Neuroendocrine Control of Female Puberty: Glial and Neuronal Interactions

Emerging evidence suggests that, in addition to neuronal inputs, growth factors of glial origin are also important in the control of mammalian puberty via a cell-cell interaction that ultimately affects the neurons that release gonadotropin-releasing hormone (GnRH), a neurohormone controlling sexual development. Among these growth factors, transforming growth factor-α (TGFα) appears to be one of the physiologic components that controls the onset of female puberty by affecting GnRH neuronal activity in a glia-mediated autocrine/paracrine manner. Specifically, TGFα induces glia to produce bioactive substances, such as prostaglandin E2 (PGE2). In turn, PGE2 directly acts on GnRH neurons to stimulate the release of GnRH. Furthermore, the neuregulin of glial origin neu differentiation factor (NDF) was found to facilitate the action of TGFα, suggesting that other growth factors may exert their biologic effects on GnRH neuronal function via a glia/neuron interaction. Another indication that glial cells may be involved in the regulation of neuroendocrine function is the presence of estrogen receptors on hypothalamic astrocytes. Thus, region-specific glial cells appear to play an integral role in the regulation of neuroendocrine function. Journal of Investigative Dermatology Symposium Proceedings 2:19–22, 199

Astrocyte-Specific Disruption of SynCAM1 Signaling Results in ADHD-Like Behavioral Manifestations

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

Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis

The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirus-mediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.This work was supported by grants from the NIH, the National Institute of Child Health and Human Development/NIH (to S.R. Ojeda), the European Society for Paediatric Endocrinology (to H. Jung), the German Research Foundation (to S. Heger), and the European Commission (PIONEER to S. Heger)

Supplements to article: A novel transcription complex that selectively modulates apoptosis of breast cancer cells through regulation of FASTKD2

The materials provided here are supplemental tables and figures to an article to be published in 'Molecular and Cellular Biology.'(This refers to the article.) We previously reported that expression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (a.k.a IRF-2BP2), the cellular target of NRIF3, was identified as a transcriptional repressor and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF2BP1 (Interferon Regulatory Factor-2 Binding Protein 1) and EAP1 (Enhanced At Puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF2BP1, and EAP1 occurs through the conserved C4 zinc-fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the Death Domain region of NRIF3 (DD1). The DIF-1 complex was found to repress FASTKD2, a putative pro-apoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown while expression of FASTKD2 leads to apoptosis of both breast and non-breast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer

Leveraging human genomic information to identify nonhuman primate sequences for expression array development

BACKGROUND: Nonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. RESULTS: We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. CONCLUSION: This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays

EAP1 regulation of GnRH promoter activity is important for human pubertal timing

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.Peer reviewe

FSH regulates acetycholine production by ovarian granulosa cells

BACKGROUND: It has been previously shown that cultured granulosa cells (GCs) derived from human ovarian preovulatory follicles contain choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. They also produce ACh and express functional muscarinic ACh receptors. ACh can act on GCs to increase proliferation, disrupt gap junctional communication, alter intracellular calcium levels, as well as expression of transcription factors, suggesting an unrecognized role of ACh in GC function. To gain further insights into the possible role of ACh in the ovary, we examined ChAT expression in the gland before and after birth, as well as in adults, and studied the regulation of ACh production by FSH. METHODS: ChAT immunohistochemistry was performed using ovarian samples of different species and ages (embryonic, postnatal and adult rats and mice, including embryonic ovaries from mice null for ChAT, neonatal and adult rhesus monkeys and adult humans). ACh was measured by HPLC and/or a fluorescence based method in rat ovaries and in a FSH receptor-expressing cell line (rat GFSHR-17) cultured with or without FSH. RESULTS: In adult rat, as well as in all other species, ovarian ChAT immunoreactivity is associated with GCs of antral follicles, but not with other structures, indicating that GCs are the only ovarian source of ACh. Indeed ACh was clearly detected in adult rat ovaries by two methods. ChAT immunoreactivity is absent from embryonic and/or neonatal ovaries (mouse/rat and monkey) and ovarian development in embryonic mice null for ChAT appears normal, suggesting that ACh is not involved in ovarian or follicular formation. Since ChAT immunoreactivity is present in GCs of large follicles and since the degree of the ChAT immunoreactivity increases as antral follicles grow, we tested whether ACh production is stimulated by FSH. Rat GFSHR-17 cells that stably express the FSH receptor, respond to FSH with an increase in ACh production. CONCLUSION: ACh and ChAT are present in GCs of growing follicles and FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs, we suggest that ACh may act in the growing ovarian follicle as a local mediator of some of the actions ascribed to FSH

TTF-1, a homeodomain-containing transcription factor, participates in the control of body fluid homeostasis by regulating angiotensinogen gene transcription in the rat subfornical organ.

In recent years, it has become increasingly evident that angiotensins synthesized in the brain contribute to regulating body fluid homeostasis. Although angiotensinogen, the unique angiotensin precursor, is produced in the brain, the factors that regulate its gene expression remain unknown. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in discrete areas of the hypothalamus. We now report that the subfornical organ, an important site of angiotensinogen synthesis, is an extra-hypothalamic site of TTF-1 expression. Double in situ hybridization histochemistry demonstrated the presence of TTF-1 mRNA in angiotensinogen-producing cells of the rat subfornical organ. RNase protection assays showed that TTF-1 and angiotensinogen mRNA levels are simultaneously increased in the subfornical organ by water deprivation. The angiotensinogen promoter contains seven presumptive TTF-1 binding motifs, four of which are recognized by the TTF-1 homeodomain. In the C6 glioma cell line, TTF-1 transactivates the angiotensinogen promoter in a dose-dependent manner. This transactivation is abolished by deletion of the TTF-1 binding motif at -125. Intracranial administration of an antisense TTF-1 oligodeoxynucleotide decreased angiotensinogen mRNA in the subfornical organ and dramatically reduced the animal's water intake while increasing urine excretion. Moreover, plasma arginine vasopressin content was decreased by the same treatment. These results demonstrate a novel role for TTF-1 in the regulation of body fluid homeostasis, exerted via the transactivational control of angiotensinogen synthesis in the subfornical organ

Elucidating the genetic architecture of reproductive ageing in the Japanese population.

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

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation