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Contents The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference ( RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus ( AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats. [ABSTRACT FROM AUTHOR]

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-likelike 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D 2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Female puberty is subject to Polycomb Group (PcG)-dependent transcriptional repression. Kiss1, a puberty-activating gene, is a key target of this silencing mechanism. Using a gain-of-function approach and a systems biology strategy we now show that EED, an essential PcG component, acts in the arcuate nucleus of the hypothalamus to alter the functional organization of a gene network involved in the stimulatory control of puberty. A central node of this network is Kdm6b, which encodes an enzyme that erases the PcG-dependent histone modification H3K27me3. Kiss1 is a first neighbor in the network; genes encoding glutamatergic receptors and potassium channels are second neighbors. By repressing Kdm6b expression, EED increases H3K27me3 abundance at these gene promoters, reducing gene expression throughout a gene network controlling puberty activation. These results indicate that Kdm6b repression is a basic mechanism used by PcG to modulate the biological output of puberty-activating gene networks.

Cats are a critical pre-clinical model for studying adeno-associated virus (AAV) vector-mediated gene therapies. A recent study has described the high prevalence of anti-AAV neutralizing antibodies among domestic cats in Switzerland. However, our knowledge of pre-existing humoral immunity against various AAV serotypes in cats is still limited. Here, we show that, although antibodies binding known AAV serotypes (AAV1 to AAV11) are prevalent in cats living in the Northeastern United States, these antibodies do not necessarily neutralize AAV infectivity. We analyzed sera from 35 client-owned, 20 feral, and 30 specific pathogen-free (SPF) cats for pre-existing AAV-binding antibodies against the 11 serotypes. Antibody prevalence was 7 to 90% with an overall median of 50%. The AAV-binding antibodies showed broad reactivities with other serotypes. Of 44 selected antibodies binding AAV2, AAV6 or AAV9, none exhibited appreciable neutralizing activities. Instead, AAV6 or AAV9-binding antibodies showed a transduction-enhancing effect. AAV6-binding antibodies were highly prevalent in SPF cats (83%), but this was primarily due to cross-reactivity with preventive vaccine-induced anti-feline panleukopenia virus antibodies. These results indicate that prevalent pre-existing immunity in cats is not necessarily inhibitory to AAV and highlight a substantial difference in the nature of AAV-binding antibodies in cats living in geographically different regions.

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Recent studies demonstrated that changes in DNA methylation (DNAm) and inactivation of two imprinted genes (MKRN3 and DLK1) alter the onset of female puberty. We aimed to investigate the association of DNAm profiling with the timing of human puberty analyzing the genome-wide DNAm patterns of peripheral blood leukocytes from ten female patients with central precocious puberty (CPP) and 33 healthy girls (15 pre- and 18 post-pubertal). For this purpose, we performed comparisons between the groups: pre- versus post-pubertal, CPP versus pre-pubertal, and CPP versus post-pubertal., Results: Analyzing the methylome changes associated with normal puberty, we identified 120 differentially methylated regions (DMRs) when comparing pre- and post-pubertal healthy girls. Most of these DMRs were hypermethylated in the pubertal group (99%) and located on the X chromosome (74%). Only one genomic region, containing the promoter of ZFP57, was hypomethylated in the pubertal group. ZFP57 is a transcriptional repressor required for both methylation and imprinting of multiple genomic loci. ZFP57 expression in the hypothalamus of female rhesus monkeys increased during peripubertal development, suggesting enhanced repression of downstream ZFP57 target genes. Fourteen other zinc finger (ZNF) genes were related to the hypermethylated DMRs at normal puberty. Analyzing the methylome changes associated with CPP, we demonstrated that the patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Forty-eight ZNF genes were identified as having hypermethylated CpG sites in CPP., Conclusion: Methylome profiling of girls at normal and precocious puberty revealed a widespread pattern of DNA hypermethylation, indicating that the pubertal process in humans is associated with specific changes in epigenetically driven regulatory control. Moreover, changes in methylation of several ZNF genes appear to be a distinct epigenetic modification underlying the initiation of human puberty.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na + , K + -ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K + ) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K + conductances functionally associated with NKA and leads to stunted neuronal growth., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Population studies elucidating the genetic architecture of reproductive ageing have been largely limited to European ancestries, restricting the generalizability of the findings and overlooking possible key genes poorly captured by common European genetic variation. Here, we report 26 loci (all P < 5 × 10 -8 ) for reproductive ageing, i.e. puberty timing or age at menopause, in a non-European population (up to 67,029 women of Japanese ancestry). Highlighted genes for menopause include GNRH1, which supports a primary, rather than passive, role for hypothalamic-pituitary GnRH signalling in the timing of menopause. For puberty timing, we demonstrate an aetiological role for receptor-like protein tyrosine phosphatases by combining evidence across population genetics and pre- and peri-pubertal changes in hypothalamic gene expression in rodent and primate models. Furthermore, our findings demonstrate widespread differences in allele frequencies and effect estimates between Japanese and European associated variants, highlighting the benefits and challenges of large-scale trans-ethnic approaches.

