Development And Regulation Of The Gonadotropin Releasing Hormone System

促性腺激素释放激素系统的发育和调节

• 批准号: 10688925
• 负责人: SUSAN WRAY
• 金额: $ 229.64万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688925
• 关键词: Address; Adult; Agonist; Animals; Anniversary; Axon; Biochemical; Bioinformatics; Biological Assay; Biological Models; Brain; CD100 antigen; Calcium; Calcium Oscillations; Cell Line; Cell Lineage; Cell model; Cells; Cellular Morphology; Communication; Coupling; Cues; DRD4 gene; Data; Destinations; Development; Dopamine; Dopamine Agonists; Dopamine Receptor; Electrophysiology (science); Etiology; Event; Exhibits; Failure; Family; Fiber; Frequencies; Functional disorder; GNRH1 gene; Gases; Gene Expression; Genes; Genetic; Genetic study; Glutamates; Goals; Gonadotropin Hormone Releasing Hormone; Heterogeneity; Human; Human Genetics; Idiopathic Hypogonadotropic Hypogonadism; Image; Impairment; In Vitro; International; Knockout Mice; Learning; Libraries; Link; Location; Measures; Mediating; Medical Genetics; Membrane; Modeling; Molecular; Monitor; Morphology; Movement; Mus; Mutate; Mutation; Neuroendocrine Cell; Neuroendocrinology; Neuronal Differentiation; Neurons; Neurosecretory Systems; Nose; Outcome; Paper; Patients; Pattern; Peptide Synthesis; Peptides; Phenotype; Play; Population; Process; Property; Prosencephalon; Proteins; Puberty; Publications; Publishing; Regulation; Reporting; Reproduction; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Route; Semaphorins; Signal Transduction; Synapses; Syndrome; System; Transcript; Transcriptional Regulation; Transgenic Animals; Tyrosine 3-Monooxygenase; Variant; Vertebrates; Visualization; Work; antagonist; base; cell motility; craniofacial development; differential expression; early embryonic stage; experimental study; farmer; hypothalamic pituitary gonadal axis; immunocytochemistry; in vitro Model; male; member; migration; molecular array; mutant; paracrine; patient screening; pituitary gonadal axis; plexin; postsynaptic neurons; prenatal; progenitor; rare genetic disorder; receptor; reproductive; reproductive function; screening; tool; transmission process

GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and two in vitro model systems nasal explants and GnRH derived cell lines. Using these animal and in vitro models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate, they also mature, and the two processes may in fact be linked. To investigate the maturation of GnRH neurons, we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of patients with reproductive dysfunction. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, four articles were published, 2 reviews and 2 primary articles. In vertebrates, Gonadotropin releasing hormone-1 (GnRH) neuroendocrine cells originate from two different lineages in the olfactory placode and migrate into the forebrain where they regulate reproduction. Three publications focused on the development of the GnRH system and one focused on regulation of GnRH neuronal activity relevant for reproductive function. Developmental articles: This paper (Welch B, Cho H-J, Ucakturk SA, Farmer, SM, Cetinkaya