Epigenetic modifier Kdm6a/Utx controls the specification of hypothalamic neuronal subtypes in a sex-dependent manner.

Kdm6a is an X-chromosome-linked H3K27me2/3 demethylase that promotes chromatin accessibility and gene transcription and is critical for tissue/cell-specific differentiation. Previous results showed higher Kdm6a levels in XX than in XY hypothalamic neurons and a female-specific requirement for Kdm6a in mediating increased axogenesis before brain masculinization. Here, we explored the sex-specific role of Kdm6a in the specification of neuronal subtypes in the developing hypothalamus. Hypothalamic neuronal cultures were established from sex-segregated E14 mouse embryos and transfected with siRNAs to knockdown Kdm6a expression (Kdm6a-KD). We evaluated the effect of Kdm6a-KD on Ngn3 expression, a bHLH transcription factor regulating neuronal sub-specification in hypothalamus. Kdm6a-KD decreased Ngn3 expression in females but not in males, abolishing basal sex differences. Then, we analyzed Kdm6a-KD effect on Ascl1, Pomc, Npy, Sf1, Gad1, and Th expression by RT-qPCR. While Kdm6a-KD downregulated Ascl1 in both sexes equally, we found sex-specific effects for Pomc, Npy, and Th. Pomc and Th expressed higher in female than in male neurons, and Kdm6a-KD reduced their levels only in females, while Npy expressed higher in male than in female neurons, and Kdm6a-KD upregulated its expression only in females. Identical results were found by immunofluorescence for Pomc and Npy neuropeptides. Finally, using ChIP-qPCR, we found higher H3K27me3 levels at Ngn3, Pomc, and Npy promoters in male neurons, in line with Kdm6a higher expression and demethylase activity in females. At all three promoters, Kdm6a-KD induced an enrichment of H3K27me3 only in females. These results indicate that Kdm6a plays a sex-specific role in controlling the expression of transcription factors and neuropeptides critical for the differentiation of hypothalamic neuronal populations regulating food intake and energy homeostasis.

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.

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.

Gonadal hormone-independent sex differences in GABAA receptor activation in rat embryonic hypothalamic neurons.

BACKGROUND AND PURPOSE: GABAA receptor functions are dependent on subunit composition, and, through their activation, GABA can exert trophic actions in immature neurons. Although several sex differences in GABA-mediated responses are known to be dependent on gonadal hormones, few studies have dealt with sex differences detected before the critical period of brain masculinisation. In this study, we assessed GABAA receptor functionality in sexually segregated neurons before brain hormonal masculinisation. EXPERIMENTAL APPROACH: Ventromedial hypothalamic neurons were obtained from embryonic day 16 rat brains and grown in vitro for 2 days. Calcium imaging and electrophysiology recordings were carried out to assess GABAA receptor functional parameters. KEY RESULTS: GABAA receptor activation elicited calcium entry in immature hypothalamic neurons mainly through L-type voltage-dependent calcium channels. Nifedipine blocked calcium entry more efficiently in male than in female neurons. There were more male than female neurons responding to GABA, and they needed more time to return to resting levels. Pharmacological characterisation revealed that propofol enhanced GABAA -mediated currents and blunted GABA-mediated calcium entry more efficiently in female neurons than in males. Testosterone treatment did not erase such sex differences. These data suggest sex differences in the expression of GABAA receptor subtypes. CONCLUSION AND IMPLICATIONS: GABA-mediated responses are sexually dimorphic even in the absence of gonadal hormone influence, suggesting genetically biased differences. These results highlight the importance of GABAA receptors in hypothalamic neurons even before hormonal masculinisation of the brain.

Estradiol-Mediated Axogenesis of Hypothalamic Neurons Requires ERK1/2 and Ryanodine Receptors-Dependent Intracellular Ca2+ Rise in Male Rats.

17ß-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos in vitro, but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca2+ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca2+ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca2+ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca2+, which often occurs in repetitive Ca2+ oscillations. This response was not observed in the absence of extracellular Ca2+ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca2+ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.

Neurite atrophy and apoptosis mediated by PERK signaling after accumulation of GM2-ganglioside.

GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca2+ depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca2+ depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.