Polycomb group (PcG) proteins control the timing of puberty by repressing the Kiss1 gene in hypothalamic arcuate nucleus (ARC) neurons. Here we identify two members of the Trithorax group (TrxG) of modifiers, mixed-lineage leukemia 1 (MLL1), and 3 (MLL3), as central components of an activating epigenetic machinery that dynamically counteracts PcG repression. Preceding puberty, MLL1 changes the chromatin configuration at the promoters of Kiss1 and Tac3, two genes required for puberty to occur, from repressive to permissive. Concomitantly, MLL3 institutes a chromatin structure that changes the functional status of a Kiss1 enhancer from poised to active. RNAi-mediated, ARC-specific Mll1 knockdown reduced Kiss1 and Tac3 expression, whereas CRISPR-Cas9-directed epigenome silencing of the Kiss1 enhancer selectively reduced Kiss1 activity. Both interventions delay puberty and disrupt reproductive cyclicity. Our results demonstrate that an epigenetic switch from transcriptional repression to activation is crucial to the regulatory mechanism controlling the timing of mammalian puberty.

Llamas are considered to be reflex ovulators. However, semen from these animals is reported to be rich in ovulation-inducing factor(s), one of which has been identified as nerve growth factor (NGF). These findings suggest that ovulation in llamas may be elicited by chemical signals contained in semen instead of being mediated by neural signals. The present study examines this notion. Llamas displaying a preovulatory follicle were assigned to four groups: group 1 received an intrauterine infusion (IUI) of PBS; group 2 received an IUI of seminal plasma; group 3 was mated to a male whose urethra had been surgically diverted (urethrostomized male); and group 4 was mated to an intact male. Ovulation (detected by ultrasonography) occurred only in llamas mated to an intact male or given an IUI of seminal plasma and was preceded by a surge in plasma LH levels initiated within an hour after coitus or IUI. In both ovulatory groups, circulating β-NGF levels increased within 15 minutes after treatment, reaching values that were greater and more sustained in llamas mated with an intact male. These results demonstrate that llamas can be induced to ovulate by seminal plasma in the absence of copulation and that copulation alone cannot elicit ovulation in the absence of seminal plasma. In addition, our results implicate β-NGF as an important mediator of seminal plasma-induced ovulation in llamas because ovulation does not occur if β-NGF levels do not increase in the bloodstream, a change that occurs promptly after copulation with an intact male or IUI of seminal plasma.

In recent years the pace of discovering the molecular and genetic underpinnings of the pubertal process has accelerated considerably. Genes required for human puberty to occur have been identified and evidence has been provided suggesting that the initiation of puberty requires coordinated changes in the output of a multiplicity of genes organized into functional networks. Recent evidence suggests that a dual mechanism of epigenetic regulation affecting the transcriptional activity of neurons involved in stimulating gonadotropin-releasing hormone release plays a fundamental role in the timing of puberty. The Polycomb group (PcG) of transcriptional silencers appears to be a major component of the repressive arm of this mechanism. PcG proteins prevent the premature initiation of female puberty by silencing the Kiss1 gene in kisspeptin neurons of the arcuate nucleus (ARC) of the hypothalamus. Because the abundance of histone marks either catalyzed by--or associated with--the Trithorax group (TrxG) of transcriptional activators increases at the time when PcG control subsides, it appears that the TrxG complex is the counteracting partner of PcG-mediated gene silencing. In this chapter, we discuss the concept that a switch from epigenetic repression to activation within ARC kisspeptin neurons is a core mechanism underlying the initiation of female puberty., (© 2016 S. Karger AG, Basel.)

