Tibolone restrains neuroinflammation in mouse experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an immune-mediated and degenerating disease in which myelin sheaths are damaged as a result of chronic progressive inflammation of the central nervous system. Tibolone [(7α,17α)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-in-3-one], a synthetic estrogenic compound with tissue-specific actions and used for menopausal hormone therapy, shows neuroprotective and antioxidant properties both in vivo and in vitro. In the present study, we analyzed whether tibolone plays a therapeutic role in experimental autoimmune encephalomyelitis (EAE) mice, a commonly used model of MS. Female C57BL/6 mice were induced with the myelin oligodendrocyte glycoprotein MOG35-55 and received s.c. tibolone (0.08 mg kg-1 ) injection every other day from the day of induction until death on the acute phase of the disease. Reactive gliosis, Toll like receptor 4 (TLR4), high mobility group box protein 1 (HMGB1), inflammasome parameters, activated Akt levels and myelin were assessed by a real-time polymerase chain reaction, immunohistochemistry, and western blot analysis. Our findings indicated that, in the EAE spinal cord, tibolone reversed the astrocytic and microglial reaction, and reduced the hyperexpression of TLR4 and HMGB1, as well as NLR family pyrin domain containing 3-caspase 1-interleukin-1ß inflammasome activation. At the same time, tibolone attenuated the Akt/nuclear factor kappa B pathway and limited the white matter demyelination area. Estrogen receptor expression was unaltered with tibolone treatment. Clinically, tibolone improved neurological symptoms without uterine compromise. Overall, our data suggest that tibolone may serve as a promising agent for the attenuation of MS-related inflammation.

High-fat diet alters stress behavior, inflammatory parameters and gut microbiota in Tg APP mice in a sex-specific manner.

Long-term high-fat diet (HFD) consumption commonly leads to obesity, a major health concern of western societies and a risk factor for Alzheimer's disease (AD). Both conditions present glial activation and inflammation and show sex differences in their incidence, clinical manifestation, and disease course. HFD intake has an important impact on gut microbiota, the bacteria present in the gut, and microbiota dysbiosis is associated with inflammation and certain mental disorders such as anxiety. In this study, we have analyzed the effects of a prolonged (18 weeks, starting at 7 months of age) HFD on male and female mice, both wild type (WT) and TgAPP mice, a model for AD, investigating the behavioral profile, gut microbiota composition and inflammatory/phagocytosis-related gene expression in hippocampus. In the open-field test, no overt differences in motor activity were observed between male and female or WT and TgAPP mice on a low-fat diet (LFD). However, HFD induced anxiety, as judged by decreased motor activity and increased time in the margins in the open-field, and a trend towards increased immobility time in the tail suspension test, with increased defecation. Intriguingly, female TgAPP mice on HFD showed less immobility and defecation compared to female WT mice on HFD. HFD induced dysbiosis of gut microbiota, resulting in reduced microbiota diversity and abundance compared with LFD fed mice, with some significant differences due to sex and little effect of genotype. Gene expression of pro-inflammatory/phagocytic markers in the hippocampus were not different between male and female WT mice, and in TgAPP mice of both sexes, some cytokines (IL-6 and IFNγ) were higher than in WT mice on LFD, more so in female TgAPP (IL-6). HFD induced few alterations in mRNA expression of inflammatory/phagocytosis-related genes in male mice, whether WT (IL-1ß, MHCII), or TgAPP (IL-6). However, in female TgAPP, altered gene expression returned towards control levels following prolonged HFD (IL-6, IL-12ß, TNFα, CD36, IRAK4, PYRY6). In summary, we demonstrate that HFD induces anxiogenic symptoms, marked alterations in gut microbiota, and increased expression of inflammatory genes, except for female TgAPP that appear to be resistant to the diet effects. Lifestyle interventions should be introduced to prevent AD onset or exacerbation by reducing inflammation and its associated symptoms; however, our results suggest that the eventual goal of developing prevention and treatment strategies should take sex into consideration.

Role of Neuroglobin in the Neuroprotective Actions of Estradiol and Estrogenic Compounds.

