Rapid stimulation of the PI3-kinase/Akt signalling pathway in developing midbrain neurones by oestrogen

Oestrogen promotes the differentiation of neurones in the central nervous system. In the rodent midbrain, the maturation of dopaminergic neurones appears to be under oestrogen control. This is supported by the fact that dopaminergic cells contain nuclear oestrogen receptors-alpha/beta (ER). Second, aromatase is transiently expressed in the developing midbrain. In previous studies, we have shown that oestrogen increases dopamine synthesis and plasticity of dopamine cells. These effects are transmitted through classical nuclear ER but require also the stimulation of nonclassical signalling pathways involving the activation of membrane receptors. This study attempted to identify nonclassical oestrogen-dependent signalling cascades which might be stimulated downstream of membrane ERs. Using cultured mouse midbrain cells, we could demonstrate by Western blotting, that oestrogen rapidly phosphorylates Akt, a kinase which is implicated in the phosphatidylinositol 3 (PI3)-kinase pathway. This effect was only seen in midbrain neurones but not astrocytes. Oestrogen-induced Akt phosphorylation was time- and dose-dependent, showing highest responses after 30 min and at a steroid concentration of 10(-8) and 10(-6) M. Immunocytochemistry for phosphorylated Akt (pAkt) demonstrated that pAkt is predominantly found in a nuclear/perinuclear position and that oestrogen exposure increased the number of pAkt-positive cells. To investigate the mechanisms which are involved in transmitting oestrogen effects on the cellular level, cells were treated with antagonists for distinct signalling pathways. The application of the nuclear ER antagonist ICI 182 780 did not abolish the oestrogen-induced Akt phosphorylation. In contrast, interrupting intracellular calcium signalling with BAPTA completely prevented this effect. The PI3-kinase inhibitor LY294002 also inhibited the activation of Akt by oestrogen. Our study clearly indicates that oestrogen can rapidly stimulate the PI3-kinase/Akt signalling cascade in differentiating midbrain neurones. This effect requires the intermediate activation of calcium-dependent signalling pathways. In conclusion, oestrogen effects in the developing midbrain appear to be connected with the PI3-kinase/Akt signalling mechanism.