Estrogen-mediated neuroprotection of primary mesencephalic dopamine neurons

The available evidence suggests that estrogen serves as a neuroprotectant in many disorders originating in the brain, including the nigrostriatal neurodegeneration observed in Parkinson's disease. While this steroid has been shown to protect dopamine neurons of the substantia nigra, estrogen's mechanisms of action at the cellular and molecular levels are poorly understood. In the present study, we investigated estrogen protection of dopaminergic neurons in primary mouse mesencephalic cultures treated with 1-methyl-4-phenylpyridinium (MPP +), the active metabolite of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which represents a toxin-induced model of Parkinson's disease that mimics the selective loss of substantia nigra dopaminergic neurons in the midbrain. Initial experiments were conducted in human brain-derived neuroblastoma dopamine expressing SH-SY5Y cells as a mechanistic tool to correlate further investigations in primary mesencephalic neurons. However, a lack of protection by estrogen in response to MPP+ exposure in the homogenous population of SH-SY5Y cells redirected the focus in heterogeneous mesencephalic cultures to investigate indirect estrogen-mediated neuroprotection. Mesencephalic dopamine cultures express both ERalpha and ERbeta with a predominance of ERalpha on both dopamine neurons and astrocytes. 17beta-estradiol protects dopamine neurons from injury induced by MPP+ in a time and ERalpha dependent manner. Interestingly, ablation of glial cells from mesencephalic cultures significantly reduced the neuroprotective effects of estrogen suggesting that the neuroprotection provided by estrogen against MPP+ toxicity is indirect and involves an interplay between at least two cell types. In an effort to identify the molecular mechanism whereby estrogen was protecting mesencephalic dopamine neurons, we examined estrogen signaling in primary astrocyte cultures to evaluate a possible indirect neuroprotective signaling mechanism initiated by estrogen. ERalpha was expressed in the plasma membrane of actrocytes and a 17beta-estradiol time course revealed a significant PI3K dependent increase in Akt phosphorylation and stimulation of the downstream transcription factor, CREB in astrocyte cultures. When these results were evaluated in mesencephalic cultures by applying estrogen-conditioned astrocyte media that was pharmacologically treated with inhibitors to the ER and PI3K, we found that estrogen-conditioned media significantly protected mesencephalic dopamine neurons devoid of glia and that the protection afforded by estrogen in dopamine neurons was completely indirect and dependent on PI3K activity in astrocytes. Thus, estrogen protects midbrain dopamine neurons against MPP + neurotoxicity via an ERalpha mediated mechanism involving indirect estrogen-mediated astrocyte signaling and PI3K stimulation. In conclusion, estrogen's indirect neuroprotective mechanism via astrocytes and the subsequent release of estrogen-regulated, astrocyte-derived proteins may represent an alternative approach in treating the neurodegeneration observed in Parkinson's disease.