Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease.

Neuroactive steroids are known neuroprotective agents and neurotransmitter regulators. We previously found that expression of the enzymes synthesizing 5α-dihydroprogesterone (5α-DHP), allopregnanolone (ALLO), and dehydroepiandrosterone sulfate (DHEAS) were reduced in the substantia nigra (SN) of Parkinson's Disease (PD) brain. Here, concentrations of a comprehensive panel of steroids were measured in human post-mortem brains of PD patients and controls. Gas chromatography-mass spectrometry (GC/MS) was used to measure steroid levels in SN (involved in early symptoms) and prefrontal cortex (PFC) (involved later in the disease) of five control (CTR) and nine PD donors, divided into two groups: PD4 (PD-Braak stages 1-4) and PD6 (PD-Braak stages 5-6). In SN, ALLO was increased in PD4 compared to CTR and 5α-DHP and ALLO levels were diminished in PD6 compared to PD4. The ALLO metabolite 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was higher in PD4 compared to CTR. In PFC, 3α5α20α-HHP was higher in PD4 compared to both CTR and PD6. The effects of 5α-DHP, ALLO and DHEAS were tested on human post-mortem brain slices of patients and controls in culture. RNA expression of genes involved in neuroprotection, neuroinflammation and neurotransmission was analysed after 5 days of incubation with each steroid. In PD6 slices, both 5α-DHP and ALLO induced an increase of the glutamate reuptake effector GLAST1, while 5α-DHP also increased gene expression of the neuroprotective TGFB. In CTR slices, ALLO caused reduced expression of IGF1 and GLS, while DHEAS reduced the expression of p75 and the anti-apoptotic molecule APAF1. Together these data suggest that a potentially protective upregulation of ALLO occurs at early stages of PD, followed by a downregulation of progesterone metabolites at later stages that may exacerbate the pathological changes, especially in SN. Neuroprotective effects of neurosteroids are thus dependent on the neuropathological stage of the disease.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

Long-lasting masculinizing effects of postnatal androgens on myelin governed by the brain androgen receptor.

The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.

Steroids in Stroke with Special Reference to Progesterone.

Both sex and steroid hormones are important to consider in human ischemic stroke and its experimental models. Stroke initiates a cascade of changes that lead to neural cell death, but also activates endogenous protective processes that counter the deleterious consequences of ischemia. Steroids may be part of these cerebroprotective processes. One option to provide cerebroprotection is to reinforce these intrinsic protective mechanisms. In the current review, we first summarize studies describing sex differences and the influence of steroid hormones in stroke. We then present and discuss our recent results concerning differential changes in endogenous steroid levels in the brains of male and female mice and the importance of progesterone receptors (PR) during the early phase after stroke. In the third part, we give an overview of experimental studies, including ours, that provide evidence for the pleiotropic beneficial effects of progesterone and its promising cerebroprotective potential in stroke. We also highlight the key role of PR signaling as well as potential additional mechanisms by which progesterone may provide cerebroprotection.

A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes.SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.

Behavioral evidence for sex steroids hypersensitivity in castrated male canaries.

In seasonally breeding songbirds such as canaries, singing behavior is predominantly under the control of testosterone and its metabolites. Short daylengths in the fall that break photorefractoriness are followed by increasing daylengths in spring that activate singing via both photoperiodic and hormonal mechanisms. However, we observed in a group of castrated male Fife fancy canaries maintained for a long duration under a short day photoperiod a large proportion of subjects that sang at high rates. This singing rate was not correlated with variation in the low circulating concentrations of testosterone. Treatment of these actively singing castrated male canaries with a combination of an aromatase inhibitor (ATD) and an androgen receptor blocker (flutamide) only marginally decreased this singing activity as compared to control untreated birds and did not affect various measures of song quality. The volumes of HVC and of the medial preoptic nucleus (POM) were also unaffected by these treatments but were relatively large and similar to volumes in testosterone-treated males. In contrast, peripheral androgen-sensitive structures such as the cloacal protuberance and syrinx mass were small, similar to what is observed in castrates. Together these data suggest that after a long-term steroid deprivation singing behavior can be activated by very low concentrations of testosterone. Singing normally depends on the activation by testosterone and its metabolites of multiple downstream neurochemical systems such as catecholamines, nonapeptides or opioids. These transmitter systems might become hypersensitive to steroid action after long term castration as they probably are at the end of winter during the annual cycle in seasonally breeding temperate zone species.

Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum.

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and ß are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/ß in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions.

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

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.

The neural androgen receptor: a therapeutic target for myelin repair in chronic demyelination.