Estradiol exerts neuroprotective actions that are mediated by the regulation of a variety of signaling pathways and homeostatic molecules. Among these is neuroglobin, which is upregulated by estradiol and translocated to the mitochondria to sustain neuronal and glial cell adaptation to injury. In this paper, we will discuss the role of neuroglobin in the neuroprotective mechanisms elicited by estradiol acting on neurons, astrocytes and microglia. We will also consider the role of neuroglobin in the neuroprotective actions of clinically relevant synthetic steroids, such as tibolone. Finally, the possible contribution of the estrogenic regulation of neuroglobin to the generation of sex differences in brain pathology and the potential application of neuroglobin as therapy against neurological diseases will be examined.

G Protein-Coupled Estrogen Receptor Immunoreactivity Fluctuates During the Estrous Cycle and Show Sex Differences in the Amygdala and Dorsal Hippocampus.

G protein-coupled estrogen receptor (GPER) in the amygdala and the dorsal hippocampus mediates actions of estradiol on anxiety, social recognition and spatial memory. In addition, GPER participates in the estrogenic regulation of synaptic function in the amygdala and in the process of adult neurogenesis in the dentate gyrus. While the distribution of the canonical estrogen receptors α and ß in the amygdala and dorsal hippocampus are well characterized, little is known about the regional distribution of GPER in these brain regions and whether this distribution is affected by sex or the stages of the estrous cycle. In this study we performed a morphometric analysis of GPER immunoreactivity in the posterodorsal medial, anteroventral medial, basolateral, basomedial and central subdivisions of the amygdala and in all the histological layers of CA1 and the dentate gyrus of the dorsal hippocampal formation. The number of GPER immunoreactive cells was estimated in these different structures. GPER immunoreactivity was detected in all the assessed subdivisions of the amygdaloid nucleus and dorsal hippocampal formation. The number of GPER immunoreactive cells was higher in males than in estrus females in the central (P = 0.001) and the posterodorsal medial amygdala (P < 0.05); higher in males than in diestrus females in the strata orients (P < 0.01) and radiatum-lacunosum-moleculare (P < 0.05) of CA1-CA3 and in the molecular layer of the dentate gyrus (P < 0.01); higher in diestrus females than in males in the basolateral amygdala (P < 0.05); higher in diestrus females than in estrus females in the central (P < 0.01), posterodorsal medial (P < 0.01) and basolateral amygdala (P < 0.01) and higher in estrus females than in diestrus females in the strata oriens (P < 0.05) and radiatum-lacunosum-moleculare (P < 0.05) of CA1-CA3 and in the molecular layer (P < 0.05) and the hilus of the dentate gyrus (P < 0.05). The findings suggest that estrogenic regulation of the amygdala and hippocampus through GPER may be different in males and in females and may fluctuate during the estrous cycle.

Microglia, neurodegeneration and loss of neuroendocrine control.

Microglia, the primary regulators of inflammatory responses in the brain, suffer deterioration during aging culminating in their inability to generate adequate adaptive responses to maintain physiological homeostasis in brain tissue. Microglia affect the function of other glial cells and neurons, including those involved in the hypothalamic control of body homeostasis. Microglial dysfunction with aging in cognitive areas such as the hippocampus is known to associate with cognitive decline; more recently, microglial alterations in the hypothalamus during midlife was suggested to participate in changes in the endocrine and metabolic control exerted by this brain region. Consequently, the feed-back loops between endocrine glands and the hypothalamus are altered. This generates a vicious circle in which the plasma levels of key neuroprotective hormones, such as gonadal hormones, insulin-like growth factor-1, growth hormone and leptin and their hypothalamic signaling are decreased, which further enhances microglial alterations and deterioration of hypothalamic function. Hypothalamic dysfunction is a risk factor for neurodegenerative diseases and these diseases in turn promote additional alterations in hypothalamic microglial cells, which are unable to cope with the neurodegenerative process, resulting in permanent damage of the neuronal-glial circuits controlling endocrine homeostasis, food intake and body metabolism. Thus, a "vicious cycle" may such be initiated.

