Gonadal hormone deprivation regulates response to tibolone in neurodegenerative pathways.

Gonadal hormone deprivation (GHD) and decline such as menopause and bilateral oophorectomy are associated with an increased risk of neurodegeneration. Yet, hormone therapies (HTs) show varying efficacy, influenced by factors such as sex, drug type, and timing of treatment relative to hormone decline. We hypothesize that the molecular environment of the brain undergoes a transition following GHD, impacting the effectiveness of HTs. Using a GHD model in mice treated with Tibolone, we conducted proteomic analysis and identified a reprogrammed response to Tibolone, a compound that stimulates estrogenic, progestogenic, and androgenic pathways. Through a comprehensive network pharmacological workflow, we identified a reprogrammed response to Tibolone, particularly within "Pathways of Neurodegeneration", as well as interconnected pathways including "cellular respiration", "carbon metabolism", and "cellular homeostasis". Analysis revealed 23 proteins whose Tibolone response depended on GHD and/or sex, implicating critical processes like oxidative phosphorylation and calcium signalling. Our findings suggest the therapeutic efficacy of HTs may depend on these variables, suggesting a need for greater precision medicine considerations whilst highlighting the need to uncover underlying mechanisms.

Sex chromosome complement interacts with gonadal hormones in determining regional-specific neuroactive steroid levels in plasma, hippocampus, and hypothalamus. A study using the four core genotype mouse model.

An important aspect of the neuromodulatory and neuroprotective actions exerted by neuroactive steroids is that they are sex-specific, as determined by the sexually dimorphic levels of these molecules in plasma and the nervous tissue. Thus, the identification of the factors that generate the sex-dimorphic levels of neuroactive steroids may be crucial from a neuroprotectant perspective. The main driver for sex determination in mammals is the SRY gene and the subsequent presence of a specific gonad: testes for males and ovaries for females, thus producing hormonal compounds, primarily androgens and estrogens, respectively. Nowadays, it is well established that despite the relevance of gonads, other factors control sexual features, and, among them, sex chromosome complement is highly relevant. In this study, neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in the hypothalamus, the hippocampus, and plasma of the four core genotype mouse model, to determine the relative contribution of sex chromosome complement and gonads in determining their sex dimorphic levels. The data obtained reveal that although gonads are the main contributing factor for sex differences in neuroactive steroid levels, the levels of some neuroactive steroids, including testosterone, are also influenced in brain and plasma by tissue-specific actions of sex chromosomes. The data presented here adds a new piece to the puzzle of steroid level regulation, which may be useful in designing sex-specific neuroprotective approaches to pathological conditions affecting the nervous system.

Interaction of gonadal hormones, dopaminergic system, and epigenetic regulation in the generation of sex differences in substance use disorders: A systematic review.

Substance use disorder (SUD) is a chronic condition characterized by pathological drug-taking and seeking behaviors. Remarkably different between males and females, suggesting that drug addiction is a sexually differentiated disorder. The neurobiological bases of sex differences in SUD include sex-specific reward system activation, influenced by interactions between gonadal hormone level changes, dopaminergic reward circuits, and epigenetic modifications of key reward system genes. This systematic review, adhering to PICOS and PRISMA-P 2015 guidelines, highlights the sex-dependent roles of estrogens, progesterone, and testosterone in SUD. In particular, estradiol elevates and progesterone reduces dopaminergic activity in SUD females, whilst testosterone and progesterone augment SUD behavior in males. Finally, SUD is associated with a sex-specific increase in the rate of opioid and monoaminergic gene methylation. The study reveals the need for detailed research on gonadal hormone levels, dopaminergic or reward system activity, and epigenetic landscapes in both sexes for efficient SUD therapy development.

Respirasome Proteins Are Regulated by Sex-Hormone Interactions in the Brain.

