FIGURE 6.

AKT activation by IGF-I is involved in neuroprotection by astrocytes. A, neuron survival was determined in cultures of neurons alone or in combination with astrocytes. These cultures were treated with H₂O₂ (50 μm), IGF-I (100 nm), or the combination. Quantification of neuron survival was assessed by counting the total number of viable β-III-tubulin⁺ (neuronal marker) cells determined by plasma membrane integrity and the absence of nuclear alterations detected with DAPI. The percentage of viable neurons was expressed relative to IGF-I-treated cultures. Deleterious effects of H₂O₂ were much stronger in neuron cultures alone than in combination with astrocytes. IGF-I co-administration increased neuron survival only in co-cultures with astrocytes (***, p < 0.001 versus H₂O₂-treated co-cultures; n = 3). B, upper, representative photomicrographs of co-cultures of neurons with astrocytes immunostained with β-III-tubulin (neurons), GFAP (astrocytes), and DAPI (cell nuclei). Astrocytes were transfected with PTENA4 mutant or a control construct prior to co-culture with neurons and treatment with H₂O₂ (100 μm), IGF-I (100 nm), or both. Lower, quantification of neuron survival was assessed by counting the total number of viable β-III-tubulin⁺ (neuronal marker) cells determined by plasma membrane integrity and the absence of nuclear alterations detected with DAPI. The percentage of viable neurons was expressed relative to vehicle treatments. H₂O₂ exposure significantly reduced neuron survival both in co-cultures with PTENA4 astrocytes and with control astrocytes (***, p < 0.001 versus control co-cultures; n = 3). IGF-I co-administration significantly prevented H₂O₂ effects on neuronal survival in co-cultures with control astrocytes (***, p < 0.001 versus H₂O₂-treated co-cultures with control construct astrocytes; n = 3), whereas in co-cultures with PTENA4-transfected astrocytes, IGF-I effects were abolished (***, p < 0.001 versus H₂O₂ + IGF-I-treated co-cultures with control construct astrocytes; n = 3). C, upper, representative photomicrographs of co-cultures of neurons with astrocytes immunostained with β-III-tubulin, GFAP, and DAPI. Astrocytes were transfected with an AKT-CA mutant or a control construct prior to co-culture with neurons and treatment with H₂O₂ (100 μm). Lower, H₂O₂ exposure significantly reduced neuron survival in co-cultures with control astrocytes (***, p < 0.001 versus control co-cultures), whereas in co-cultures with AKT-CA-astrocytes, the H₂O₂ effect was partially prevented (*, p < 0.05 versus H₂O₂-treated co-cultures with control construct astrocytes). Error bars represent S.E.