Lithium chloride stimulates PLP and MBP expression in oligodendrocytes via Wnt/ß-catenin and Akt/CREB pathways.

Deciphering the molecular pathways involved in myelin gene expression is a major point of interest to better understand re/myelination processes. In this study, we investigated the role of Lithium Chloride (LiCl), a drug largely used for the treatment of neurological disorders, on the two major central myelin gene expression (PLP and MBP) in mouse oligodendrocytes. We show that LiCl enhances the expression of both PLP and MBP, by increasing mRNA amount and promoter activities. We investigated whether Wnt/ß-catenin and/or Akt/CREB pathways are modulated by LiCl to regulate myelin gene expression. We showed that ß-catenin is required both for PLP and MBP basal promoter activities and for LiCl-induced myelin gene stimulation. Furthermore, while CREB functionality does not influence PLP expression, MBP promoter activity depends on Akt/CREB activation. Finally, we show that LiCl can stimulate oligodendrocyte morphological maturation, and promote remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Our data provide mechanistic evidences that Akt/CREB together with ß-catenin participate in the transcriptional control of PLP and MBP exerted by LiCl. Therefore, the use of LiCl to balance between ß-catenin and CREB effectors could be considered as an efficient remyelinating strategy.

Distribution of membrane progesterone receptor alpha in the male mouse and rat brain and its regulation after traumatic brain injury.

Progesterone has been shown to exert pleiotropic actions in the brain of both male and females. In particular, after traumatic brain injury (TBI), progesterone has important neuroprotective effects. In addition to intracellular progesterone receptors, membrane receptors of the hormone such as membrane progesterone receptor (mPR) may also be involved in neuroprotection. Three mPR subtypes (mPRα, mPRß, and mPRγ) have been described and mPRα is best characterized pharmacologically. In the present study we investigated the distribution, cellular localization and the regulation of mPRα in male mouse and rat brain. We showed by reverse transcription-PCR that mPRα is expressed at similar levels in the male and female mouse brain suggesting that its expression may not be influenced by steroid levels. Treatment of males by estradiol or progesterone did not modify the level of expression of mPRα as shown by Western blot analysis. In situ hybridization and immunohistochemistry analysis showed a wide expression of mPRα in particular in the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum. Double immunofluorescence and confocal microscopy analysis showed that mPRα is expressed by neurons but not by oligodendrocytes and astrocytes. In the rat brain, the distribution of mPRα was similar to that observed in mouse brain; and after TBI, mPRα expression was induced in oligodendrocytes, astrocytes and reactive microglia. The wide neuroanatomical distribution of mPRα suggests that this receptor may play a role beyond neuroendocrine and reproductive functions. However, in the absence of injury its role might be restricted to neurons. The induction of mPRα after TBI in microglia, astrocytes and oligodendrocytes, points to a potential role in mediating the modulatory effects of progesterone in inflammation, ion and water homeostasis and myelin repair in the injured brain.

Membrane progesterone receptors localization in the mouse spinal cord.

The recent molecular cloning of membrane receptors for progesterone (mPRs) has tremendous implications for understanding the multiple actions of the hormone in the nervous system. The three isoforms which have been cloned from several species, mPRalpha, mPRbeta and mPRgamma, have seven-transmembrane domains, are G protein-coupled and may thus account for the rapid modulation of many intracellular signaling cascades by progesterone. However, in order to elucidate the precise functions of mPRs within the nervous system it is first necessary to determine their expression patterns and also to develop new pharmacological and molecular tools. The aim of the present study was to profile mPR expression in the mouse spinal cord, where progesterone has been shown to exert pleiotropic actions on neurons and glial cells, and where the hormone can also be locally synthesized. Our results show a wide distribution of mPRalpha, which is expressed in most neurons, astrocytes, oligodendrocytes, and also in a large proportion of NG2(+) progenitor cells. This mPR isoform is thus likely to play a major role in the neuroprotective and promyelinating effects of progesterone. On the contrary, mPRbeta showed a more restricted distribution, and was mainly present in ventral horn motoneurons and in neurites, consistent with an important role in neuronal transmission and plasticity. Interestingly, mPRbeta was not present in glial cells. These observations suggest that the two mPR isoforms mediate distinct and specific functions of progesterone in the spinal cord. A significant observation was their very stable expression, which was similar in both sexes and not influenced by the presence or absence of the classical progesterone receptors. Although mPRgamma mRNA could be detected in spinal cord tissue by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization analysis did not allow us to verify and to map its presence, probably due to its relatively low expression. The present study is the first precise map of the regional and cellular distribution of mPR expression in the nervous system, a prior requirement for in vivo molecular and pharmacological strategies aimed to elucidate their precise functions. It thus represents a first important step towards a new understanding of progesterone actions in the nervous system within a precise neuroanatomical context.

The membrane-associated progesterone-binding protein 25-Dx: expression, cellular localization and up-regulation after brain and spinal cord injuries.

Progesterone has neuroprotective effects in the injured and diseased spinal cord and after traumatic brain injury (TBI). In addition to intracellular progesterone receptors (PR), membrane-binding sites of progesterone may be involved in neuroprotection. A first putative membrane receptor of progesterone, distinct from the classical intracellular PR isoforms, with a single membrane-spanning domain, has been cloned from porcine liver. Homologous proteins were cloned in rats (25-Dx), mice (PGRMC1) and humans (Hpr.6). We will refer to this progesterone-binding protein as 25-Dx. The distribution and regulation of 25-Dx in the nervous system may provide some clues to its functions. In spinal cord, 25-Dx is localized in cell membranes of dorsal horn neurons and ependymal cells lining the central canal. A role of 25-Dx in mediating the protective effects of progesterone in the spinal cord is supported by the observation that its mRNA and protein are up-regulated by progesterone in dorsal horn of the injured spinal cord. In contrast, the classical intracellular PRs were down-regulated under these conditions. In brain, 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, ependymal cells of the ventricular walls, and the meninges. Interestingly, it is co-expressed with vasopressin in neurons of the paraventricular, supraoptic and retrochiasmatic nuclei. In response to TBI, 25-Dx expression is up-regulated in neurons and induced in astrocytes. The expression of 25-Dx in structures involved in cerebrospinal fluid production and osmoregulation, and its up-regulation after brain damage, point to a potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after TBI. Our observations suggest that progesterone's actions may involve different signaling mechanisms depending on the pathophysiological context, and that 25-Dx may be involved in the neuroprotective effect of progesterone in the injured brain and spinal cord.

Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry.

Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 24 h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(4)-androstenedione, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.