Effects of neuroactive steroids on myelin of peripheral nervous system

Peripheral nervous system (PNS) possess both classical (e.g. progesterone receptor, PR, androgen receptor, AR) and non-classical (e.g. GABA_A receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids. Our data have indicated that neuroactive steroids, like for instance, progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3-diol, stimulate both in vivo and in vitro (Schwann cell cultures), the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). It is important to highlight that the mechanisms by which neuroactive steroids exert their effects on the expression of Po and PMP22 involve different kind of receptors depending on the steroid and on the myelin protein considered. In particular, at least in culture of Schwann cells, the expression of Po seems to be under the control of PR, while that of PMP22 needs the GABA_A receptor.

Because Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the PNS, the present observations might suggest the utilization of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin.     

Steroid metabolism and effects in central and peripheral glial cells.

Hormonal steroids participate in the control of a large number of functions of the central nervous system (CNS); recent data show that they may also intervene at the level of the peripheral nervous system (PNS). Both the CNS and the PNS metabolize endogenous as well as exogenous steroids; one of the major enzymatic system is represented by the 5alpha-reductase-3alpha-hydroxysteroid complex. This is a versatile system, since every steroid possessing the delta 4-3keto configuration (e.g., testosterone, progesterone, deoxycorticosterone) may be a substrate. High levels of 5alpha-reductase are found in the white matter of the CNS and in purified myelin. The observation that, in addition to neurons, glia may be a target for steroid action is an important recent finding. The effects of progesterone, testosterone, corticoids, and their respective 5alpha and 3alpha-5alpha derivatives on the expression of glial genes are presented and discussed. It has also been found that progesterone and/or its 5alpha-reduced metabolites increase the mRNA for the two major proteins of peripheral myelin, the glycoprotein Po and the peripheral myelin protein 22, in the sciatic nerve of normal and aged animals and in Schwann cells. The hypothesis has been put forward that glycoprotein Po might be under the control of progestagens acting mainly via the progesterone receptor, and that peripheral myelin protein 22 might be controlled via an interaction of steroids with the gamma-aminobutyric acid (GABA)ergic system. It is known that tetrahydroprogesterone, the 3alpha-5alpha-reduced metabolite of progesterone, interacts with the GABA(A) receptor. Our recent data show that several subunits of this receptor are present in sciatic nerve as well as in Schwann cells that reside in this nerve. These data open multiple possibilities for new therapeutic approaches to demyelinating diseases.     

Steroid effects on the gene expression of peripheral myelin proteins

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA(A) receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).     

Therapeutic administration of progesterone antagonist in a model of Charcot-Marie-Tooth disease (CMT-1A)

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy. The predominant subtype, CMT-1A, accounts for more than 50% of all cases and is associated with an interstitial chromosomal duplication of 17p12 (refs. 2,3). We have generated a model of CMT-1A by introducing extra copies of the responsible disease gene, Pmp22 (encoding the peripheral myelin protein of 22 kDa), into transgenic rats. Here, we used this model to test whether progesterone, a regulator of the myelin genes Pmp22 and myelin protein zero (Mpz) in cultured Schwann cells, can modulate the progressive neuropathy caused by moderate overexpression of Pmp22. Male transgenic rats (n = 84) were randomly assigned into three treatment groups: progesterone, progesterone antagonist (onapristone) and placebo control. Daily administration of progesterone elevated the steady-state levels of Pmp22 and Mpz mRNA in the sciatic nerve, resulting in enhanced Schwann cell pathology and a more progressive clinical neuropathy. In contrast, administration of the selective progesterone receptor antagonist reduced overexpression of Pmp22 and improved the CMT phenotype, without obvious side effects, in wild-type or transgenic rats. Taken together, these data provide proof of principle that the progesterone receptor of myelin-forming Schwann cells is a promising pharmacological target for therapy of CMT-1A.     

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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) x 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 5alpha-dihydroprogesterone (5alpha-DHP) and to the GABAA receptor-active metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, allopregnanolone). The 5alpha-reductase inhibitor L685-273 partially inhibited the effect of PROG, and 3alpha,5alpha-THP (2(-5) x 10(-5) M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3alpha,5alpha-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.     

Induction of myelin gene expression in Schwann cell cultures by an interleukin-6 receptor-interleukin-6 chimera.

Expression of myelin basic protein (MBP) and Po gene products is induced during the final postnatal maturation of Schwann cells and reinduced during nerve regeneration. We show that a chimeric protein containing interleukin-6 fused to its soluble receptor (IL6RIL6 chimera) induces MBP and Po RNAs and proteins in cultures of dorsal root ganglia (DRG) from 14 day old mouse embryos. Activation of gp130 signaling by IL6RIL6 appears comparable to cyclic AMP elevating agents to induce the myelin gene products in DRG and in pure Schwann cell cultures.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination

Some steroids are synthesized within the central and peripheral nervous system, mostly by glial cells. These are known as neurosteroids. In the brain, certain neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3alpha,5alpha-reduced metabolite 3alpha, 5alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.     

