Susan Wray

This review focuses on the potential for intervention in hormone disorders by replacing neuroendocrine cells with engineered cells that avoid amplifying dysfunctional brain–body feedback by independently sending healthy signals to the body. This paradigm presents opportunities to develop treatment strategies for common disorders often overlooked in favor of symptom reduction, particularly in women's health. Numerous neuroendocrine cells integrate signals from the hormone control centers of the brain and release their signals to the anterior pituitary via the hypophyseal portal system. Subsequent release of pituitary hormones carries the signals throughout the body. Hormones produced by peripheral organs form feedback loops, sending their signals to multiple brain circuits to be processed and delivered back to the hormone control centers. This brain–body feedback system maintains homeostasis, reproductive and metabolic function, and affects social behavior. Dysfunction of these feedback systems presents a unique target for neuron replacement therapy. Therapeutic potential is introduced by using neuroendocrine replacement cells, modified in vitro do to form synthetic gene circuits that will override the dysfunctional brain–body feedback in vivo and act as hormone regulators. Specific strategies for reversing disorders in the hormone systems are presented, which serve as alternatives to hormone therapy.

Pituitary adenomas constitute up to 20% of primary brain tumors, yet somatic mutations are only found in 15% of pituitary adenomas. Epigenomic dysregulation has been proposed as a tumorigenic mechanism in pituitary adenomas causing Cushing’s disease (CD). We created paired datasets of human CD adenomas and en-route margin adult human pituitary glands, and assayed their chromatin accessibility, DNA methylation, transcriptomic, proteomic, and phospho-proteomic landscapes. In CD adenomas, we found epigenetic reactivation of a neurodevelopmental phosphoprotein program typically lost in the postnatal pituitary gland. CD cells overexpressed PPP1R17 , an endogenous inhibitor of the protein phosphatase PP2A. Mechanistically, PPP1R17 overexpression in normal murine pituitary cells recapitulated the adenoma phenotype, and the resulting hyperproliferation was reversed in a PP2A-dependent manner by fingolimod, an FDA-approved small molecule, both in vitro and in vivo. Our findings highlight kinase-phosphatase imbalance as a targetable mechanism in CD.

Many clinical studies have identified correlations between thyroid dysfunction and reproductive issues, yet the underlying mechanisms behind this interaction remain poorly understood. In this study, we investigated the effect of triiodothyronine (T3) on the activity of gonadotropin-releasing hormone (GnRH) neurons, a key regulator of the central reproductive axis. Dual labeling confirmed that GnRH neurons express thyroid receptor (TR)α and integrin αVβ3 receptors mediating genomic and nongenomic effects of thyroid hormones, respectively. Using calcium imaging in an ex vivo model, we show that T3 induces a rapid and sustained increase of calcium oscillation frequency in GnRH neurons. No change in response was detected after application of T4. The T3 stimulatory effect was not inhibited by a TR-specific antagonist (1-850) but was mimicked by membrane-impermeable T3-BSA, indicating a mechanism independent of nuclear TR signaling. In contrast, the blockade of membrane αVβ3 integrins (with cilengitide) prevented the T3-induced increase in GnRH neurons calcium peak oscillation frequency. Further investigation using modulators of intracellular calcium and calcium entry revealed that binding to αVβ3 integrin can induce distinct calcium responses depending on the ligand, with T3 triggering a complex response involving multiple channels and calcium sources, possibly with compensatory mechanisms. In sum, these results demonstrate for the first time a direct effect of thyroid hormones on GnRH neuronal activity, with T3 stimulating calcium oscillations through the nongenomic αVβ3 integrin pathway. Understanding this thyroid-reproductive axis interaction will help clarify the mechanisms linking thyroid dysfunction to reproductive disorders and pave the way for targeted therapeutic interventions.

Polycystic ovary syndrome (PCOS) is a heterogenous disorder characterized by reproductive and metabolic abnormalities. PCOS etiology remains poorly understood, although the hypothalamus is suspected to play a central role in many cases. Human genetic studies have also shown an association with the transcription factor‐coding gene GATA4 , but without providing a functional link. Here, we show that adult Greywick female mice may bridge this gap. These mice phenocopy PCOS with partial penetrance, due to the serendipitous insertion of a Gata4 promoter‐driven transgene in a strong enhancer region. Resulting robust transgene expression in subsets of hypothalamic neurons and glia impairs endogenous Gata4 expression, resulting in misexpression of genes linked to the control of fertility and food intake. We also show that this previously overlooked role of GATA4 in the hypothalamus can be replicated by conditional knockout approaches. Overall, this study sheds light not only on PCOS etiology but also on the role played by GATA4 in the central control of reproduction.

