The steroid hormones corticosterone and testosterone are supplied to the central nervous system by endocrine glands, the adrenals and gonads. In contrast, the 3 beta-hydroxy-delta 5-derivatives of cholesterol, pregnenolone and dehydroepiandrosterone, accumulate in the rat brain through mechanisms independent of peripheral sources. Immunohistochemical studies have been performed with specific antibodies to bovine adrenal cytochrome P-450scc, which is involved in cholesterol side-chain cleavage and pregnenolone formation. The enzyme was localized in the white matter throughout the brain. Scarce clusters of cell bodies were also stained in the entorhinal and cingulate cortex and in the olfactory bulb. These observations strongly support the existence of "neurosteroids," which have been posited on the basis of biochemical, physiological, and behavioral studies.
Evidence has been accumulated showing that neurosteroids, particularly progesterone (PROG) and its metabolites, may participate in myelination and remyelination in the peripheral nervous system, but very few studies have been undertaken in the central nervous system (CNS). The aim of this work was to investigate the capacities of synthesis and metabolism of PROG at three important stages of the oligodendroglial lineage: oligodendrocyte pre-progenitors (OPP), oligodendrocyte progenitors (OP), and fully differentiated oligodendrocytes (OL). Experiments have been conducted in vitro using highly purified primary cell cultures from rat brain. Cells were incubated with (3)H-pregnenolone ((3)H-PREG), the immediate precursor of PROG, or with (3)H-PROG, and steroids metabolites were then identified by thin layer chromatography and high-performance liquid chromatography (HPLC). mRNA expression of key steroidogenic enzymes was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that only OPP and OP, but not OL, expressed 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA and were able to synthesize PROG from PREG. In the three cell types studied, PROG was metabolized by the type 1 isoform of 5 alpha-reductase into 5 alpha-dihydroprogesterone (5 alpha-DHPROG). This enzyme exhibited a 5-fold higher activity in OL than in OPP and OP. 5 alpha-DHPROG was further transformed either into 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THPROG), known as a positive allosteric modulator of the GABA(A) receptor, or into the 3 beta-isomer. The 3 alpha,5 alpha-THPROG synthesis was 10 times higher in OPP than in the other cell studied, while the 3 beta,5 alpha-THPROG production did not change with cell differentiation. PROG synthesis and metabolism and the dramatic changes in neurosteroidogenesis observed during the oligodendroglial differentiation may contribute to oligodendrocyte development or the myelination process.
The earliest identified neonatal neural progenitors are cells that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). One of these progenitors is the early PSA-NCAM+ progenitor (ePSA-NCAM+ progenitor; Gago et al. [2003] Mol Cell Neurosci 22:162-178), which corresponds to a multipotential cell with a default differentiation through glial lineages. The ePSA-NCAM+ progenitor can synthesize the neurosteroid progesterone (PROG) and its reduced metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, or allopregnanolone; Gago et al. [ 2001] Glia 36:295-308). The latter is a potent positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors. In the present work, we demonstrate that PROG and 3alpha,5alpha-THP both stimulate ePSA-NCAM+ progenitor proliferation. PROG exerted its mitogenic effect indirectly, through its conversion to 3alpha,5alpha-THP, since it could be abolished by an inhibitor of the 5alpha-reductase (L685-273) and mimicked by 3alpha,5alpha-THP. A dose-response curve revealed a bell-shaped effect of 3alpha,5alpha-THP on ePSA-NCAM+ progenitor proliferation, with greatest stimulation at nanomolar concentrations. The mitogenic effect of 3 alpha,5 alpha-THP was mediated by GABAA receptors, insofar as it could be blocked by the selective antagonist bicuculline. ePSA-NCAM+ progenitors indeed expressed mRNAs for GABAA receptor subunits, and GABA enhanced cell proliferation, an effect that was also bicuculline sensitive. Moreover, these cells synthesized GABA, which was involved in a tonic stimulation of their proliferation. These results reveal complex autocrine/paracrine loops in the control of ePSA-NCAM+ progenitor proliferation, involving both neurosteroid and GABA signaling, and suggest a novel key role for 3alpha,5alpha-THP in the development of the nervous system.
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