The neuroactive steroids dehydroepiandrosterone (DHEA) its sulfate ester DHEA sulfate (DHEAS) and allopregnanolone (Allo) made by the CNS and the adrenals appear to exert a protective effect in hippocampal and cortical neuron ischemia- and excitotoxicity-induced injury. effect of DHEA DHEAS and Allo was compared to that of a long list of structurally related compounds and was found to be structure-specific confined primarily to conformation 3β-OH-Δ5 for androstenes and 3α-OH for pregnanes. Indeed 3 Δ4 or C7 hydroxylated androstenes GTx-024 and 3β pregnanes were ineffective. The prosurvival effect of DHEA(S) and Allo was for his or her action because Bcl-2 antisense oligonucleotides reversed their effects. Finally DHEA(S) and Allo triggered cAMP response element-binding protein and NF-κB upstream effectors of antiapoptotic Bcl-2 protein manifestation. They also triggered the antiapoptotic kinase PKCα/β a posttranslational activator of Bcl-2 protein. Our findings suggest that decrease of DHEA(S) and Allo during ageing or stress may leave the adrenal medulla unprotected against proapoptotic difficulties. The neuroactive steroids dehydroepiandrosterone GTx-024 (DHEA) its sulfate ester DHEA sulfate (DHEAS) and allopregnanolone (Allo) are produced in the brain and the GTx-024 adrenals (1-3). Their production rate and levels in serum and adrenals decrease gradually with improving age (4-7). Physical or emotional stress may decrease them characteristic paradigms being major depression (8) and chronic swelling (9). The decrease of their levels is associated with neuronal dysfunction and degeneration (10-12) most probably because these steroids guard CNS neurons against noxious providers (13-15). Indeed both DHEA and DHEAS [DHEA(S)] protects rat hippocampal neurons against = 3 < 0.001). For assessment serum supplementation for 12 h showed an apoptosis rate of 0.61 ± 0.04. Inhibition of apoptosis in chromaffin cells was retained for at least 48 h. Fig. 1. DHEA(S) and Allo safeguarded rat chromaffin cells in tradition against serum deprivation-induced apoptosis. Freshly isolated rat chromaffin cells were cultured either in total or serum-free press comprising 10-7 M DHEA DHEAS or Allo for numerous ... Based on these data additional experiments were carried out by using the well established model of chromaffin cell apoptosis the Personal computer12 rat pheochromocytoma cell collection (20). As expected serum deprivation experienced a deleterious effect on Personal computer12 cell ethnicities. FACS analysis exposed that 25% of Personal computer12 cells managed in serum-free medium underwent apoptosis within 24 h (Fig. 2= 6 < 0.001). Therefore all three steroids tested strongly inhibited serum deprivation-induced apoptosis by >50% to the degree that their protecting effects were also easily visualized under optical microscopy. For comparison serum supplementation for 24 h showed an apoptosis rate of 0.047 ± 0.008 resulting as expected in higher protection. The antiapoptotic effects were dose-dependent with EC50 at 1.8 1.1 and 1.5 nM for DHEA DHEAS and Allo respectively (Fig. 2depicts a mean 40% inhibition of serum deprivation-induced apoptosis in PC12 cells exposed to Vezf1 three steroids. Indeed the percentage of apoptotic cells cultured in serum-free medium in the absence of steroids was 24.6% compared to 15.1% 16.9% and 10.8% for DHEA DHEAS and Allo respectively. This profile of FACS analysis was highly reproducible in at least three independent experiments. The Antiapoptotic Effect of DHEA(S) and Allo Was Structure-Specific. To assess the specificity of the cytoprotective action of DHEA DHEAS and Allo a host of structurally related compounds were also tested in parallel to our steroids. Structure-activity analysis revealed the following data. (depicts their effect on the transcriptional level. Fig. 3. DHEA(S) and Allo induced the expression of the antiapoptotic Bcl-2 proteins in serum-deprived PC12 cells. Cells were cultured for 2-12 h either in complete or serum-free media containing 10-7 M DHEA DHEAS or Allo. Cellular extracts containing … To confirm these data further experiments were carried out GTx-024 by using Bcl-2 antisense oligonucleotides which reversed the antiapoptotic cytoprotective effects of DHEA(S) and Allo (Fig. 4= 3 < 0.005) (Fig. 5< 0.005) (Fig. 5= 3 < 0.05) (Fig. 6). In serum-deprived cells exposed to steroids for 10 min levels of phosphorylated PKCα/β were maintained to those seen in the presence of serum (DHEA 3.69 ± 0.2; DHEAS 4.12 GTx-024 ± 0.1; Allo 4.96 ± 0.3; < 0.05) (Fig. 6). The ability of neuroactive steroids to restore PKCα/β phosphorylation under serum deprivation.