Myelin regeneration is a major therapeutic goal in demyelinating diseases, and the failure to remyelinate rapidly has profound consequences for the health of axons and for brain function. However, there is no efficient treatment for stimulating myelin repair, and current therapies are limited to anti-inflammatory agents. Males are less likely to develop multiple sclerosis than females, but often have a more severe disease course and reach disability milestones at an earlier age than females, and these observations have spurred interest in the potential protective effects of androgens. Here, we demonstrate that testosterone treatment efficiently stimulates the formation of new myelin and reverses myelin damage in chronic demyelinated brain lesions, resulting from the long-term administration of cuprizone, which is toxic for oligodendrocytes. In addition to the strong effect of testosterone on myelin repair, the number of activated astrocytes and microglial cells returned to low control levels, indicating a reduction of neuroinflammatory responses. We also identify the neural androgen receptor as a novel therapeutic target for myelin recovery. After the acute demyelination of cerebellar slices in organotypic culture, the remyelinating actions of testosterone could be mimicked by 5α-dihydrotestosterone, a metabolite that is not converted to oestrogens, and blocked by the androgen receptor antagonist flutamide. Testosterone treatment also failed to promote remyelination after chronic cuprizone-induced demyelination in mice with a non-functional androgen receptor. Importantly, testosterone did not stimulate the formation of new myelin sheaths after specific knockout of the androgen receptor in neurons and macroglial cells. Thus, the neural brain androgen receptor is required for the remyelination effect of testosterone, whereas the presence of the receptor in microglia and in peripheral tissues is not sufficient to enhance remyelination. The potent synthetic testosterone analogue 7α-methyl-19-nortestosterone, which has been developed for long-term male contraception and androgen replacement therapy in hypogonadal males and does not stimulate prostate growth, also efficiently promoted myelin repair. These data establish the efficacy of androgens as remyelinating agents and qualify the brain androgen receptor as a promising drug target for remyelination therapy, thus providing the preclinical rationale for a novel therapeutic use of androgens in males with multiple sclerosis.

Chronic administration of XBD173 ameliorates cognitive deficits and neuropathology via 18 kDa translocator protein (TSPO) in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of ß-amyloid peptide (Aß). It affects cognition and leads to memory impairment. The mitochondrial translocator protein (TSPO) plays an essential role in maintaining mitochondrial homeostasis and has been implicated in several neuronal disorders or neuronal injuries. Ligands targeting the mitochondrial translocator protein (18 kDa), promote neurosteroidogenesis and may be neuroprotective. To study whether the TSPO ligand XBD173 may exert early neuroprotective effects in AD pathology we investigated the impact of XBD173 on amyloid toxicity and neuroplasticity in mouse models of AD. We show that XBD173 (emapunil), via neurosteroid-mediated signaling and delta subunit-containing GABAA receptors, prevents the neurotoxic effect of Aß on long-term potentiation (CA1-LTP) in the hippocampus and prevents the loss of spines. Chronic but not acute administration of XBD173 ameliorates spatial learning deficits in transgenic AD mice with arctic mutation (ArcAß). The heterozygous TSPO-knockout crossed with the transgenic arctic mutation model of AD mice (het TSPOKO X ArcAß) treated with XBD173 does not show this improvement in spatial learning suggesting TSPO is needed for procognitive effects of XBD173. The neuroprotective profile of XBD173 in AD pathology is further supported by a reduction in plaques and soluble Aß levels in the cortex, increased synthesis of neurosteroids, rescued spine density, reduction of complement protein C1q deposits, and reduced astrocytic phagocytosis of functional synapses both in the hippocampus and cortex. Our findings suggest that XBD173 may exert therapeutic effects via TSPO in a mouse model of AD.

The different natural estrogens promote endothelial healing through distinct cell targets.

The main estrogen, 17ß-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4's action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.

Interplay between LXR and Wnt/ß-catenin signaling in the negative regulation of peripheral myelin genes by oxysterols.

Oxysterols are reactive molecules generated from the oxidation of cholesterol. Their implication in cholesterol homeostasis and in the progression of neurodegenerative disorders is well known, but few data are available for their functions in the peripheral nervous system. Our aim was to study the influence of oxysterols on myelin gene expression and myelin sheath formation in peripheral nerves. We show by gas chromatography/mass spectrometry that Schwann cells and sciatic nerves contain 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol and that they express their biosynthetic enzymes and receptors (liver X receptors LXRα and LXRß). We demonstrate that oxysterols inhibit peripheral myelin gene expression [myelin protein zero (MPZ) and peripheral myelin protein-22 (PMP22)] in a Schwann cell line. This downregulation is mediated by either LXRα or LXRß, depending on the promoter context, as suggested by siRNA strategy and chromatin immunoprecipitation assays in Schwann cells and in the sciatic nerve of LXR knock-out mice. Importantly, the knock-out of LXR in mice results in thinner myelin sheaths surrounding the axons. Oxysterols repress myelin genes via two mechanisms: by binding of LXRs to myelin gene promoters and by inhibiting the Wnt/ß-catenin pathway that is crucial for the expression of myelin genes. The Wnt signaling components (Disheveled, TCF/LEF, ß-catenin) are strongly repressed by oxysterols. Furthermore, the recruitment of ß-catenin at the levels of the MPZ and PMP22 promoters is decreased. Our data reveal new endogenous mechanisms for the negative regulation of myelin gene expression, highlight the importance of oxysterols and LXR in peripheral nerve myelination, and open new perspectives of treating demyelinating diseases with LXR agonists.