Molecular mechanisms and cellular events involved in the neuroprotective actions of estradiol. Analysis of sex differences.

Estradiol, either from peripheral or central origin, activates multiple molecular neuroprotective and neuroreparative responses that, being mediated by estrogen receptors or by estrogen receptor independent mechanisms, are initiated at the membrane, the cytoplasm or the cell nucleus of neural cells. Estrogen-dependent signaling regulates a variety of cellular events, such as intracellular Ca2+ levels, mitochondrial respiratory capacity, ATP production, mitochondrial membrane potential, autophagy and apoptosis. In turn, these molecular and cellular actions of estradiol are integrated by neurons and non-neuronal cells to generate different tissue protective responses, decreasing blood-brain barrier permeability, oxidative stress, neuroinflammation and excitotoxicity and promoting synaptic plasticity, axonal growth, neurogenesis, remyelination and neuroregeneration. Recent findings indicate that the neuroprotective and neuroreparative actions of estradiol are different in males and females and further research is necessary to fully elucidate the causes for this sex difference.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

Lipotoxic Effects of Palmitic Acid on Astrocytes Are Associated with Autophagy Impairment.

Obesity is associated with an increase in the brain levels of saturated free fatty acids, such as palmitic acid (PA). Previous studies have shown that PA exerts proinflammatory actions and reduces cell viability in astrocyte cultures. In this study, we have assessed whether an alteration in autophagy is involved in the effects of PA on astrocytes. Primary astrocytes were obtained from the cerebral cortex of male and female CD1 mouse pups and were incubated for 4.5 or 24 h with 250-500 µM PA. PA increased the levels of LC3-II, an autophagosome marker, and reduced LC3-II flux in astrocytes, suggesting a blockade of autophagy. This effect was observed both after 4.5 and 24 h of treatment with PA. PA had additional effects after treatment for 24 h, increasing the expression of proinflammatory cytokines, decreasing cell viability, and increasing the levels of an endoplasmic reticulum stress marker. In addition, PA decreased the expression of estrogen receptors, but only in female astrocytes. However, the treatment with estradiol, estrogen receptor agonists, or inhibitor of estradiol synthesis did not counteract the action of PA on cell viability. Rapamycin, an autophagy inducer, was unable to prevent the effect of PA on cell viability. In addition, hydroxychloroquine, an autophagy blocker, did not cause per se astrocyte death. These findings suggest that the effect of PA on autophagy is not sufficient to induce astrocyte loss, which is only observed when prolonged PA treatment causes other alterations in astrocytes, such as increased inflammation and endoplasmic reticulum stress.

Neuroactive steroids, neurosteroidogenesis and sex.

The nervous system is a target and a source of steroids. Neuroactive steroids are steroids that target neurons and glial cells. They include hormonal steroids originated in the peripheral glands, steroids locally synthesized by the neurons and glial cells (neurosteroids) and synthetic steroids, some of them used in clinical practice. Here we review the mechanisms of synthesis, metabolism and action of neuroactive steroids, including the role of epigenetic modifications and the mitochondria in their sex specific actions. We examine sex differences in neuroactive steroid levels under physiological conditions and their role in the establishment of sex dimorphic structures in the nervous system and sex differences in its function. In addition, particular attention is paid to neuroactive steroids under pathological conditions, analyzing how pathology alters their levels and their role as neuroprotective factors, considering the influence of sex in both cases.

Ovarian Hormone-Dependent Effects of Dietary Lipids on APP/PS1 Mouse Brain.