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

Background: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown., Materials and Methods: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays., Results and Conclusion: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Epithelial ovarian cancer is one of the most lethal of gynecological malignancies. Due to its lack of early symptoms, detection usually occurs when the tumor is no longer confined to the ovary. We previously identified Fbxw15, a gene encoding an F-box protein in the mouse ovary, and showed that its expression is developmentally regulated. Here we report the molecular analysis of its human homologue, FBXW12 in epithelial ovarian tumors. To search for FBXW12 gene mutations, we PCR-amplified and sequenced the coding region of FBXW12, the gene's 5-untranslated region and the proximal promoter in each of 30 EOC tumors. Promoter methylation was determined by DNA bisulfite conversion, followed by methylation specific PCR. FBXW12 intracellular localization was identified by means of immunohistochemistry. A complete deletion of the gene's coding region, the 5'-UTR and the proximal promoter, was observed in 3 EOC samples. Eight of the remaining 27, had a deletion of the 5'-UTR, and the proximal promoter. FBXW12 mRNA was detected in 2 of the 19 samples without deletions. The methylation specific PCR results demonstrated CpGs methylation in the FBXW12 proximal promoter. Immunohistochemistry assay revealed that within the normal ovary, FBXW12 has an oocyte specific expression, whereas in EOC samples it is present in the ovarian surface epithelium. Our results indicate that the FBXW12 gene is deleted in approximately ten percent of the EOC cases studied; such deletions comprised either the FBXW12 promoter or the mRNA-encoding region. Moreover, FBXW12 could be epigenetically silenced by CpGs methylation in some of these EOC cases.

Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Background: ADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary., Methods: We studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined., Results: Immunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 μm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles., Conclusions: The results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary.

Substantial progress has been made in recent years toward deciphering the molecular and genetic underpinnings of the pubertal process. The availability of powerful new methods to interrogate the human genome has led to the identification of genes that are essential for puberty to occur. Evidence has also emerged suggesting that the initiation of puberty requires the coordinated activity of gene sets organized into functional networks. At a cellular level, it is currently thought that loss of transsynaptic inhibition, accompanied by an increase in excitatory inputs, results in the pubertal activation of GnRH release. This concept notwithstanding, a mechanism of epigenetic repression targeting genes required for the pubertal activation of GnRH neurons was recently identified as a core component of the molecular machinery underlying the central restraint of puberty. In this chapter we will discuss the potential contribution of various mechanisms of epigenetic regulation to the hypothalamic control of female puberty., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Polycystic ovarian syndrome (PCOS), the most common female endocrine disorder of unknown etiology, is characterized by reproductive abnormalities and associated metabolic conditions comprising insulin resistance, type 2 diabetes mellitus, and dyslipidemia. We previously reported that transgenic overexpression of nerve growth factor (NGF), a marker of sympathetic hyperactivity, directed to the ovary by the mouse 17α-hydroxylase/C17-20 lyase promoter (17NF mice), results in ovarian abnormalities similar to those seen in PCOS women. To investigate whether ovarian overproduction of NGF also induces common metabolic alterations of PCOS, we assessed glucose homeostasis by glucose tolerance test, plasma insulin levels, and body composition by dual-energy x-ray absorptiometry scan in young female 17NF mice and wild-type mice. 17NF mice exhibited increased body weight and alterations in body fat distribution with a greater accumulation of visceral fat compared with sc fat (P < .01). 17NF mice also displayed glucose intolerance (P < .01), decreased insulin-mediated glucose disposal (P < .01), and hyperinsulinemia (P < .05), which, similar to PCOS patients, occurred independently of body weight. Additionally, 17NF mice exhibited increased sympathetic outflow observed as increased interscapular brown adipose tissue temperature. This change was evident during the dark period (7 pm to 7 am) and occurred concomitant with increased interscapular brown adipose tissue uncoupling protein 1 expression. These findings suggest that overexpression of NGF in the ovary may suffice to cause both reproductive and metabolic alterations characteristic of PCOS and support the hypothesis that sympathetic hyperactivity may contribute to the development and/or progression of PCOS.