Normal spermatogenesis in a man with mutant luteinizing hormone.

Men with mutations in LHB, the gene encoding the beta subunit of luteinizing hormone (LHB), have azoospermia with absent or few fetal Leydig cells. We report a mutation in LHB in a man and his sister. The man presented with absence of virilization, undetectable luteinizing hormone, and a low serum testosterone level. He had complete spermatogenesis with a normal sperm count. The mutant luteinizing hormone had a low level of partial activity in vitro. We concluded that the residual luteinizing hormone activity, resulting in the expression of steroidogenic enzymes in few mature Leydig cells producing small amounts of intratesticular testosterone (20.2 ng per gram), was sufficient for complete and quantitatively normal spermatogenesis.

Aging Is Associated With Lower Neuroactive Steroids and Worsened Outcomes Following Cerebral Ischemia in Male Mice.

Ischemic stroke is a leading cause of disability and death, and aging is the main nonmodifiable risk factor. Following ischemia, neuroactive steroids have been shown to play a key role in cerebroprotection. Thus, brain steroid concentrations at the time of injury as well as their regulation after stroke are key factors to consider. Here, we investigated the effects of age and cerebral ischemia on steroid levels, behavioral outcomes, and neuronal degeneration in 3- and 18-month-old C57BL/6JRj male mice. Ischemia was induced by middle cerebral artery occlusion for 1 hour followed by reperfusion (MCAO/R) and analyses were performed at 6 hours after MCAO. Extended steroid profiles established by gas chromatography coupled with tandem mass spectrometry revealed that (1) brain and plasma concentrations of the main 5α-reduced metabolites of progesterone, 11-deoxycorticosterone, and corticosterone were lower in old than in young mice; (2) after MCAO/R, brain concentrations of progesterone, 5α-dihydroprogesterone, and corticosterone increased in young mice; and (3) after MCAO/R, brain concentrations of 5α-reduced metabolites of progesterone, 3α5α-tetrahydrodeoxycorticosterone, and 3ß5α-tetrahydrodeoxycorticosterone were lower in old than in young mice. After ischemia, old mice showed increased sensori-motor deficits and more degenerating neurons in the striatum than young mice. Altogether, these findings strongly suggest that the decreased capacity of old mice to metabolize steroids toward the 5α-reduction pathway comparatively to young mice may contribute to the worsening of their stroke outcomes.

Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status.

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

A novel dual mode-of-action anti-hyperalgesic compound in rats which is neuroprotective and promotes neuroregeneration.

Chronic neuropathic pain (CNP) can result from surgery or traumatic injury, but also from peripheral neuropathies caused by diseases, viral infections, or toxic treatments. Opioids, although very effective for acute pain, do not prevent the development of CNP, and are considered as insufficient treatment. Therefore, there is high need for effective and safe non-opioid options to treat, prevent and eventually reverse CNP. A more effective approach to alleviating CNP would constitute a treatment that acts concurrently on various mechanisms involved in relieving pain symptoms and preventing or reversing chronification by enhancing both neuroprotection and neuroregeneration. We have identified and characterized GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-caboxylic acid amide), a novel drug which is able to activate both voltage-gated potassium channels of the Kv7 family and the mitochondrial translocator protein 18 kDa (TSPO). The dual mode-of-action (MoA) of GRT-X was indicated in in vitro studies and in vivo in a rat model of diabetic neuropathy. In this model, mechanical hyperalgesia was dose-dependently inhibited. After severe crush lesion of cervical spinal nerves in rats, GRT-X promoted survival, speeded up regrowth of sensory and motor neurons, and accelerated recovery of behavioral and neuronal responses to heat, cold, mechanical and electrical stimuli. These properties may reduce the likelihood of chronification of acute pain, and even potentially relieve established CNP. The absence of a conditioned place preference in rats suggests lack of abuse potential. In conclusion, GRT-X offers a promising preclinical profile with a novel dual MoA.

