7α-hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11ß-hydroxysteroid dehydrogenase in E(t)C cerebellar neurons.

The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E(t)C cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11ß-hydroxysteroid dehydrogenase (11ß-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E(t)C cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E(t)C neuronal cells exhibit 11ß-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11ß-HSD2 dehydrogenase activity. Consequently, E(t)C cells incubated with DHEA did not yield 7-oxo- or 7ß-OH-DHEA. Our findings are supported by the reductive environment of E(t)C cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11ß-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E(t)C neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11ß-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11ß-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in E(t)C neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites.

Brain microglia express steroid-converting enzymes in the mouse.

In the CNS, steroid hormones play a major role in the maintenance of brain homeostasis and it's response to injury. Since activated microglia are the pivotal immune cell involved in neurodegeneration, we investigated the possibility that microglia provide a discrete source for the metabolism of active steroid hormones. Using RT-PCR, our results showed that mouse microglia expressed mRNA for 17beta-hydroxysteroid dehydrogenase type 1 and steroid 5alpha-reductase type 1, which are involved in the metabolism of androgens and estrogens. Microglia also expressed the peripheral benzodiazepine receptor and steroid acute regulatory protein; however, the enzymes required for de novo formation of progesterone and DHEA from cholesterol were not expressed. To test the function of these enzymes, primary microglia cultures were incubated with steroid precursors, DHEA and AD. Microglia preferentially produced delta-5 androgens (Adiol) from DHEA and 5alpha-reduced androgens from AD. Adiol behaved as an effective estrogen receptor agonist in neuronal cells. Activation of microglia with pro-inflammatory factors, LPS and INFgamma did not affect the enzymatic properties of these proteins. However, PBR ligands reduced TNFalpha production signifying an immunomodulatory role for PBR. Collectively, our results suggest that microglia utilize steroid-converting enzymes and related proteins to influence inflammation and neurodegeneration within microenvironments of the brain.

Characterization of a cerebellar granule progenitor cell line, EtC.1, and its responsiveness to 17-beta-estradiol.

Mouse cerebellar development occurs at late embryonic stages and through the first few weeks of postnatal life. Hormones such as 17-beta-estradiol (E2) have been implicated in cerebellar development, through the expression of E2 receptors (ER). However, the role of E2 in the development and function of cerebellar neurons has yet to be fully elucidated. To gain insight into E2's actions on the developing cerebellum, we characterized a cloned neuronal cell line, E(t)C.1, derived from late embryonic cerebellum for its neural properties and responsiveness to E2. Our results revealed that E(t)C.1 cells express markers characteristic of neural progenitor cells such as Nestin, Musashi, and Doublecortin (DCX), and of the granule cell lineage such as Math1 and Zipro1. The ER alpha and beta (ERalpha and ERbeta) were also identified in this cell line. Functionality of ERs was verified using an Estrogen Response Element (ERE)-Luciferase reporter plasmid. E2 modulated ERalpha, FMRP, and IL-6, which were expressed in these cells. However, E2 did not induce changes in neural proteins nor induce maturation of E(t)C.1 cells. CREB and ERK(1/2) protein kinases were not modulated by E2 either. Interestingly, E(t)C.1 expressed active p450 Aromatase (P450arom), which was confirmed by the aromatization of androstenedione (AD) to E2 and other estrogen metabolites. Collectively, our results show that the E(t)C.1 cell line may serve as a model to study early development of cerebellar progenitor granule cells, and their responsiveness to E2.

Selective conversion by microglia of dehydroepiandrosterone to 5-androstenediol-A steroid with inherent estrogenic properties.

The well-established neuroprotective effect of dehydroepiandrosterone (DHEA) has been attributed to its metabolism in the brain to provide estrogens known to be neuroprotective and to enhance memory and learning in humans and animals. However, our previous work showed that the conversion of DHEA to 4-androstenedione (AD), the precursor of estrone (E(1)) and estradiol (E(2)), is very low in several different types of neural cells, and that the main product is 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). In this study, we found that microglia are an exception and produce mainly 5-androstene-3beta,17beta-diol (Delta(5)-Adiol), a C(19) steroid with estrogen-like activity from DHEA. Virtually, no other products, including testosterone (T) were detected by TLC or HPLC in incubations of (3)H-labeled DHEA with the BV2 microglial cell line. Microglia are important brain cells that are thought to play a house-keeping role during the steady state, and that are crucial to the brain's immune reaction to injury and the healing process. Our findings suggest that the microglia-produced Delta(5)-Adiol might have a role in modulating estrogen-sensitive neuroplastic events in the brain, in the absence of adequate local synthesis of estrone and estradiol.

Dehydroepiandrosterone (DHEA) metabolism in the brain: identification by liquid chromatography/mass spectrometry of the delta-4-isomer of DHEA and related steroids formed from androstenedione by mouse BV2 microglia.

Studies to elucidate the role of dehydroepiandrosterone (DHEA) metabolism in neuroprotection have compared its relative 7-hydroxylation against estrogen formation by way of 4-androstenedione (AD) in various rodent brain cell lines. In all cases, the 7alpha- and 7beta-hydroxy epimers of DHEA were found to be the dominant products with one notable exception. BV2 mouse microglia were virtually unable to hydroxylate DHEA at C-7 and converted AD to a major unknown metabolite not observed with mouse BHc hippocampal cells. In this paper, we describe the identification of this compound based on its physical properties and analysis by TLC and HPLC. Its identity as 3beta-hydroxy-4-androstene-17-one, the Delta(4)-isomer of DHEA, was confirmed by mass spectrometry (LC/MS), as well as by reverse isotope dilution analysis involving co-crystallization with the synthetic steroid. Possible mechanisms for the formation of this isomer of DHEA by BV2 microglia are proposed, together with that of other C-19 steroids detected which include testosterone (T), 5alpha-dihydrotestosterone and 5alpha-androstanedione.

Metabolism of dehydroepiandrosterone by rodent brain cell lines: relationship between 7-hydroxylation and aromatization.

The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.