Context: Gordon Holmes syndrome (GHS) is characterized by cerebellar ataxia/atrophy and normosmic hypogonadotropic hypogonadism (nHH). The underlying pathophysiology of this combined neurodegeneration and nHH remains unknown., Objective: We aimed to provide insight into the disease mechanism in GHS., Methods: We studied a cohort of 6 multiplex families with GHS through autozygosity mapping and whole-exome sequencing., Results: We identified 6 patients from 3 independent families carrying loss-of-function mutations in PNPLA6, which encodes neuropathy target esterase (NTE), a lysophospholipase that maintains intracellular phospholipid homeostasis by converting lysophosphatidylcholine to glycerophosphocholine. Wild-type PNPLA6, but not PNPLA6 bearing these mutations, rescued a well-established Drosophila neurodegenerative phenotype caused by the absence of sws, the fly ortholog of mammalian PNPLA6. Inhibition of NTE activity in the LβT2 gonadotrope cell line diminished LH response to GnRH by reducing GnRH-stimulated LH exocytosis, without affecting GnRH receptor signaling or LHβ synthesis., Conclusion: These results suggest that NTE-dependent alteration of phospholipid homeostasis in GHS causes both neurodegeneration and impaired LH release from pituitary gonadotropes, leading to nHH.

Premature ovarian failure (POF) affects 1% of women in reproductive age, but its etiology remains uncertain. Whereas kisspeptins, the products of Kiss1 that act via Kiss1r (aka, Gpr54), are known to operate at the hypothalamus to control GnRH/gonadotropin secretion, additional actions at other reproductive organs, including the ovary, have been proposed. Yet, their physiological relevance is still unclear. We present here a series of studies in Kiss1r haplo-insufficient and null mice suggesting a direct role of kisspeptin signaling in the ovary, the defect of which precipitates a state of primary POF. Kiss1r hypomorph mice displayed a premature decline in ovulatory rate, followed by progressive loss of antral follicles, oocyte loss, and a reduction in all categories of preantral follicles. These alterations were accompanied by reduced fertility. Because of this precocious ovarian ageing, mice more than 48 weeks of age showed atrophic ovaries, lacking growing follicles and corpora lutea. This phenomenon was associated with a drop in ovarian Kiss1r mRNA expression, but took place in the absence of a decrease in circulating gonadotropins. In fact, FSH levels increased in aged hypomorph animals, reflecting loss of follicular function. In turn, Kiss1r-null mice, which do not spontaneously ovulate and have arrested follicular development, failed to show normal ovulatory responses to standard gonadotropin priming and required GnRH prestimulation during 1 week in order to display gonadotropin-induced ovulation. Yet, the magnitude of such ovulatory responses was approximately half of that seen in control immature wild-type animals. Altogether, our data are the first to demonstrate that Kiss1r haplo-insufficiency induces a state of POF, which is not attributable to defective gonadotropin secretion. We also show that the failure of follicular development and ovulation linked to the absence of Kiss1r cannot be fully rescued by (even extended) gonadotropin replacement. These findings suggest a direct ovarian role of kisspeptin signaling, the perturbation of which may contribute to the pathogenesis of POF.

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.

In 2013, considerable progress was made towards deciphering the molecular foundations of puberty. Loss of transcriptional repression was identified as a core mechanism underlying the onset of puberty, and this loss was found to be precipitated by epigenetic cues. It was also discovered that nutritional deprivation delays puberty by repressing reproductive neuroendocrine function.