Programming of the reproductive axis by hormonal and genetic manipulation in mice

In mammals, the reproductive function is controlled by the hypothalamic­pituitary­gonadal axis. During development, mechanisms mediated by gonadal steroids exert an imprinting at the hypothalamic­pituitary level, by establishing sexual differences in the circuits that control male and female reproduction. In rodents, the testicular production of androgens increases drastically during the fetal/neonatal stage. This process is essential for the masculinization of the reproductive tract, genitals and brain. The conversion of androgens to estrogens in the brain is crucial for the male sexual differentiation and behavior. Conversely, feminization of the brain occurs in the absence of high levels of gonadal steroids during the perinatal period in females. Potential genetic contribution to the differentiation of brain cells through direct effects of genes located on sex chromosomes is also relevant. In this review, we will focus on the phenotypic alterations that occur on the hypothalamic­pituitary­gonadal axis of transgenic mice with persistently elevated expression of the human chorionic gonadotropin hormone (hCG). Excess of endogenously synthesized gonadal steroids due to a constant hCG stimulation is able to disrupt the developmental programming of the hypothalamic­pituitary axis in both transgenic males and females. Locally produced estrogens by the hypothalamic aromatase might play a key role in the phenotype of these mice. The 'four core genotypes' mouse model demonstrated a potential influence of sex chromosome genes in brain masculinization before critical periods of sex differentiation. Thus, hormonal and genetic factors interact to regulate the local production of the neurosteroids necessary for the programming of the male and female reproductive function.

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.

Regulation of aromatase expression in the anterior amygdala of the developing mouse brain depends on ERß and sex chromosome complement.

During development sex differences in aromatase expression in limbic regions of mouse brain depend on sex chromosome factors. Genes on the sex chromosomes may affect the hormonal regulation of aromatase expression and this study was undertaken to explore that possibility. Male E15 anterior amygdala neuronal cultures expressed higher levels of aromatase (mRNA and protein) than female cultures. Furthermore, treatment with oestradiol (E2) or dihydrotestosterone (DHT) increased Cyp19a1 expression and aromatase protein levels only in female neuronal cultures. The effect of E2 on aromatase expression was not imitated by oestrogen receptor (ER) α agonist PPT or the GPER agonist G1, but it was fully reproduced by DPN, a specific ligand of ERß. By contrast, the effect of DHT on aromatase expression was not blocked by the anti-androgen flutamide, but completely abrogated by the ERß antagonist PHTPP. Experiments using the four core genotype model showed a sex chromosome effect in ERß expression (XY > XX) and regulation by E2 or DHT (only XX respond) in amygdala neurons. In conclusion, sex chromosome complement governs the hormonal regulation of aromatase expression through activation of ERß in developing mouse brain.

Molecular mechanisms involved in the regulation of neuritogenesis by estradiol: Recent advances.

This review analyzes the signaling mechanisms activated by estradiol to regulate neuritogenesis in several neuronal populations. Estradiol regulates axogenesis by the activation of the mitogen activated protein kinase (MAPK) cascade through estrogen receptor α located in the plasma membrane. In addition, estradiol regulates MAPK signaling via the activation of protein kinase C and by increasing the expression of brain derived neurotrophic factor and tyrosine kinase receptor B. Estradiol also interacts with the signaling of insulin-like growth factor-I receptor through estrogen receptor α, modulating the phosphoinositide-3 kinase signaling pathway, which contributes to the stabilization of microtubules. Finally, estradiol modulates dendritogenesis by the inhibition of Notch signaling, by a mechanism that, at least in hippocampal neurons, is mediated by G-protein coupled receptor 30. This article is part of a Special Issue entitled 'Neurosteroids'.

ERK activation in the amygdala and hippocampus induced by fear conditioning in ethanol withdrawn rats: modulation by MK-801.

The extracellular signal-regulated kinase (ERK) pathway, which can be activated by NMDA receptor stimulation, is involved in fear conditioning and drug addiction. We have previously shown that withdrawal from chronic ethanol administration facilitated the formation of contextual fear memory. In order to explore the neural substrates and the potential mechanism involved in this effect, we examined: 1) the ERK1/2 activation in the central (CeA) and basolateral (BLA) nuclei of the amygdala and in the dorsal hippocampus (dHip), 2) the effect of the NMDA receptor antagonist MK-801 on fear conditioning and ERK activation and 3) the effect of the infusion of U0126, a MEK inhibitor, into the BLA on fear memory formation in ethanol withdrawn rats. Rats made dependent via an ethanol-containing liquid diet were subjected to contextual fear conditioning on day 3 of ethanol withdrawal. High basal levels of p-ERK were found in CeA and dHip from ethanol withdrawn rats. ERK activation was significantly increased both in control (60min) and ethanol withdrawn rats (30 and 60min) in BLA after fear conditioning. Pre-training administration of MK-801, at a dose that had no effect on control rats, prevented the increase in ERK phosphorylation in BLA and attenuated the freezing response 24h later in ethanol withdrawn rats. Furthermore, the infusion of U0126 into the BLA, but not the CeA, before fear conditioning disrupted fear memory formation. These results suggest that the increased fear memory can be linked to changes in ERK phosphorylation, probably due to NMDA receptor activation in BLA in ethanol withdrawn rats.