We have previously shown that progesterone (PROG) is synthesized by Schwann cells and promotes myelin formation in the peripheral nervous system (PNS). We now report that this neurosteroid also stimulates myelination in organotypic slice cultures of 7-day-old (P7) rat and mouse cerebellum. Myelination was evaluated by immunofluorescence analysis of the myelin basic protein (MBP). After 7 days in culture (7DIV), we found that adding PROG (2-5 · 10 )5 M) to the culture medium caused a fourfold increase in MBP expression when compared to control slices. The effect of PROG on MBP expression involves the classical intracellular PROG receptor (PR): the selective PR agonist R5020 significantly increased MBP expression and the PR antagonist mifepristone (RU486) completely abolished the effect of PROG on this MBP expression. Moreover, treatment of P7-cerebellar slice cultures from PR knockout (PRKO) mice with PROG had no significant effect on MBP expression. PROG was metabolized in the cerebellar slices to 5a-dihydroprogesterone (5a-DHP) and to the GABA A receptor-active metabolite 3a,5a-tetrahydroprogesterone (3a,5a-THP, allopregnanolone). The 5a-reductase inhibitor L685-273 partially inhibited the effect of PROG, and 3a,5a-THP (2-5 · 10 )5 M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3a,5a-THP involved GABA A receptors, as it could be inhibited by the selective GABA A receptor antagonist bicuculline. These findings suggest that progestins stimulate MBP expression and consequently suggest an increase in CNS myelination via two signalling systems, the intracellular PR and membrane GABA A receptors, and they confirm a new role of GABA A receptors in myelination.

The utility and safety of postmenopausal hormone replacement therapy has recently been put into question by large clinical trials. Their outcome has been extensively commented upon, but discussions have mainly been limited to the effects of estrogens. In fact, progestagens are generally only considered with respect to their usefulness in preventing estrogen stimulation of uterine hyperplasia and malignancy. In addition, various risks have been attributed to progestagens and their omission from hormone replacement therapy has been considered, but this may underestimate their potential benefits and therapeutic promises. A major reason for the controversial reputation of progestagens is that they are generally considered as a single class. Moreover, the term progesterone is often used as a generic one for the different types of both natural and synthetic progestagens. This is not appropriate because natural progesterone has properties very distinct from the synthetic progestins. Within the nervous system, the neuroprotective and promyelinating effects of progesterone are promising, not only for preventing but also for reversing age-dependent changes and dysfunctions. There is indeed strong evidence that the aging nervous system remains at least to some extent sensitive to these beneficial effects of progesterone. The actions of progesterone in peripheral target tissues including breast, blood vessels, and bones are less well understood, but there is evidence for the beneficial effects of progesterone. The variety of signaling mechanisms of progesterone offers exciting possibilities for the development of more selective, efficient, and safe progestagens. The recognition that progesterone is synthesized by neurons and glial cells requires a reevaluation of hormonal aging.

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.

Enhancing the endogenous capacity of myelin repair is a major therapeutic challenge in demyelinating diseases such as multiple sclerosis. We found that progesterone and the synthetic 19-norprogesterone derivative 16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione (Nestorone) promote the remyelination of axons by oligodendrocytes after lysolecithin-induced demyelination in organotypic cultures of cerebellar slices taken from postnatal rats or mice. The intracellular progesterone receptors (PR) mediate the proremyelinating actions of Nestorone, because they are not observed in slices from PR knockout mice. Notably, Nestorone was less efficient in heterozygous mice, expressing reduced levels of PR, suggesting PR haploinsufficiency in myelin repair. Using mice expressing the enhanced green fluorescent protein (EGFP) under the control of the proteolipid gene promoter, we showed that both progesterone and Nestorone strongly increased the reappearance of cells of the oligodendroglial lineage in the demyelinated slices. In contrast to Nestorone, the pregnane derivative medroxyprogesterone acetate had no effect. The increase in oligodendroglial cells by Nestorone resulted from enhanced NG2(+) and Olig2(+) oligodendrocyte progenitor cell (OPC) recruitment. In cocultures of lysolecithin-demyelinated cerebellar slices from wild-type mice apposed to brain stem slices of proteolipid gene promoter-EGFP mice, Nestorone stimulated the migration of OPC towards demyelinated axons. In this coculture paradigm, Nestorone indeed markedly increased the number of EGFP(+) cells migrating into the demyelinated cerebellar slices. Our results show that Nestorone stimulates the recruitment and maturation of OPC, two steps which are limiting for efficient myelin repair. They may thus open new perspectives for the use of progestins, which selectively target PR, to promote the endogenous regeneration of myelin.

In rat striatal slices, 2-chloroadenosine, which had no direct effect on inositol phosphate formation, potentiated in a dose-dependent manner the accumulation of inositol phosphates induced either by carbamylcholine (10(-3) M) or by noradrenaline (10(-4) M). Experiments made on pure populations of striatal neurons or striatal glial cells in primary culture from mouse embryos indicated that 2-chloroadenosine potentiated the noradrenaline-elicited phosphoinositide breakdown in striatal glial cultures but did not modify the responses evoked either by noradrenaline or by carbamylcholine in striatal neuronal cultures. However, 2-chloroadenosine enhanced both the carbamylcholine and the noradrenaline-induced accumulation of inositol phosphates in neuroglial cocultures just as it did in rat striatal slices. The potentiation by 2-chloroadenosine of the carbamylcholine response, which is neuron specific, involved a cooperative effect between neurons and glial cells and, as shown by additional experiments, required a brief contact only between the 2 types of cells. The potentiating effect of 2-chloroadenosine was blocked completely by a nonselective A1, A2 adenosine antagonist isobutylmethylxanthine either on rat striatal slices or on mouse embryonic cocultures (noradrenaline and carbamylcholine responses) or on mouse embryonic glial cultures (noradrenaline response). These data indicate the involvement of an extracellular membrane-bound adenosine receptor, possibly of the A1 subtype since N6-cyclohexyladenosine, an A1 adenosine receptor agonist, was more efficient than 5'-N-ethylcarboxamide-adenosine, a rather selective A2 adenosine receptor agonist. We propose that 2-chloroadenosine acts through an adenosine receptor located on glial cells and induces the synthesis of a substance that improves the coupling between carbamylcholine or noradrenaline and phospholipase C located in glial cells or neurons.