The importance of the Kiss1 gene in the control of reproductive development is well documented. However, much less is known about the transcriptional regulation of Kiss1 expression in the hypothalamus. Critical for these studies is an accurate identification of the site(s) where Kiss1 transcription is initiated. Employing 5'-RACE PCR, we detected a transcription start site (TSS1) used by the hypothalamus of rats, mice, nonhuman primates and humans to initiate Kiss1 transcription. In rodents, an exon 1 encoding 5'-untranslated sequences is followed by an alternatively spliced second exon, which encodes 5'-untranslated regions of two different lengths and contains the translation initiation codon (ATG). In nonhuman primates and humans, exon 2 is not alternatively spliced. Surprisingly, in rat mediobasal hypothalamus (MBH), but not preoptic area (POA), an additional TSS (TSS2) located upstream from TSS1 generates an exon 1 longer (377 bp) than the TSS1-derived exon 1 (98 bp). The content of TSS1-derived transcripts increased at puberty in the POA and MBH of female rats. It also increased in the MBH after ovariectomy, and this change was prevented by estrogen. In contrast, no such changes in TSS2-derived transcript abundance were detected. Promoter assays showed that the proximal TSS1 promoter is much more active than the putative TSS2 promoter, and that only the TSS1 promoter is regulated by estrogen. These differences appear to be related to the presence of a TATA box and binding sites for transcription factors activating transcription and interacting with estrogen receptor-α in the TSS1, but not TSS2, promoter., (© 2014 S. Karger AG, Basel)

This article is part of a Special Issue "Puberty and Adolescence". Puberty is a major developmental milestone controlled by the interaction of genetic factors and environmental cues of mostly metabolic and circadian nature. An increased pulsatile release of the decapeptide gonadotropin releasing hormone (GnRH) from hypothalamic neurosecretory neurons is required for both the initiation and progression of the pubertal process. This increase is brought about by coordinated changes that occur in neuronal and glial networks associated with GnRH neurons. These changes ultimately result in increased neuronal and glial stimulatory inputs to the GnRH neuronal network and a reduction of transsynaptic inhibitory influences. While some of the major players controlling pubertal GnRH secretion have been identified using gene-centric approaches, much less is known about the system-wide control of the overall process. Because the pubertal activation of GnRH release involves a diversity of cellular phenotypes, and a myriad of intracellular and cell-to-cell signaling molecules, it appears that the overall process is controlled by a highly coordinated and interactive regulatory system involving hundreds, if not thousands, of gene products. In this article we will discuss emerging evidence suggesting that these genes are arranged as functionally connected networks organized, both internally and across sub-networks, in a hierarchical fashion. According to this concept, the core of these networks is composed of transcriptional regulators that, by directing expression of downstream subordinate genes, provide both stability and coordination to the cellular networks involved in initiating the pubertal process. The integrative response of these gene networks to external inputs is postulated to be coordinated by epigenetic mechanisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The timing of puberty is controlled by many genes. The elements coordinating this process have not, however, been identified. Here we show that an epigenetic mechanism of transcriptional repression times the initiation of female puberty in rats. We identify silencers of the Polycomb group (PcG) as principal contributors to this mechanism and show that PcG proteins repress Kiss1, a puberty-activating gene. Hypothalamic expression of two key PcG genes, Eed and Cbx7, decreased and methylation of their promoters increased before puberty. Inhibiting DNA methylation blocked both events and resulted in pubertal failure. The pubertal increase in Kiss1 expression was accompanied by EED loss from the Kiss1 promoter and enrichment of histone H3 modifications associated with gene activation. Preventing the eviction of EED from the Kiss1 promoter disrupted pulsatile gonadotropin-releasing hormone release, delayed puberty and compromised fecundity. Our results identify epigenetic silencing as a mechanism underlying the neuroendocrine control of female puberty.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the MECP2. Several genes have been shown to be MECP2 targets. We previously identified FXYD1 (encoding phospholemman; a protein containing the motif phenylalanine-X-tyrosine-aspartate), a gene encoding a transmembrane modulator of the Na, K-ATPase (NKA) enzyme, as one of them. In the absence of MECP2, FXYD1 expression is increased in the frontal cortex (FC) of both RTT patients and Mecp2(Bird) null mice. Here, we show that Fxyd1 mRNA levels are also increased in the FC and hippocampus (HC) of male mice carrying a truncating mutation of the Mecp2 gene (Mecp2(308)). To test the hypothesis that some of the behavioral phenotypes seen in these Mecp2 mutants could be ameliorated by genetically preventing the Fxyd1 response to MECP2 deficiency, we crossed Fxyd1 null male mice with Mecp2(308) heterozygous females and behaviorally tested the adult male offspring. Mecp2(308) mice had impaired HC-dependent novel location recognition, and this impairment was rescued by deletion of both Fxyd1 alleles. No other behavioral or sensorimotor impairments were rescued. These results indicate that reducing FXYD1 levels improves a specific cognitive impairment in MECP2-deficient mice., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