The formation of senile plaques through amyloid-ß peptide (Aß) aggregation is a hallmark of Alzheimer's disease (AD). Irrespective of its actual role in the synaptic alterations and cognitive impairment associated with AD, different therapeutic approaches have been proposed to reduce plaque formation. In rodents, daily intake of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs) is required for neural development, and there is experimental and epidemiological evidence that their inclusion in the diet has positive effects on several neurodegenerative diseases. Similarly, estradiol appears to reduce senile plaque formation in primary mouse cell cultures, human cortical neurons and mouse AD models, and it prevents Aß toxicity in neural cell lines. We previously showed that differences in dietary n-6/n-3 LC-PUFAs ratios modify the lipid composition in the cerebral cortex of female mice and the levels of amyloid precursor protein (APP) in the brain. These effects depended in part on the presence of circulating estradiol. Here we explored whether this potentially synergistic action between diet and ovarian hormones may influence the progression of amyloidosis in an AD mouse model. Our results show that a diet with high n-3 LC-PUFA content, especially DHA (22:6n-3), reduces the hippocampal accumulation of Aß1 - 4 0, but not amyloid Aß1 - 42 in female APPswe/PS1 E9A mice, an effect that was counteracted by the loss of the ovaries and that depended on circulating estradiol. In addition, this interaction between dietary lipids and ovarian function also affects the composition of the brain lipidome as well as the expression of certain neuronal signaling and synaptic proteins. These findings provide new insights into how ovarian hormones and dietary composition affect the brain lipidome and amyloid burden. Furthermore, they strongly suggest that when designing dietary or pharmacological strategies to combat human neurodegenerative diseases, hormonal and metabolic status should be specifically taken into consideration as it may affect the therapeutic response.

Estradiol Activates PI3K/Akt/GSK3 Pathway Under Chronic Neurodegenerative Conditions Triggered by Perinatal Asphyxia.

Perinatal asphyxia (PA) remains as one of the most important causes of short-term mortality, psychiatric and neurological disorders in children, without an effective treatment. In previous studies we have observed that the expression of different neurodegenerative markers increases in CA1 hippocampal area of 4-months-old male rats born by cesarean section and exposed for 19 min to PA. We have also shown that a late treatment with 17ß estradiol (daily dose of 250 µg/kg for 3 days) was able to revert the brain alterations observed in those animals. Based on these previous results, the main aim of the present study was to explore the mechanism by which the estrogenic treatment is involved in the reversion of the chronic neurodegenerative conditions induced by PA. We demonstrated that estradiol treatment of adult PA exposed animals induced an increase in estrogen receptor (ER) α and insulin-like growth factor receptor (IGF-1R) protein levels, an activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 beta/ß-catenin signaling pathway and an increase in Bcl-2/Bax ratio in the hippocampus in comparison to PA exposed animals treated with vehicle. Taking together, our data suggest that the interaction between ERα and IGF-IR, with the subsequent downstream activation, underlies the beneficial effects of estradiol observed in late treatment of PA.

The Synthetic Steroid Tibolone Decreases Reactive Gliosis and Neuronal Death in the Cerebral Cortex of Female Mice After a Stab Wound Injury.

Previous studies have shown that estradiol reduces reactive gliosis after a stab wound injury in the cerebral cortex. Since the therapeutic use of estradiol is limited by its peripheral hormonal effects, it is of interest to determine whether synthetic estrogenic compounds with tissue-specific actions regulate reactive gliosis. Tibolone is a synthetic steroid that is widely used for the treatment of climacteric symptoms and/or the prevention of osteoporosis. In this study, we have assessed the effect of tibolone on reactive gliosis in the cerebral cortex after a stab wound brain injury in ovariectomized adult female mice. By 7 days after brain injury, tibolone reduced the number of glial fibrillary acidic protein (GFAP) immunoreactive astrocytes, the number of ionized calcium binding adaptor molecule 1 (Iba1) immunoreactive microglia, and the number of microglial cells with a reactive phenotype in comparison to vehicle-injected animals. These effects on gliosis were associated with a reduction in neuronal loss in the proximity to the wound, suggesting that tibolone exerts beneficial homeostatic actions in the cerebral cortex after an acute brain injury.

A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling.

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

Neural-derived estradiol regulates brain plasticity.