The existence of sex differences in disease incidence is attributed, in part, to sex differences in metabolism. Uncovering the precise mechanism driving these differences is an extraordinarily complex process influenced by genetics, endogenous hormones, sex-specific lifetime events, individual differences and external environmental/social factors. In fact, such differences may be subtle, but across a life span, increase susceptibility to a pathology. Whilst research persists in the hope of discovering an elegant biological mechanism to underpin sex differences in disease, here, we show, for the first time, that such a mechanism may be subtle in nature but influenced by multiple sex-specific factors. A proteomic dataset was generated from a gonadectomized mouse model treated with Tibolone, a menopausal hormone therapy. Following functional enrichment analysis, we identified that Alzheimer's disease and the electron transport chain-associated pathways were regulated by sex-hormone interactions. Specifically, we identified that the expression of three respirasome proteins, NDUFA2, NDUFA7 and UQCR10, is significantly altered by compounding factors that contribute to sex differences. These proteins function in bioenergetics and produce reactive oxygen species, which are each dysregulated in many diseases with sex differences in incidence. We show sex-specific reprogrammed responses to Tibolone following gonadectomy, which primarily influence the expression of proteins contributing to metabolic pathways. This further infers that metabolic differences may underpin the observed sex differences in disease, but also that hormone therapy research now has potential in exploring sex-specific interventions to produce an effective method of prevention or treatment.

Genetics and Epigenetics of the X and Y Chromosomes in the Sexual Differentiation of the Brain.

For many decades to date, neuroendocrinologists have delved into the key contribution of gonadal hormones to the generation of sex differences in the developing brain and the expression of sex-specific physiological and behavioral phenotypes in adulthood. However, it was not until recent years that the role of sex chromosomes in the matter started to be seriously explored and unveiled beyond gonadal determination. Now we know that the divergent evolutionary process suffered by X and Y chromosomes has determined that they now encode mostly dissimilar genetic information and are subject to different epigenetic regulations, characteristics that together contribute to generate sex differences between XX and XY cells/individuals from the zygote throughout life. Here we will review and discuss relevant data showing how particular X- and Y-linked genes and epigenetic mechanisms controlling their expression and inheritance are involved, along with or independently of gonadal hormones, in the generation of sex differences in the brain.

X-linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of hypothalamic neurons.

Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17ß-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization.

Amyloid-ß1-40 differentially stimulates proliferation, activation of oxidative stress and inflammatory responses in male and female hippocampal astrocyte cultures.

Alzheimer's disease (AD) is the most common form of dementia and has a higher incidence in women. The main component of the senile plaques characteristic of AD is amyloid-beta (Aß), with surrounding astrocytes contributing to the degenerative process. We hypothesized that the sex difference in the incidence of AD could be partially due to differential astrocytic responses to Aß. Thus, the effect of Aß1-40 on cell viability, the inflammatory response, and oxidative status was studied in cultures of hippocampal astrocytes from male and female rats. Aß1-40 increased astrocyte viability in both female and male cultures by activating proliferation and survival pathways. Pro-inflammatory and anti-inflammatory responses were induced in astrocytes from both sexes. Aß1-40 did not affect endoplasmic reticulum stress although it induced oxidative stress in male and female astrocytes. Interestingly, male astrocytes had an increase in cell number and significantly lower cell death in response to Aß1-40. Conversely, astrocytes from females displayed a greater inflammatory response after the Aß1-40 challenge. These results suggest that the inflammatory and oxidative environment induced by Aß1-40 in female astrocytes may contribute to enhance the vulnerability to AD and warrants further studies to unveil the mechanisms underlying sex differences in astrocytic responses.

G Protein-Coupled Estrogen Receptor Immunoreactivity in the Rat Hypothalamus Is Widely Distributed in Neurons, Astrocytes, and Oligodendrocytes, Fluctuates during the Estrous Cycle, and Is Sexually Dimorphic.

INTRODUCTION: The membrane-associated G protein-coupled estrogen receptor 1 (GPER) mediates the regulation by estradiol of arginine-vasopressin immunoreactivity in the supraoptic and paraventricular hypothalamic nuclei of female rats and is involved in the estrogenic control of hypothalamic regulated functions, such as food intake, sexual receptivity, and lordosis behavior. OBJECTIVE: To assess GPER distribution in the rat hypothalamus. METHODS: GPER immunoreactivity was assessed in different anatomical subdivisions of five selected hypothalamic regions of young adult male and cycling female rats: the arcuate nucleus, the lateral hypothalamus, the paraventricular nucleus, the supraoptic nucleus, and the ventromedial hypothalamic nucleus. GPER immunoreactivity was colocalized with NeuN as a marker of mature neurons, GFAP as a marker of astrocytes, and CC1 as a marker of mature oligodendrocytes. RESULTS: GPER immunoreactivity was detected in hypothalamic neurons, astrocytes, and oligodendrocytes. Sex and regional differences and changes during the estrous cycle were detected in the total number of GPER-immunoreactive cells and in the proportion of neurons, astrocytes, and oligodendrocytes that were GPER-immunoreactive. CONCLUSIONS: These findings suggest that estrogenic regulation of hypothalamic function through GPER may be different in males and females and may fluctuate during the estrous cycle in females.