Background/aim: TTF1 and EAP1 are transcription factors that modulate gonadotropin-releasing hormone expression. We investigated the contribution of TTF1 and EAP1 genes to central pubertal disorders., Patients and Methods: 133 patients with central pubertal disorders were studied: 86 with central precocious puberty and 47 with normosmic isolated hypogonadotropic hypogonadism. The coding region of TTF1 and EAP1 were sequenced. Variations of polyglutamine and polyalanine repeats in EAP1 were analyzed by GeneScan software. Association of TTF1 and EAP1 to genes implicated in timing of puberty was investigated by meta-network framework GeneMANIA and Cytoscape software., Results: Direct sequencing of the TTF1 did not reveal any mutation or polymorphisms. Four EAP1 synonymous variants were identified with similar frequencies among groups. The most common EAP1 5'-distal polyalanine genotype was the homozygous 12/12, but the genotype 12/9 was identified in 2 central precocious puberty sisters without functional alteration in EAP1 transcriptional activity. TTF1 and EAP1 were connected, via genetic networks, to genes implicated in the control of menarche., Conclusion: No TTF1 or EAP1 germline mutations were associated with central pubertal disorders. TTF1 and EAP1 may affect puberty by changing expression in response to other members of puberty-associated gene networks, or by differentially affecting the expression of gene components of these networks., (© 2013 S. Karger AG, Basel.)

Study Question: Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology?, Summary Answer: DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL., What Is Known Already: DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined., Study Design, Size, Duration: Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients., Participants/materials, Setting, Methods: Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²⁺, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity., Main Results and the Role of Chance: Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²⁺ levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments., Limitations, Reasons for Caution: Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available., Wider Implications of the Findings: In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates., Study Funding/competing Interest(s): This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).

Thyroid transcription factor 1 (TTF1), a member of the Nkx family of transcription factors required for basal forebrain morphogenesis, functions in the postnatal hypothalamus as a transcriptional regulator of genes encoding neuromodulators and hypophysiotrophic peptides. One of these peptides is gonadotrophin-releasing hormone (GnRH). In the present study, we show that Ttf1 mRNA abundance varies in a diurnal and melatonin-dependent fashion in the preoptic area of the rat, with maximal Ttf1 expression attained during the dark phase of the light/dark cycle, preceding the nocturnal peak in GnRH mRNA content. GnRH promoter activity oscillates in a circadian manner in GT1-7 cells, and this pattern is enhanced by TTF1 and blunted by small interfering RNA-mediated Ttf1 gene silencing. TTF1 transactivates GnRH transcription by binding to two sites in the GnRH promoter. Rat GnRH neurones in situ contain key proteins components of the positive (BMAL1, CLOCK) and negative (PER1) limbs of the circadian oscillator, and these proteins repress Ttf1 promoter activity in vitro. By contrast, Ttf1 transcription is activated by CRY1, a clock component required for circadian rhythmicity. In turn, TTF1 represses transcription of Rev-erbα, a heme receptor that controls circadian transcription within the positive limb of the circadian oscillator. These findings suggest that TTF1 is a component of the molecular machinery controlling circadian oscillations in GnRH gene transcription., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 Blackwell Publishing Ltd.)