In addition to be an ovarian hormone, estradiol is a neurosteroid synthesized by neural cells. The brain is a steroidogenic tissue that metabolizes testosterone to estradiol. The last step in the synthesis of estradiol is catalyzed by the enzyme aromatase, which is widely expressed in the brain of male and female animals and humans. Studies that have manipulated the expression or the activity of aromatase have revealed that brain-derived estradiol acts as a neuromodulator and regulates different forms of brain plasticity in male and female animals. The regulation of neuroplastic events by brain-derived estradiol probably participates in the effects of brain aromatase on behavior and cognition.

Interaction of sex chromosome complement, gonadal hormones and neuronal steroid synthesis on the sexual differentiation of mammalian neurons.

Female mouse hippocampal and hypothalamic neurons growing in vitro show a faster development of neurites than male mouse neurons. This sex difference in neuritogenesis is determined by higher expression levels of the neuritogenic factor neurogenin 3 in female neurons. Experiments with the four core genotype mouse model, in which XX and XY animals with male gonads and XX and XY animals with female gonads are generated, indicate that higher levels of neurogenin 3 in developing neurons are determined by the presence of the XX chromosome complement. Female XX neurons express higher levels of estrogen receptors than male XY neurons. In female XX neurons, neuronal derived estradiol increases neurogenin 3 expression and neuritogenesis. In contrast, neuronal-derived estradiol is not able to upregulate neurogenin 3 in male XY neurons, resulting in decreased neuritogenesis compared to female neurons. However, exogenous testosterone increases neurogenin 3 expression and neuritogenesis in male XY neurons. These findings suggest that sex differences in neuronal development are determined by the interaction of sex chromosomes, neuronal derived estradiol and gonadal hormones.

Oestradiol synthesized by female neurons generates sex differences in neuritogenesis.

Testosterone produced by the foetal testis is converted by male neurons to oestradiol, which masculinizes neuronal morphology. Female neurons are known to synthesize oestradiol in absence of exogenous testosterone. However, the role of neuronal oestradiol on the differentiation of foetal female neurons is unknown. Here we show that, due to endogenous neuronal oestradiol synthesis, female hippocampal neurons have higher expression of the neuritogenic protein Neurogenin 3 and enhanced neuritogenesis than males. Exogenous application of testosterone or its metabolite dihydrotestosterone increases Neurogenin 3 expression and promotes neuritogenesis in males, but reduces these parameters in females. Together our data indicate that gonadal-independent oestradiol synthesis by female neurons participates in the generation of sex differences in hippocampal neuronal development.

Profiling Neuroactive Steroid Levels After Traumatic Brain Injury in Male Mice.

The incidence of traumatic brain injuries (TBIs) in humans has rapidly increased in the last ten years. The most common causes are falls and car accidents. Approximately 80 000-90 000 persons per year will suffer some permanent disability as a result of the lesion, and one of the most common symptoms is the decline of hormone levels, also known as post-TBI hormonal deficiency syndrome. This issue has become more and more important, and many studies have focused on shedding some light on it. The hormonal decline affects not only gonadal steroid hormones but also neuroactive steroids, which play an important role in TBI recovery by neuroprotective and neurotrophic actions. The present work used an adolescent close-head murine model to analyze brain and plasma neurosteroid level changes after TBI and to establish correlations with edema and neurological impairments, 2 of the hallmarks of TBI. Our results showed changes in brain pregnenolone, testosterone, dihydrotestosterone (DHT), and 3α-diol levels whereas in plasma, the changes were present in progesterone, DHT, 3α-diol, and 3ß-diol. Within them, pregnenolone, progesterone, DHT, and 3α-diol levels positively correlated with edema formation and neurological score, whereas testosterone inversely correlated with these 2 variables. These findings suggest that changes in the brain levels of some neuroactive steroids may contribute to the alterations in brain function caused by the lesion and that plasma levels of some neuroactive steroids could be good candidates of blood markers to predict TBI outcome.

Adverse effects of 5α-reductase inhibitors: What do we know, don't know, and need to know?

