaptations. These results point out the importance of AKT activation intended for astrocyte survival during oxidative stress and reinforce the idea that modulation of astrocytes by IGF-I forms part of the brain responses to oxidative damage. == Experimental Procedures == == == == == == Animals and Reagents == Postnatal day a few and 7 Wistar rats were used (Harlan, Spain). All efforts were made to minimize suffering and reduce the number of animals. Animals were kept under light/dark conditions following European Union guidelines (directive 86/609/EEC) and handled according to institutionally approved procedures. Antibodies to phospho-AKT (Ser-473), phospho-p38MAPK (Thr-180/Tyr-182), p38MAPK, c-Jun N-terminal kinases (JNKs), FOXO3, phospho (Thr-32)-FOXO3, PTEN, and phospho-PTEN (Ser-380/Thr-382/383) were from Cell Signaling Technology (Danvers, MA). IGF-I receptor (C-20), AKT1/2 (H-136), R-RAS, and phospho-JNK (Thr-183/Tyr-185).