Current evidence suggests that the acquisition of female reproductive capacity and the maintenance of mature reproductive function are related processes transcriptionally regulated by gene networks operating within the neuroendocrine brain. One of these genes, termed enhanced at puberty 1 (EAP1), encodes an upstream regulator of these processes. Selective inhibition of EAP1 expression in discrete regions of the rat and nonhuman primate (NHP) hypothalamus, via targeted delivery of RNA interference, either disrupts (rats) or abolishes (monkeys) reproductive cycles. The striking loss of menstrual cyclicity resulting from knocking down hypothalamic EAP1 expression suggests that diminished EAP1 function may contribute to disorders of the menstrual cycle of neuroendocrine origin. Here we show that a single-nucleotide polymorphism in the 5'-flanking region of EAP1 gene is associated with increased incidence of amenorrhea/oligomenorrhea in NHP. In the presence of the risk allele, binding of the transcription factor mothers against decapentaplegic homolog 3 (SMAD3) to its recognition site contained within the polymorphic sequence in the monkey EAP1 promoter is reduced. The risk allele also diminishes the increase in EAP1 promoter activity elicited by TGFβ1, a peptide that activates a SMAD3/4-mediated signaling pathway to regulate gene transcription. These findings indicate that common genetic variation in the EAP1 locus increases the susceptibility of NHP to loss/disruption of menstrual cyclicity. They also raise the possibility that polymorphisms in EAP1 may increase the risk of functional hypothalamic amenorrhea in humans.

Mammalian reproductive cyclicity requires the periodic discharge of GnRH from hypothalamic neurons into the portal vessels connecting the neuroendocrine brain to the pituitary gland. GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The transcriptional control of this process is not well understood, but it appears to involve several genes. One of them, termed enhanced at puberty 1 (EAP1), has been postulated to function in the female hypothalamus as an upstream regulator of neuroendocrine reproductive function. RNA interference-mediated inhibition of EAP1 expression, targeted to the preoptic region, delays puberty and disrupts estrous cyclicity in rodents, suggesting that EAP1 is required for the normalcy of these events. Here, we show that knocking down EAP1 expression in a region of the medial basal hypothalamus that includes the arcuate nucleus, via lentiviral-mediated delivery of RNA interference, results in cessation of menstrual cyclicity in female rhesus monkeys undergoing regular menstrual cycles. Neither lentiviruses encoding an unrelated small interfering RNA nor the placement of viral particles carrying EAP1 small interfering RNA outside the medial basal hypothalamus-arcuate nucleus region affected menstrual cycles, indicating that region-specific expression of EAP1 in the hypothalamus is required for menstrual cyclicity in higher primates. The cellular mechanism by which EAP1 exerts this function is unknown, but the recent finding that EAP1 is an integral component of a powerful transcriptional-repressive complex suggests that EAP1 may control reproductive cyclicity by inhibiting downstream repressor genes involved in the neuroendocrine control of reproductive function.

SynCAM1 is an adhesion molecule involved in synaptic differentiation and organization. SynCAM1 is also expressed in astroglial cells where it mediates astrocyte-to astrocyte and glial-neuronal adhesive communication. In astrocytes, SynCAM1 is functionally linked to erbB4 receptors, which are involved in the control of both neuronal/glial development and mature neuronal and glial function. Here we report that mice carrying a dominant-negative form of SynCAM1 specifically targeted to astrocytes (termed GFAP-DNSynCAM1 mice) exhibit disrupted diurnal locomotor activity with enhanced and more frequent episodes of activity than control littermates during the day (when the animals are normally sleeping) accompanied by shorter periods of rest. GFAP-DNSynCAM1 mice also display high levels of basal activity in the dark period (the rodent's awake/active time) that are attenuated by the psychostimulant D,L-amphetamine, and reduced anxiety levels in response to both avoidable and unavoidable provoking stimuli. These results indicate that disruption of SynCAM1-dependent astroglial function results in behavioral abnormalities similar to those described in animals model of attention-deficit hyperactive disorder (ADHD), and suggest a hitherto unappreciated contribution of glial cells to the pathophysiology of this disorder.