Aging and sex: Impact on microglia phagocytosis.

Microglia dysfunction and activation are important hallmarks of the aging brain and are concomitant with age-related neurodegeneration and cognitive decline. Age-associated changes in microglia migration and phagocytic capacity result in maladaptive responses, chronic neuroinflammation, and worsened outcomes in neurodegenerative disorders. Given the sex bias in the incidence, prevalence, and therapy response of most neurological disorders, we have here examined whether the phagocytic activity of aged microglia is different in males and females. With this aim, the phagocytosis activity of male and female cells was compared in an in vitro aged microglia model and in microglia isolated from adult (5-month-old) or aged (18-month-old) mice. In both models, the phagocytosis of neural debris increased with aging in male and female cells and was higher in aged female microglia than in aged male cells. However, female aged microglia lost its ability to adapt its phagocytic activity to inflammatory conditions. These findings suggest that microglia phagocytosis of neural debris may represent a previously unexplored neuroprotective characteristic of aged microglia that may contribute to the generation of sex differences in the manifestation of neurodegenerative diseases.

Tibolone as Hormonal Therapy and Neuroprotective Agent.

Tibolone (TIB), a selective tissue estrogenic activity regulator (STEAR) in clinical use by postmenopausal women, activates hormonal receptors in a tissue-specific manner. Estrogenic activity is present mostly in the brain, vagina, and bone, while the inactive forms predominate in the endometrium and breast. Conflicting literature on TIB's actions has been observed. While it has benefits for vasomotor symptoms, bone demineralization, and sexual health, a higher relative risk of hormone-sensitive cancer has been reported. In the brain, TIB can improve mood and cognition, neuroinflammation, and reactive gliosis. This review aims to discuss the systemic effects of TIB on peri- and post-menopausal women and its role in the brain. We suggest that TIB is a hormonal therapy with promising neuroprotective properties.

Estradiol-dependent axogenesis and Ngn3 expression are determined by XY sex chromosome complement in hypothalamic neurons.

Hypothalamic neurons show sex differences in neuritogenesis, female neurons have longer axons and higher levels of the neuritogenic factor neurogenin 3 (Ngn3) than male neurons in vitro. Moreover, the effect of 17-ß-estradiol (E2) on axonal growth and Ngn3 expression is only found in male-derived neurons. To investigate whether sex chromosomes regulate these early sex differences in neuritogenesis by regulating the E2 effect on Ngn3, we evaluated the growth and differentiation of hypothalamic neurons derived from the "four core genotypes" mouse model, in which the factors of "gonadal sex" and "sex chromosome complement" are dissociated. We showed that sex differences in neurite outgrowth are determined by sex chromosome complement (XX > XY). Moreover, E2 increased the mRNA expression of Ngn3 and axonal length only in XY neurons. ERα/ß expressions are regulated by sex chromosome complement; however, E2-effect on Ngn3 expression in XY neurons was only fully reproduced by PPT, a specific ligand of ERα, and prevented by MPP, a specific antagonist of ERα. Together our data indicate that sex chromosomes regulate early development of hypothalamic neurons by orchestrating not only sex differences in neuritogenesis, but also regulating the effect of E2 on Ngn3 expression through activation of ERα in hypothalamic neurons.

Physiopathological role of the enzymatic complex 5α-reductase and 3α/ß-hydroxysteroid oxidoreductase in the generation of progesterone and testosterone neuroactive metabolites.