Steroids are important physiological orchestrators of endocrine as well as peripheral and central nervous system functions. One of the key processes for regulation of these molecules lies in their enzymatic processing by a family of 5α-reductase (5α-Rs) isozymes. By catalyzing a key rate-limiting step in steroidogenesis, this family of enzymes exerts a crucial role not only in the physiological control but also in pathological events. Indeed, both 5α-R inhibition and supplementation of 5α-reduced metabolites are currently used or have been proposed as therapeutic strategies for a wide array of pathological conditions. In particular, the potent 5α-R inhibitors finasteride and dutasteride are used in the treatments of benign prostatic hyperplasia (BPH), as well as in male pattern hair loss (MPHL) known as androgenetic alopecia (AGA). Recent preclinical and clinical findings indicate that 5α-R inhibitors evoke not only beneficial, but also adverse effects. Future studies should investigate the biochemical and physiological mechanisms that underlie the persistence of the adverse sexual side effects to determine why a subset of patients is afflicted with such persistence or irreversible adverse effects. Also a better focus of clinical research is urgently needed to better define those subjects who are likely to be adversely affected by such agents. Furthermore, research on the non-sexual adverse effects such as diabetes, psychosis, depression, and cognitive function are needed to better understand the broad spectrum of the effects these drugs may elicit during their use in treatment of AGA or BPH. In this review, we will summarize the state of art on this topic, overview the key unresolved questions that have emerged on the pharmacological targeting of these enzymes and their products, and highlight the need for further studies to ascertain the severity and duration of the adverse effects of 5α-R inhibitors, as well as their biological underpinnings.

Sex chromosome complement determines sex differences in aromatase expression and regulation in the stria terminalis and anterior amygdala of the developing mouse brain.

Aromatase, which converts testosterone in estradiol, is involved in the generation of brain sex dimorphisms. Here we used the "four core genotypes" mouse model, in which the effect of gonadal sex and sex chromosome complement is dissociated, to determine if sex chromosomes influence the expression of brain aromatase. The brain of 16 days old XY mouse embryos showed higher aromatase expression in the stria terminalis and the anterior amygdaloid area than the brain of XX embryos, independent of gonadal sex. Furthermore, estradiol or dihydrotestosterone increased aromatase expression in cultures of anterior amygdala neurons derived from XX embryos, but not in those derived from XY embryos. This effect was also independent of gonadal sex. The expression of other steroidogenic molecules, estrogen receptor-α and androgen receptor was not influenced by sex chromosomes. In conclusion, sex chromosomes determine sex dimorphisms in aromatase expression and regulation in the developing mouse brain.

The Selective Estrogen Receptor Modulator Raloxifene Regulates Arginine-Vasopressin Gene Expression in Human Female Neuroblastoma Cells Through G Protein-Coupled Estrogen Receptor and ERK Signaling.

The selective estrogen receptor modulator raloxifene reduces blood pressure in hypertensive postmenopausal women. In the present study we have explored whether raloxifene regulates gene expression of arginine vasopressin (AVP), which is involved in the pathogenesis of hypertension. The effect of raloxifene was assessed in human female SH-SY5Y neuroblastoma cells, which have been recently identified as a suitable cellular model to study the estrogenic regulation of AVP. Raloxifene, within a concentration ranging from 10(-10) M to 10(-6) M, decreased the mRNA levels of AVP in SH-SY5Y cells with maximal effect at 10(-7) M. This effect of raloxifene was imitated by an agonist (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone of G protein-coupled estrogen receptor-1 (GPER) and blocked by an antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline of GPER and by GPER silencing. Raloxifene induced a time-dependent increase in the level of phosphorylated ERK1 and ERK2, by a mechanism blocked by the GPER antagonist. The treatment of SH-SY5Y cells with either a MAPK/ERK kinase 1/2-specific inhibitor (1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadine) or a protein kinase C inhibitor (sotrastaurin) blocked the effects of raloxifene on the phosphorylation of ERK1/2 and the regulation of AVP mRNA levels. These results reveal a mechanism mediating the regulation of AVP expression by raloxifene, involving the activation of GPER, which in turn activates protein kinase C, MAPK/ERK kinase, and ERK. The regulation of AVP by raloxifene and GPER may have implications for the treatment of blood hypertension(.).