Vasoactive intestinal peptide (VIP) is an important component of the suprachiasmatic nucleus (SCN) which relays circadian information to neuronal populations, including GnRH neurons. Human and animal studies have shown an impact of disrupted daily rhythms (chronic shift work, temporal food restriction, clock gene disruption) on both male and female reproduction and fertility. To date, how VIP modulates GnRH neurons remains unknown. Calcium imaging and electrophysiology on primary GnRH neurons in explants and adult mouse brain slice, respectively, were used to address this question. We found VIP excites GnRH neurons via the VIP receptor, VPAC2. The downstream signaling pathway uses both Gs protein/adenylyl cyclase/protein kinase A (PKA) and phospholipase C/phosphatidylinositol 4,5-bisphosphate (PIP₂) depletion. Furthermore, we identified a UCL2077-sensitive target, likely contributing to the slow afterhyperpolarization current (I_AHP), as the PKA and PIP₂ depletion target, and the KCa3.1 channel as a specific target. Thus, VIP/VPAC2 provides an example of Gs protein-coupled receptor-triggered excitation in GnRH neurons, modulating GnRH neurons likely via the slow I_AHP. The possible identification of KCa3.1 in the GnRH neuron slow I_AHP may provide a new therapeutical target for fertility treatments.

Postnatal development of functional pituitary gonadotrophs is necessary for maturation of the hypothalamic-pituitary-gonadal axis, puberty, and reproduction. Here we examined the role of PI4-kinase A, which catalyzes the biosynthesis of PI4P in mouse reproduction by knocking out this enzyme in cells expressing the gonadotropin-releasing hormone (GnRH) receptor. Knockout (KO) mice were infertile, reflecting underdeveloped gonads and reproductive tracts and lack of puberty. The number and distribution of hypothalamic GnRH neurons and Gnrh1 expression in postnatal KOs were not affected, whereas Kiss1/kisspeptin expression was increased. KO of PI4-kinase A also did not alter embryonic establishment and neonatal development and function of the gonadotroph population. However, during the postnatal period, there was a progressive loss of expression of gonadotroph-specific genes, including Fshb, Lhb, and Gnrhr, accompanied by low gonadotropin synthesis. The postnatal gonadotroph population also progressively declined, reaching approximately one-third of that observed in controls at 3 months of age. In these residual gonadotrophs, GnRH-dependent calcium signaling and calcium-dependent membrane potential changes were lost, but intracellular administration of inositol-14,5-trisphosphate rescued this signaling. These results indicate a key role for PI4-kinase A in the postnatal development and maintenance of a functional gonadotroph population.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis.

In vertebrates, gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility by regulating gonadotrophs in the anterior pituitary. While it is known that acetylcholine (ACh) influences GnRH secretion, whether the effect is direct or indirect, and the specific ACh receptor (AChR) subtype(s) involved remain unclear. Here, we determined 1) whether ACh can modulate GnRH cellular activity and 2) a source of ACh afferents contacting GnRH neurons. Calcium imaging was used to assay GnRH neuronal activity. With GABAergic and glutamatergic transmission blocked, subtype-specific AChR agonists and antagonists were applied to identify direct regulation of GnRH neurons. ACh and nicotine caused a rise in calcium that declined gradually back to baseline after 5-6 min. This response was mimicked by an alpha3-specific agonist. In contrast, muscarine inhibited GnRH calcium oscillations, and blocking M2 and M4 together prevented this inhibition. Labeling for choline acetyltransferase (ChAT) and GnRH revealed ChAT fibers contacting GnRH neurons, primarily in the medial septum (MS), and in greater number in females than males. ChAT positive cells in the MS are known to express p75NGFRs. Labeling for p75NGFR, ChAT and GnRH indicated that ChAT fibers contacting GnRH cells originate from cholinergic cells within these same rostral areas. Together, these results indicate that cholinergic cells in septal areas can directly regulate GnRH neurons.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by absent pubertal development and infertility, often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2. POU6F2 encodes two distinct isoforms. In mouse, pituitary and gonads expressed both isoforms, but only isoform1 was detected in GnRH cells. Although the function of isoform2 is well known, using bioinformatics and cells assays on a human-derived GnRH cell line, we demonstrate isoform1 can also act as a transcriptional regulator, decreasing GNRH1 expression. The impact of two POU6F2 variants (MT1 and MT2) was then examined. MT1, but not MT2, reduced transcriptional activity of either isoform, preventing Hes5 promoter activation by isoform2 and repression of GnRH transcripts by isoform1. GnRH transcription increases as the cells migrate into the brain. Augmentation earlier can disrupt normal GnRH cell migration, consistent with POU6F2 variants contributing to IHH pathogenesis.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both LH and FSH from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on G_i/o protein coupling, while the D1/5R-mediated excitation required G_s protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons. Significance Statement The convergence of information on neurons secreting gonadotropin-releasing hormone (GnRH) shape their secretory profile and consequently, fertility. Dopamine inhibits GnRH neurons, yet the specific dopamine receptor subtype(s) involved remain unclear. Using single RT-PCR, immunofluorescence, and dopamine receptor-specific pharmacological tools, we show here that dopamine D4 receptor (D4R) plays a role in the dopaminergic inhibition of GnRH neurons, adding a new tool for the modulation of reproductive function.