The enzymatic complex 5α-reductase (5α-R) and 3α/3ß-hydroxysteroid oxidoreductase (HSOR) is expressed in the nervous system, where it transforms progesterone (PROG) and testosterone (T) into neuroactive metabolites. These metabolites regulate myelination, brain maturation, neurotransmission, reproductive behavior and the stress response. The expression of 5α-R and 3α-HSOR and the levels of PROG and T reduced metabolites show regional and sex differences in the nervous system and are affected by changing physiological conditions as well as by neurodegenerative and psychiatric disorders. A decrease in their nervous tissue levels may negatively impact the course and outcome of some pathological events. However, in other pathological conditions their increased levels may have a negative impact. Thus, the use of synthetic analogues of these steroids or 5α-R modulation have been proposed as therapeutic approaches for several nervous system pathologies. However, further research is needed to fully understand the consequences of these manipulations, in particular with 5α-R inhibitors.

The synthetic steroid tibolone exerts sex-specific regulation of astrocyte phagocytosis under basal conditions and after an inflammatory challenge.

BACKGROUND: Tibolone is a synthetic steroid used in clinical practice for the treatment of climacteric symptoms and osteoporosis. Active metabolites of tibolone, generated in target tissues, have an affinity for estrogen and androgen receptors. Astrocytes are direct targets for estrogenic compounds and previous studies have shown that tibolone protects brain cortical neurons in association with a reduction in reactive astrogliosis in a mouse model of traumatic brain injury. Since phagocytosis is a crucial component of the neuroprotective function exerted by astrocytes, in the present study, we have assessed whether tibolone regulates phagocytosis in primary astrocytes incubated with brain-derived cellular debris. METHODS: Male and female astrocyte cell cultures were obtained from newborn (P0-P2) female and male Wistar rats. Astrocytic phagocytosis was first characterized using carboxylate beads, Escherichia coli particles, or brain-derived cellular debris. Then, the effect of tibolone on the phagocytosis of Cy3-conjugated cellular debris was quantified by measuring the intensity of Cy3 dye-emitted fluorescence in a given GFAP immunoreactive area. Before the phagocytosis assays, astrocytes were incubated with tibolone in the presence or absence of estrogen or androgen receptor antagonists or an inhibitor of the enzyme that synthesizes estradiol. The effect of tibolone on phagocytosis was analyzed under basal conditions and after inflammatory stimulation with lipopolysaccharide. RESULTS: Tibolone stimulated phagocytosis of brain-derived cellular debris by male and female astrocytes, with the effect being more pronounced in females. The effect of tibolone in female astrocytes was blocked by a selective estrogen receptor ß antagonist and by an androgen receptor antagonist. None of these antagonists affected tibolone-induced phagocytosis in male astrocytes. In addition, the inhibition of estradiol synthesis in the cultures enhanced the stimulatory effect of tibolone on phagocytosis in male astrocytes but blocked the effect of the steroid in female cells under basal conditions. However, after inflammatory stimulation, the inhibition of estradiol synthesis highly potentiated the stimulation of phagocytosis by tibolone, particularly in female astrocytes. CONCLUSIONS: Tibolone exerts sex-specific regulation of phagocytosis in astrocytes of both sexes, both under basal conditions and after inflammatory stimulation.

Lipotoxicity, neuroinflammation, glial cells and oestrogenic compounds.

The high concentrations of free fatty acids as a consequence of obesity and being overweight have become risk factors for the development of different diseases, including neurodegenerative ailments. Free fatty acids are strongly related to inflammatory events, causing cellular and tissue alterations in the brain, including cell death, deficits in neurogenesis and gliogenesis, and cognitive decline. It has been reported that people with a high body mass index have a higher risk of suffering from Alzheimer's disease. Hormones such as oestradiol not only have beneficial effects on brain tissue, but also exert some adverse effects on peripheral tissues, including the ovary and breast. For this reason, some studies have evaluated the protective effect of oestrogen receptor (ER) agonists with more specific tissue activities, such as the neuroactive steroid tibolone. Activation of ERs positively affects the expression of pro-survival factors and cell signalling pathways, thus promoting cell survival. This review aims to discuss the relationship between lipotoxicity and the development of neurodegenerative diseases. We also elaborate on the cellular and molecular mechanisms involved in neuroprotection induced by oestrogens.

