Martine El-Etr

Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum

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 5α‐dihydroprogesterone (5α‐DHP) and to the GABAA receptor‐active metabolite 3α,5α‐tetrahydroprogesterone (3α,5α‐THP, allopregnanolone). The 5α‐reductase inhibitor L685‐273 partially inhibited the effect of PROG, and 3α,5α‐THP (2–5 × 10−5 M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3α,5α‐THP involved GABAA receptors, as it could be inhibited by the selective GABAA 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 GABAA receptors, and they confirm a new role of GABAA receptors in myelination.

Systemic progesterone administration results in a partial reversal of the age-associated decline in CNS remyelination following toxin-induced demyelination in male rats.

In order to establish the effects of systemically administered progesterone on central nervous system (CNS) remyelination, a toxin‐induced model of CNS demyelination was used in which the rate of remyelination is age‐dependent. The rapid remyelination in young adult rats allowed an assessment of potential adverse effects of progesterone while the slow remyelination in older adult rats allowed an assessment of its potentially beneficial effects. There was no significant difference in the rate of remyelination between young control and treated animals. However, a modest but significant increase in the extent of oligodendrocyte remyelination in response to progesterone (and a comparable significant decrease in the proportion of axons that remained demyelinated) was observed in older rats 5 weeks after lesion induction. We also found a significant increase in the proportion of Schwann cell remyelinated axons between 3 and 5 weeks after lesion induction that was not apparent in the control animals. These results indicate that progesterone does not inhibit CNS remyelination and that it has a positive modulating effect on oligodendrocyte remyelination in circumstances where it is occurring sub‐optimally.

Progesterone and the oligodendroglial lineage: Stage-dependent biosynthesis and metabolism

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 3H‐pregnenolone (3H‐PREG), the immediate precursor of PROG, or with 3H‐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 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 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 5α‐reductase into 5α‐dihydroprogesterone (5α‐DHPROG). This enzyme exhibited a 5‐fold higher activity in OL than in OPP and OP. 5α‐DHPROG was further transformed either into 3α,5α‐tetrahydroprogesterone (3α,5α‐THPROG), known as a positive allosteric modulator of the GABAA receptor, or into the 3β‐isomer. The 3α,5α‐THPROG synthesis was 10 times higher in OPP than in the other cell studied, while the 3β,5α‐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. GLIA 36:295–308, 2001.

Steroid hormones in multiple sclerosis

The possible influence of steroid hormones in multiple sclerosis (MS) has been a matter of great interest. A first illustration comes from the analyses of the influence of gender on susceptibility to MS and on MS severity. A series of arguments emerge in favour of a possible influence of steroid hormones in MS. The menstrual cycle, and even more pregnancy, may influence the clinical evolution of MS. In the PRIMS study, there was a dramatic decrease in the relapse rate during pregnancy, especially in the third trimester, with a rebound increase in the 3 months post partum. Animal studies have provided further confirmatory results. Many experiments have shown that sex steroids may have immunological effects, in preventing or treating experimental allergic encephalomyelitis. They could also have an effect on myelinating and remyelinating the peripheral and possibly the central nervous system. These clinical and experimental data have led to consider sexual steroids as potential therapeutic tools for preventing relapses in women with MS, in particular in the post-partum period.

Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3—P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 μM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GRNes/Cre). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from Purkinje cell degeneration (pcd) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of pcd/pcd Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.

Genomic and membrane actions of progesterone: implications for reproductive physiology and behavior

Progesterone, produced by the ovaries and adrenal glands, regulates reproductive behavior and the surge of luteinizing hormone which precedes ovulation by acting on neurons located in different parts of the hypothalamus. The study of the activation of these reproductive functions in female rats has allowed to explore the different mechanisms of progesterone action in the brain. It has allowed to demonstrate that new actions of the hormone, which have been observed in particular in vitro systems, are also operational in vivo, and may thus be biologically relevant. This mainly concerns the direct actions of progesterone on receptors of neurotransmitters such as oxytocin and GABA. Activation of the progesterone receptor in the absence of ligand by phosphorylation may also play a role.

Progesterone and Nestorone Facilitate Axon Remyelination: A Role for Progesterone Receptors

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.

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARα-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARα independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

3α,5α-tetrahydroprogesterone (allopregnanolone) and γ-aminobutyric acid: Autocrine/paracrine interactions in the control of neonatal PSA-NCAM+ progenitor proliferation

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 3α,5α‐tetrahydroprogesterone (3α,5α‐THP, or allopregnanolone; Gago et al. [ 2001 ] Glia 36:295–308). The latter is a potent positive allosteric modulator of γ‐aminobutyric acid type A (GABAA) receptors. In the present work, we demonstrate that PROG and 3α,5α‐THP both stimulate ePSA‐NCAM+ progenitor proliferation. PROG exerted its mitogenic effect indirectly, through its conversion to 3α,5α‐THP, since it could be abolished by an inhibitor of the 5α‐reductase (L685‐273) and mimicked by 3α,5α‐THP. A dose‐response curve revealed a bell‐shaped effect of 3α,5α‐THP on ePSA‐NCAM+ progenitor proliferation, with greatest stimulation at nanomolar concentrations. The mitogenic effect of 3α,5α‐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 3α,5α‐THP in the development of the nervous system.

Progesterone and nestorone promote myelin regeneration in chronic demyelinating lesions of corpus callosum and cerebral cortex

Multiple Sclerosis affects mainly women and consists in intermittent or chronic damages to the myelin sheaths, focal inflammation, and axonal degeneration. Current therapies are limited to immunomodulators and antiinflammatory drugs, but there is no efficient treatment for stimulating the endogenous capacity of myelin repair. Progesterone and synthetic progestins have been shown in animal models of demyelination to attenuate myelin loss, reduce clinical symptoms severity, modulate inflammatory responses and partially reverse the age‐dependent decline in remyelination. Moreover, progesterone has been demonstrated to promote myelin formation in organotypic cultures of cerebellar slices. In the present study, we show that progesterone and the synthetic 19‐nor‐progesterone derivative Nestorone® promote the repair of severe chronic demyelinating lesions induced by feeding cuprizone to female mice for up to 12 weeks. Progesterone and Nestorone increase the density of NG2+ oligodendrocyte progenitor cells and CA II+ mature oligodendrocytes and enhance the formation of myelin basic protein (MBP)‐ and proteolipid protein (PLP)‐immunoreactive myelin. However, while demyelination in response to cuprizone was less marked in corpus callosum than in cerebral cortex, remyelination appeared earlier in the former. The remyelinating effect of progesterone was progesterone receptor (PR)‐dependent, as it was absent in PR‐knockout mice. Progesterone and Nestorone also decreased (but did not suppress) neuroinflammatory responses, specifically astrocyte and microglial cell activation. Therefore, some progestogens are promising therapeutic candidates for promoting the regeneration of myelin. GLIA 2015;63:104–117