Progesterone as a neurosteroid: synthesis and actions in rat glial cellsProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The central nervous system (CNS) and the peripheral nervous system (PNS) are targets for steroid hormones where they regulate important neuronal functions. Some steroid hormones are synthesized within the nervous system, either de novo from cholesterol, or by the metabolism of precursors originating from the circulation, and they were termed ‘neurosteroids'. The sex steroid progesterone can also be considered as a neurosteroid since its synthesis was demonstrated in rat glial cell cultures of the CNS (oligodendrocytes and astrocytes) and of the PNS (Schwann cells). Both types of glial cells express steroid hormone receptors, ER, GR and PR. As in target tissue, e.g. the uterus, PR is estrogen-inducible in brain glial cell cultures. In the PNS, similar PR-induction could not be seen in pure Schwann cells derived from sciatic nerves. However, a significant PR-induction by estradiol was demonstrated in Schwann cells cocultured with dorsal root ganglia (DRG), and we will present evidence that neuronal signal(s) are required for this estrogen-mediated PR-induction. Progesterone has multiple effects on glial cells, it influences growth, differentiation and increases the expression of myelin-specific proteins in oligodendrocytes, and potentiates the formation of new myelin sheaths by Schwann cells in vivo. Progesterone and progesterone analogues also promotes myelination of DRG-Neurites in tissue culture, strongly suggesting a role for this neurosteroid in myelinating processes in the CNS and in the PNS.     

Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Progesterone Stimulates the Activity of the Promoters of Peripheral Myelin Protein-22 and Protein Zero Genes in Schwann Cells

Abstract: To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P₀) genes. PROG stimulated the P₀ promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P₀ and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

周围神经损伤后神经组织中Par-3蛋白表达和分布的变化

目的观察极性蛋白Par-3在损伤后神经组织中的表达和分布,探讨Par-3蛋白在周围神经损伤后髓鞘再生中的作用。方法 32只Sprague Dawley大鼠随机分为正常对照组、损伤组(坐骨神经损伤后第1、2、4、8周)。制备坐骨神经挤压伤模型,分别于损伤后各时间点,采用免疫组织化学法检测坐骨神经损伤远端Par-3蛋白的表达和分布。结果正常大鼠坐骨神经组织中即存在Par-3蛋白,但表达量少,且仅分布于Schwann细胞核内。坐骨神经损伤后,Par-3蛋白的表达和分布发生变化。损伤后1周,Par-3蛋白表达开始升高,Par-3散在分布于Schwann细胞核和细胞浆内。损伤后2周,神经组织中的Par-3蛋白达峰值,在Schwann细胞浆内呈不对称性分布似包绕轴突,呈新月形或C形。损伤后4周和8周,Par-3蛋白表达显著降低,神经组织中Par-3蛋白主要分布于Schwann细胞核内,胞浆内很少。结果 极性蛋白Par-3可能参与周围神经损伤后Schwann细胞的髓鞘再生。     

Vestibular Schwann cells are a distinct subpopulation of peripheral glia with specific sensitivity to growth factors and extracellular matrix components

Vestibular nerve Schwann cells are predisposed to develop schwannoma. While knowledge concerning this condition has greatly improved, little is known about properties of normal vestibular Schwann cells. In an attempt to understand this predisposition, we evaluated cell density regulation and proliferative features of these cells taken from 6-day-old rats. Data were compared to those obtained with sciatic Schwann cells. In both vestibular and sciatic 7-day-old cultures, Schwann cells appear as bipolar or flattened cells. However, sciatic and vestibular cells greatly differ in other aspects: on poly-L-lysine coating, sciatic cells specifically synthesize myelin basic protein, while expression of P0 mRNAs is restricted to some vestibular cells. Laminin increases sciatic cell density but not that of vestibular cells. Fibronectin selectively enhances the proliferation of vestibular Schwann cells and lacks an effect on sciatic ones. Comparison of cell density changes between sciatic and vestibular cells shows that they are sensitive to two different sets of growth factors. Progesterone and FGF-2 combined with forskolin selectively enhance the cell density of sciatic glia, while IGF-1 and GDNF specifically increase vestibular cell density. Furthermore, BrdU incorporation assays indicate that GDNF is also a mitogen for vestibular cells. Altogether, vestibular Schwann cells display phenotypic features and responsiveness to exogenous signals that are significantly different from sciatic Schwann cells, suggesting that vestibular glia form a subpopulation of Schwann cells.     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.