Neuroprotective Effects of Testosterone in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

Patients suffering of amyotrophic lateral sclerosis (ALS) present motoneuron degeneration leading to muscle atrophy, dysphagia, and dysarthria. The Wobbler mouse, an animal model of ALS, shows a selective loss of motoneurons, astrocytosis, and microgliosis in the spinal cord. The incidence of ALS is greater in men; however, it increases in women after menopause, suggesting a role of sex steroids in ALS. Testosterone is a complex steroid that exerts its effects directly via androgen (AR) or Sigma-1 receptors and indirectly via estrogen receptors (ER) after aromatization into estradiol. Its reduced-metabolite 5α-dihydrotestosterone acts via AR. This study analyzed the effects of testosterone in male symptomatic Wobblers. Controls or Wobblers received empty or testosterone-filled silastic tubes for 2 months. The cervical spinal cord from testosterone-treated Wobblers showed (1) similar androgen levels to untreated control and (2) increased levels of testosterone, and its 5α-reduced metabolites, 5α- dihydrotestosterone, and 3ß-androstanediol, but (3) undetectable levels of estradiol compared to untreated Wobblers. Testosterone-treated controls showed comparable steroid concentrations to its untreated counterpart. In testosterone- treated Wobblers a reduction of AR, ERα, and aromatase and high levels of Sigma-1 receptor mRNAs was demonstrated. Testosterone treatment increased ChAT immunoreactivity and the antiinflammatory mediator TGFß, while it lessened vacuolated motoneurons, GFAP+ astrogliosis, the density of IBA1+ microgliosis, proinflammatory mediators, and oxidative/nitrosative stress. Clinically, testosterone treatment in Wobblers slowed the progression of paw atrophy and improved rotarod performance. Collectively, our findings indicate an antiinflammatory and protective effect of testosterone in the degenerating spinal cord. These results coincided with a high concentration of androgen-reduced derivatives after testosterone treatment suggesting that the steroid profile may have a beneficial role on disease progression.

Placental endocrine function shapes cerebellar development and social behavior.

Compromised placental function or premature loss has been linked to diverse neurodevelopmental disorders. Here we show that placenta allopregnanolone (ALLO), a progesterone-derived GABA-A receptor (GABAAR) modulator, reduction alters neurodevelopment in a sex-linked manner. A new conditional mouse model, in which the gene encoding ALLO's synthetic enzyme (akr1c14) is specifically deleted in trophoblasts, directly demonstrated that placental ALLO insufficiency led to cerebellar white matter abnormalities that correlated with autistic-like behavior only in male offspring. A single injection of ALLO or muscimol, a GABAAR agonist, during late gestation abolished these alterations. Comparison of male and female human preterm infant cerebellum also showed sex-linked myelination marker alteration, suggesting similarities between mouse placental ALLO insufficiency and human preterm brain development. This study reveals a new role for a placental hormone in shaping brain regions and behaviors in a sex-linked manner. Placental hormone replacement might offer novel therapeutic opportunities to prevent later neurobehavioral disorders.

Conditional inactivation of androgen receptor gene in the nervous system: effects on male behavioral and neuroendocrine responses.

Testosterone (T) profoundly influences central sexual differentiation and functions. In the brain, T signals either directly through androgen receptor (AR) or indirectly through estrogen receptor (ER) following aromatization into E2 (17-beta-estradiol). As T, through AR, also controls peripheral male sexual differentiation, the relative contribution of central AR in T-mediated regulation of behavioral and neuroendocrine responses still remains unclear. To address this question, we generated, by using Cre-loxP technology, mice selectively lacking AR expression in the nervous system. The mutant male urogenital tract was normally developed, and mice were able to produce offspring. Nonetheless, sexual motivation and performance as well as aggressive behaviors were affected. Only a low percentage of males displayed a complete sexual behavior and offensive attacks. The latency to show masculine behaviors was increased and copulation length prolonged. Erectile activity during mating was also altered. These alterations occurred despite increased levels of T and its metabolites, and an unaffected number of ERalpha-immunoreactive cells. Olfactory preference and neuronal activation, mapped by Fos immunoreactivity, following exposure to estrus female-soiled bedding were also normal. At comparable T levels, greater differences in masculine behaviors were observed between gonadectomized control and mutant males. AR invalidation in the nervous system also disrupted the somatotropic axis since mutant males exhibited growth retardation and decreased serum levels of insulin-like growth factor I. Our findings show that central AR is required in T-induced regulation of male-typical behaviors and gonadotrope and somatotropic axes. This genetic model offers a unique opportunity in the understanding of AR's role in cerebral functions of T.