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.

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.

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.

Resistance profile of HIV-1 quasispecies in patients under treatment failure using single molecule, real-time sequencing.

OBJECTIVE: Short-read next-generation sequencing (NGS) has been implemented to study the resistance profile of HIV as it provides a higher sensitivity than Sanger sequencing. However, short-reads only generates a consensus view of the viral population rather than a reconstruction of the viral haplotypes. In this study, we evaluated the resistance profile of HIV quasispecies in patients undergoing treatment failure using SMRT sequencing. DESIGN: Whole-pol RT-PCR was performed on viral RNA extracted from plasma samples of 38 HIV-positive individuals undergoing treatment failure, and sequenced in the RSII instrument. Error correction and viral haplotype phasing was performed with the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm. Presence of resistance mutations reported by the IAS-USA in 2017 was assessed using an in-house script. RESULTS: The SMRT sequencing-based test detected 131/134 resistance mutations previously detected using a Sanger sequencing-based test. However, the SMRT test also identified seven additional mutations present at an estimated frequency lower than 30%. The intra-host phylogenetic analysis showed that seven samples harbored at least one resistance variant at 20--80% frequency. The haplotype-resolved sequencing revealed viral diversification and selection of new resistance during suboptimal treatment, an overall trend toward selection and accumulation of new resistance mutations, as well as the co-existence of resistant and susceptible variants. CONCLUSION: Our results validate the SMRT sequencing-based test for detection of HIV drug resistance. In addition, this method unraveled the complex dynamic of HIV quasispecies during treatment failure, which might have several implications on clinical management.

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.

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.