| Abstract |
The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEA sulfate (DHEAS), and allopregnanolone (Allo) are produced by the CNS and the adrenals and their levels decrease gradually with advancing age. The decline of their levels was associated to neuronal dysfunction and degeneration, most probably because these steroids protect neurons against noxious agents. Indeed, experimental evidence suggest that they protect hippocampal and cortical neurons against ischemia- and excitotoxicity- induced injury. We have studied their protective role on the adrenal medulla, an important part of the sympathetic nervous system, and the tissue adjacent to their primary peripheral site of production. DHEA, DHEAS, and Allo protect rat neural-crest-derived chromaffin cells and the rat pheochromocytoma PC12 cell line, an established model for the study of adrenomedullary cell apoptosis and survival, against serum deprivation-induced apoptosis. Their effects are time- and dose-dependent, with EC50 1.8, 1.1, and 1.5 nM, respectively. The pro-survival effect of DHEA(S) and Allo appears to be NMDA-, GABAA-, sigma1-, or estrogen receptor- independent. It involves the antiapoptotic Bcl-2 proteins, their role being sine qua non for their action because Bcl-2 antisense oligonucleotides reverses their effects. DHEA(S) and Allo activate pro-survival transcription factors CREB and NF-κB, upstream effectors of anti-apoptotic Bcl-2 protein expression. They also activate the anti-apoptotic kinase PKCα/β, a post-translational activator of Bcl-2 protein and induce the phosphorylation-activation of two major pro-survival kinase pathways, PI3K-Akt and Src-ERK-MEK. Since zona reticularis and adrenal medulla are closely proximal with anatomically interwoven borders, we have also examined the possible paracrine effects of neurosteroids on catecholamine production from sympathoadrenal cells, using the PC12 cell model. DHEA, DHEAS, and Allo increase acutely (peak effect between 1030 min) and dose-dependently (EC50 in the nM range) catecholamine levels (norepinephrine and dopamine). It appears that the acute effect of these steroids involve actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10-6 M phallacidin, an actin filament stabilizer, completely prevents steroid-induced catecholamine secretion. DHEAS and Allo, but not DHEA, also affect catecholamine synthesis. Indeed, DHEAS and Allo increase catecholamine levels at 24 h, an effect blocked by AMPT and NSD1015, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis is supported by finding that DHEAS and Allo increase both the mRNA and protein levels of tyrosine hydroxylase. The exact nature of the receptor systems mediating these rapid actions of DHEA(S) on PC12 cells is unknown. These effects appear to be independent of NMDA or GABAA receptors since PC12 cells express negligible levels of non functional NMDA or GABAA receptors, and do not respond to their agonists. The rapid onset of DHEA and DHEAS actions on PC12 cells supports the hypothesis that these neuroactive steroids may utilize specific membrane receptors. Our findings show that non permeable DHEA-BSA maintains its ability to protect PC12 cells against apoptosis and suggest the presence of DHEA specific membrane binding sites (Kd in the nM range) on membranes isolated from PC12, human chromaffin and rat hippocampal cells. The putative membrane DHEA binding sites are functionally coupled to G-proteins, because Pertussis toxin totally blocks the anti-apoptotic effects of DHEA-BSA. In contrast to Estradiol and Progesterone, Testosterone and Corticosterone displace DHEA membrane binding, acting as antagonists by blocking the anti-apoptotic effect of DHEA-BSA, as well as its stimulatory effect on prosurvival Bcl-2/Bcl-xL proteins and Src kinase. These findings suggest that DHEA may exert neuroprotective effects via specific membrane binding sites, independent of NMDA or GABAA receptors. To investigate the structure-activity relationships of dehydroepiandrosterone (DHEA) and allopregnanolone (ALLO) as pro-survival factors, their anti-apoptotic effect was compared to that of a long list of structurally related compounds. Their pro-survival actions were found to be structure-specific, confined mainly to conformation 3β-OH-Δ5 for androstenes and 3α-OH for pregnanes. Indeed, 3-keto, Δ4, or C7 hydroxylated androstenes and 3β pregnanes were ineffective. Additionally, one of the synthetic analogs tested (TC50) was highly effective in protecting sympathoadrenal cells against apoptosis with an EC50: 0,087 nM, compared to DHEA (1 nM). TC50 binds with high affinity to recently described membrane DHEA binding sites (KD at picomolar concentration), and mimics DHEA in inducing pro-survival anti-apoptotic Bcl-2 proteins. In conclusion, our findings suggest that neurosteroids such as DHEA may act as anti-apoptotic, neuroprotective factors via membrane specific binding sites, downstream activating major pro-survival kinases (Src, MEK/ERK, PI3K/Akt) and production of anti-apoptotic Bcl-2 proteins. Finally, our findings with synthetic analogs suggest that TC50 might prove a lead molecule for the synthesis of novel neuroprotective compounds.
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