S, Abaci A, Akkus G, Simsek E, Kotan LD, Turan I, Yuksel B, Wray S, Topaloglu AK. PLXNB1 Mutations in the Etiology of Idiopathic Hypogonadotropic Hypogonadism. J. of Neuroendocrinology, 2022) examines mutations identified in human patients exhibiting delayed or absent puberty. Idiopathic hypogonadotropic hypogonadism (IHH) comprises a group of rare genetic disorders characterized by pubertal failure caused by GnRH deficiency. Genetic factors involved in semaphorin/plexin signaling have been identified in patients with IHH. PlexinB1, a member of the plexin family receptors, serves as the receptor for semaphorin 4D. In mice, perturbations in Sema4D/PlexinB1 signaling leads to improper GnRH development, highlighting the importance of investigating PlexinB1 mutations in IHH families. In total, 336 IHH patients from 290 independent families were included in the present study. Six PLXNB1 rare sequence variants (p.N361S, p.V608A, p.R636C, p.V672A, p.R1031H, and p.C1318R) are described in eight IHH patients from seven independent families. These variants were examined using bioinformatic modeling and compared to mutants reported in PLXNA1. Based on these analyses, the variant p.R1031H was assayed for alterations in cell morphology, PlexinB1 expression, and migration using a GnRH cell line and Boyden chambers. Experiments showed reduced membrane expression and impaired migration in cells expressing this variant compared to the wild-type. Our results provide clinical, genetic, molecular/cellular, and modeling evidence to implicate variants in PLXNB1 in the etiology of IHH. This review (Shan Y and Wray S. Hidden pitfalls in deciphering the gonadotropin releasing hormone neuroendocrine cell lineage. J. Neuroendocrinology, 2021) addresses the lineage of GnRH cells. To this day, the identity of GnRH progenitors remains unclear. However, the visualization of different developmental markers in subsets of GnRH neurons during early embryonic stages raised the possibility of at least two GnRH subpopulations. This observation led directly to a second question. Does visualization of different developmental markers in subsets of GnRH neurons reflect functional heterogeneity? This question remains unanswered, but as we learn more about the GnRH system, functional GnRH subpopulations becomes critically important to understanding GnRH function. This review addresses the development of the neuroendocrine GnRH system, specifically the heterogeneity of the GnRH neuroendocrine population. In this review (Duittoz A, Forni PE, Giacobini P, Golan M, Mollard P, Negrn AL, Radovick S, Wray S. Development of the Gonadotropin releasing hormone system. J. Neuroendocrinology, 2022), I assembled an international group of researchers working on the development of the GnRH system for a special issue celebrating the 50th anniversary of the discovery of GnRH. This review summarizes our current understanding of the development of the GnRH system, including discussion on open questions regarding (1) transcriptional regulation of the Gnrh1 gene; (2) prenatal development of the GnRH1 system in rodents and humans; and (3) paracrine and synaptic communication during migration of the GnRH cells. Regulation of GnRH neuronal activity: This paper (Dairaghi L, Constantin S, Oh A, Shostak D, Wray, S. The Dopamine D4 receptor regulates gonadotropin-releasing hormone neuron excitability in male mice. eNeuro, 2022)clarifies how dopamine regulated GnRH neuronal activity. While it was 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 Gai/o protein coupling, while the D1/5R-mediated excitation required Gas protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

GnRH 神经元对生殖至关重要，源自鼻基板并迁移到大脑中，成为下丘脑-垂体-性腺轴的组成部分。我们研究正常/转基因动物中 GnRH 神经元分化、迁移和轴突靶向的机制，以及两个体外模型系统鼻外植体和 GnRH 衍生细胞系。利用这些动物和体外模型，我们的工作还解决了 GnRH 神经元中（内在和跨突触）GnRH 基因表达、肽合成和分泌的调节机制。使用多种方法来识别和理解在引导 GnRH 神经元到达中枢神经系统最终位置方面发挥作用的多种分子和因子。这些包括对从迁移细胞和非迁移细胞获得的文库进行差异筛选，检查迁移路线上关键位置差异表达的分子，对基因敲除小鼠中 GnRH 系统发育的形态学检查，以及体外分子扰动和随后监测 GnRH 神经元运动。当 GnRH 神经元迁移时，它们也会成熟，这两个过程实际上可能是相关的。为了研究 GnRH 神经元的成熟，我们使用钙成像、电生理学和生化措施来检查 GnRH 神经元活性和肽分泌。此外，我们还与实验室合作对生殖功能障碍患者进行人类基因筛查。一旦发现突变，我们就会分析小鼠的表达模式并进行生物测定，以确定突变基因对 GnRH 发育的影响。 过去一年，发表了 4 篇文章，2 篇评论和 2 篇主要文章。在脊椎动物中，促性腺激素释放激素 1 (GnRH) 神经内分泌细胞起源于嗅基板中的两个不同谱系，并迁移到前脑，在那里调节繁殖。三份出版物重点关注 GnRH 系统的发展，一份重点关注与生殖功能相关的 GnRH 神经元活动的调节。 开发文章： 本文（Welch B、Cho H-J、Ucakturk SA、Farmer、SM、Cetinkaya S、Abaci A、Akkus G、Simsek E、Kotan LD、Turan I、Yuksel B、Wray S、Topaloglu AK。特发性低促性腺激素性性腺功能减退症病因中的 PLXNB1 突变。J. of Neuroendocrinology， 2022）检查了在青春期延迟或缺失的人类患者中发现的突变。特发性低促性腺激素性性腺功能减退症 (IHH) 包括一组罕见的遗传性疾病，其特征是 GnRH 缺乏引起的青春期发育迟缓。已在 IHH 患者中鉴定出参与 semaphorin/plexin 信号转导的遗传因素。 PlexinB1 是 plexin 家族受体的成员，是 semaphorin 4D 的受体。在小鼠中，Sema4D/PlexinB1 信号传导的扰动会导致 GnRH 发育不当，这凸显了研究 IHH 家族中 PlexinB1 突变的重要性。本研究总共纳入了来自 290 个独立家庭的 336 名 IHH 患者。来自 7 个独立家族的 8 名 IHH 患者中描述了 6 种 PLXNB1 罕见序列变异（p.N361S、p.V608A、p.R636C、p.V672A、p.R1031H 和 p.C1318R）。使用生物信息学模型检查这些变体，并与 PLXNA1 中报告的突变体进行比较。基于这些分析，使用 GnRH 细胞系和 Boyden 小室分析了变体 p.R1031H 的细胞形态、PlexinB1 表达和迁移的变化。实验表明，与野生型相比，表达该变体的细胞膜表达减少，迁移受损。我们的结果提供了临床、遗传、分子/细胞和建模证据，表明 PLXNB1 变异与 IHH 病因有关。 这篇综述（Shan Y 和 Wray S. 破译促性腺激素释放激素神经内分泌细胞谱系的隐藏陷阱。J. Neuroendocrinology，2021）讨论了 GnRH 细胞谱系。迄今为止，GnRH 祖细胞的身份仍不清楚。然而，早期胚胎阶段 GnRH 神经元亚群中不同发育标记的可视化提出了至少两个 GnRH 亚群的可能性。这一观察直接引出了第二个问题。 GnRH 神经元亚群中不同发育标志物的可视化是否反映了功能异质性？这个问题仍然没有答案，但随着我们对 GnRH 系统的了解越来越多，功能性 GnRH 亚群对于理解 GnRH 功能变得至关重要。这篇综述讨论了神经内分泌 GnRH 系统的发展，特别是异质性 GnRH 神经内分泌群体。 在这篇综述中（Duittoz A, Forni PE, Giacobini P, Golan M, Mollard P, Negrn AL, Radovick S, Wray S. 促性腺激素释放激素系统的发展。J. Neuroendocrinology, 2022），我召集了一个国际研究小组致力于 GnRH 系统的开发，以庆祝发现 50 周年特刊。 促性腺激素释放激素。这篇综述总结了我们目前对 GnRH 系统发展的理解，包括对以下开放性问题的讨论：（1）Gnrh1 基因的转录调控； (2) 啮齿动物和人类 GnRH1 系统的产前发育； (3) GnRH 细胞迁移过程中的旁分泌和突触通讯。 GnRH 神经元活动的调节： 本文（Dairaghi L、Constantin S、Oh A、Shostak D、Wray、S。多巴胺 D4 受体调节雄性小鼠促性腺激素释放激素神经元兴奋性。eNeuro，2022）阐明了多巴胺如何调节 GnRH 神经元活动。虽然已知多巴胺调节 GnRH 神经元，但所涉及的特定多巴胺受体亚型仍不清楚。先前对成年啮齿动物的研究报道了含有酪氨酸羟化酶（TH）（儿茶酚胺能细胞标记物）的纤维并置到 GnRH 神经元上，并且外源性多巴胺通过多巴胺 D1 样和/或 D2 样受体抑制突触后 GnRH 神经元。我们的 GnRH 神经元微阵列数据揭示了高水平的 Drd4 转录物，即多巴胺 D4 受体 (D4R)。单细胞 RT-PCR 和免疫细胞化学证实 GnRH 细胞分别表达 Drd4 转录物和蛋白质。钙成像发现，当 GABA 能和谷氨酸能传递被阻断时，应用亚型特异性多巴胺受体激动剂和拮抗剂期间 GnRH 神经元活性发生变化。多巴胺、多巴胺与D1/5R特异性或D2/3R特异性拮抗剂或D4R特异性激动剂可降低钙振荡频率。相反，D1/5R 特异性激动剂增加了钙振荡的频率。 D4R介导的抑制依赖于Gai/o蛋白偶联，而D1/5R介导的兴奋则需要Gas蛋白偶联。总之，这些结果表明 D4R 在 GnRH 神经元的多巴胺能抑制中发挥重要作用。