Sex differences and gonadal hormone regulation of brain cardiolipin, a key mitochondrial phospholipid.

Cardiolipin (CL) is a phospholipid that is almost exclusively located in the inner mitochondrial membrane of eukaryotic cells. As a result of its unique structure and distribution, CL establishes non-covalent bonds with a long list of proteins involved in ATP production, mitochondria biogenesis, mitophagy and apoptosis. Thus, the amount of CL, as well as its fatty acid composition and location, strongly impacts upon mitochondrial-dependent functions and therefore the metabolic homeostasis of different tissues. The brain is particularly sensitive to mitochondrial dysfunction as a result of its high metabolic demand. Several mitochondrial related-neurodegenerative disorders, as well as physiological ageing, show altered CL metabolism. Furthermore, mice lacking enzymes involved in CL synthesis show cognitive impairments. CL content and metabolism are regulated by gonadal hormones in the developing and adult brain. In neuronal cultures, oestradiol increases CL content, whereas adult ovariectomy decreases CL content and alters CL metabolism in the hippocampal mitochondria. Transient sex differences in brain CL metabolism have been detected during development. At birth, brain CL has a higher proportion of unsaturated fatty acids in the brain of male mice than in the brain of females. In addition, the expression of enzymes involved in CL de novo and recycling synthetic pathways is higher in males. Most of these sex differences are abolished by the neonatal androgenisation of females, suggesting a role for testosterone in the generation of sex differences in brain CL. The regulation of brain CL by gonadal hormones may be linked to their homeostatic and protective actions in neural cells, as well as the manifestation of sex differences in neurodegenerative disorders.

Sex differences in the peripubertal response to a short-term, high-fat diet intake.

Obesity is one of the most important health problems facing developed countries because being overweight is associated with a higher incidence of type 2 diabetes, cardiovascular disease and cancer, as well as other comorbidities. Although increased weight gain results from a combination of poor dietary habits and decreased energy expenditure, not all individuals have equal propensities to gain weight or to develop secondary complications of obesity. This is partially a result not only of genetics, including sex, but also the time during which an individual is exposed to an obesogenic environment. In the present study, we have compared the response of male and female mice to short-term exposure to a high-fat diet (HFD) or a low-fat diet during the peripubertal period (starting at 42 days of age) because this is a stage of dramatic hormonal and metabolic modifications. After 1 week on a HFD, there was no significant increase in body weight, although females significantly increased their energy intake. Serum leptin levels increased in both sexes, even though no change in fat mass was detected. Glyceamia and homeostasis model assessment increased in males, suggesting a rapid change in glucose metabolism. Hypothalamic pro-opiomelanocortin mRNA levels were significantly higher in females on a HFD compared to all other groups, which may be an attempt to reduce their increased energy intake. Hypothalamic inflammation and gliosis have been implicated in the development of secondary complications of obesity; however, no indication of activation of inflammatory processes or gliosis was found in response to 1 week of HFD in the hypothalamus, hippocampus or cerebellum of these young mice. These results indicate that there are both sex and age effects in the response to poor dietary intake because peripubertal male and female mice respond differently to short-term dietary changes and this response is different from that reported in adult rodents.

Sex differences in steroid levels and steroidogenesis in the nervous system: Physiopathological role.

The nervous system, in addition to be a target for steroid hormones, is the source of a variety of neuroactive steroids, which are synthesized and metabolized by neurons and glial cells. Recent evidence indicates that the expression of neurosteroidogenic proteins and enzymes and the levels of neuroactive steroids are different in the nervous system of males and females. We here summarized the state of the art of neuroactive steroids, particularly taking in consideration sex differences occurring in the synthesis and levels of these molecules. In addition, we discuss the consequences of sex differences in neurosteroidogenesis for the function of the nervous system under healthy and pathological conditions and the implications of neuroactive steroids and neurosteroidogenesis for the development of sex-specific therapeutic interventions.

Sex differences in the brain expression of steroidogenic molecules under basal conditions and after gonadectomy.

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18 kDa (TSPO), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenases (3ß-HSD), 5α-reductases (5α-R) and 3α-hydroxysteroid oxidoreductases (3α-HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α-HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α-R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α-R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α-HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function.