Tropomyosin-related kinase (TRK) receptor B (TRKB) mediates the supportive actions of neurotrophin 4/5 and brain-derived neurotrophic factor on early ovarian follicle development. Absence of TRKB receptors reduces granulosa cell (GC) proliferation and delays follicle growth. In the present study, we offer mechanistic insights into this phenomenon. DNA array and quantitative PCR analysis of ovaries from TrkB-null mice revealed that by the end of the first week of postnatal life, Jagged1, Hes1, and Hey2 mRNA abundance is reduced in the absence of TRKB receptors. Although Jagged1 encodes a NOTCH receptor ligand, Hes1 and Hey2 are downstream targets of the JAGGED1-NOTCH2 signaling system. Jagged1 is predominantly expressed in oocytes, and the abundance of JAGGED1 is decreased in TrkB(-/-) oocytes. Lack of TRKB receptors also resulted in reduced expression of c-Myc, a NOTCH target gene that promotes entry into the cell cycle, but did not alter the expression of genes encoding core regulators of cell-cycle progression. Selective restoration of JAGGED1 synthesis in oocytes of TrkB(-/-) ovaries via lentiviral-mediated transfer of the Jagged1 gene under the control of the growth differentiation factor 9 (Gdf9) promoter rescued c-Myc expression, GC proliferation, and follicle growth. These results suggest that neurotrophins acting via TRKB receptors facilitate early follicle growth by supporting a JAGGED1-NOTCH2 oocyte-to-GC communication pathway, which promotes GC proliferation via a c-MYC-dependent mechanism.

To date, a therapy for Huntington's disease (HD), a genetic, neurodegenerative disorder, remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Consequently, patients exhibit a hyperkinetic movement disorder. RNA interference (RNAi) offers therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene. We have previously demonstrated that partial suppression of both wild-type and mutant HTT in the striatum prevents behavioral and neuropathological abnormalities in rodent models of HD. However, given the role of HTT in various cellular processes, it remains unknown whether a partial suppression of both alleles will be safe in mammals whose neurophysiology, basal ganglia anatomy, and behavioral repertoire more closely resembles that of a human. Here, we investigate whether a partial reduction of HTT in the normal non-human primate putamen is safe. We demonstrate that a 45% reduction of rhesus HTT expression in the mid- and caudal putamen does not induce motor deficits, neuronal degeneration, astrogliosis, or an immune response. Together, these data suggest that partial suppression of wild-type HTT expression is well tolerated in the primate putamen and further supports RNAi as a therapy for HD.

Astrocytes in the hypothalamus release prostaglandin E(2) (PGE(2)) in response to cell-cell signaling initiated by neurons and glial cells. Upon release, PGE(2) stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patch-clamp recording we demonstrate that PGE(2) exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A (PKA) inhibitors. The acute blockade of prostaglandin synthesis by indomethacin (INDO) or the selective inhibition of astrocyte metabolism by fluoroacetate (FA) suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE(2) release due to defective erbB signaling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE(2) acts as a gliotransmitter within the GnRH neurosecretory system.

Kisspeptin, the product of the KiSS1 gene, has emerged as a key component of the mechanism by which the hypothalamus controls puberty and reproductive development. It does so by stimulating the secretion of gonadotropin releasing hormone (GnRH). Little is known about the transcriptional control of the KiSS1 gene. Here we show that a set of proteins postulated to be upstream components of a hypothalamic network involved in controlling female puberty regulates KiSS1 transcriptional activity. Using RACE-PCR we determined that transcription of KiSS1 mRNA is initiated at a single transcription start site (TSS) located 153-156bp upstream of the ATG translation initiation codon. Promoter assays performed using 293 MSR cells showed that the KiSS1 promoter is activated by TTF1 and CUX1-p200, and repressed by EAP1, YY1, and CUX1-p110. EAP1 and CUX-110 were also repressive in GT1-7 cells. All four TFs are recruited in vivo to the KiSS1 promoter and are expressed in kisspeptin neurons. These results suggest that expression of the KiSS1 gene is regulated by trans-activators and repressors involved in the system-wide control of mammalian puberty., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)