Deciphering the functional role of MKRN3 in puberty and reproduction

描述(申请人提供)：下丘脑-垂体-性腺(HPG)轴调节青春期启动和生殖功能。这个轴在人类生命的胚胎和新生儿阶段是活跃的，但在童年时期被抑制，然后最终重新激活，首先被检测到促性腺激素释放激素(GnRH)脉冲的幅度和频率增加，以启动青春期。促性腺激素释放激素(GnRH)脉冲性释放的重新出现导致垂体促性腺激素、黄体生成素和卵泡的分泌增加 促性腺激素(FSH)，以及随之而来的性腺功能激活。青春期的时机受到遗传、营养、环境和社会经济因素的复杂交互作用的影响。HPG轴的早期激活导致促性腺激素依赖性或中枢性早熟(CPP)，临床上定义为女孩在8岁之前和男孩在9岁之前发展第二性征。青春期的时间与后续疾病的风险有关；女孩月经初潮年龄较早与乳腺癌、子宫内膜癌、肥胖症、2型糖尿病和心血管疾病的风险增加有关。人类青春期发育时间的遗传决定因素，特别是CPP，在很大程度上是未知的。我们最近利用整个外显子组测序，在CPP患者中发现了印记的MKRN3基因的功能缺失突变，该基因编码Makorin Ringinger Protein 3。Mkrn3在幼年小鼠弓状核中高表达，在青春期开始前显著降低。基于这些数据，我们假设MKRN3在休眠的青春期前阶段对GnRH的释放起到“刹车”的作用，这是第一个被发现具有这种抑制作用的肽。然而，人们对这种蛋白质的功能知之甚少。该项目的长期目标是阐明MKRN3控制青春期开始的时间的分子、细胞和生理机制，以及MKRN3突变导致中央生殖轴早期激活的机制。在第一个目标中，我们将在CPP患者中发现新的MKRN3缺陷，并扩大我们对MKRN3缺陷患者表型的理解。在第二个目标中，我们将通过一系列的表达和功能研究来确定MKRN3在活体中的繁殖作用。在第三个目标中，我们将通过在神经元细胞系中进行捕获蛋白质组学，以及比较具有和不具有MKRN3功能的人诱导多能干细胞来源的神经前体细胞的基因表达谱，在体外确定MKRN3的合作伙伴和/或靶点。这些目标的成功完成将有助于我们理解GnRH重新激活的新调节者MKRN3在神经内分泌控制青春期时机中的作用，并可能在神经元的分化、发育和成熟中发挥更广泛的作用。完全了解MRKN3在GnRH调节中的作用也可能发现参与生殖神经内分泌控制的新因素，并导致开发新的工具来管理生殖障碍。

DESCRIPTION (provided by applicant): The hypothalamic-pituitary-gonadal (HPG) axis regulates puberty initiation and reproductive function. This axis is active during the embryonic and neonatal stages of human life but then suppressed during childhood, then ultimately re-activated, first detected as an increase in amplitude and frequency of gonadotropin-releasing hormone (GnRH) pulses, to initiate puberty. The re-emergence of pulsatile GnRH release leads to increases in the secretion of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), and consequent activation of gonadal function. Pubertal timing is influenced by complex interactions of genetic, nutritional, environmental and socioeconomic factors. Early activation of the HPG axis results in gonadotropin- dependent or central precocious puberty (CPP), clinically defined by the development of secondary sexual characteristics before age 8 years in girls and 9 years in boys. The timing of puberty is associated with risks of subsequent disease; earlier age of menarche in girls is associated with increased risks of breast cancer, endometrial cancer, obesity, type 2 diabetes and cardiovascular disease. The genetic determinants of the timing of human pubertal development and in particular CPP are largely unknown. We recently identified loss- of-function mutations in the imprinted MKRN3 gene, encoding makorin ring finger protein 3, in patients with CPP, using whole exome sequencing. Mkrn3is highly expressed in the arcuate nucleus of juvenile mice, with a marked reduction prior to puberty onset. Based on these data, we hypothesize that MKRN3 acts as a "brake" for GnRH release during the dormant prepubertal phase, the first peptide identified to have such an inhibitory role. However, little is known about the function of this protein. The long-term goal of this project is to elucidate the molecular, cellular, and physiologic mechanisms by which MKRN3 controls the timing of puberty onset, and by which MKRN3 mutations result in early activation of the central reproductive axis. In the first aim, we will identify novel MKRN3 defects in patients with CPP and expand our understanding of the phenotype of patients with MKRN3 deficiency. In the second aim, we will determine the reproductive role of Mkrn3 in vivothrough a series of expression and functional studies in mice with and without functional Mkrn3. In the third aim, we will identify MKRN3 partners and/or targets in vitro by performing capture proteomics in a neuronal cell line as well as comparing gene expression profiles in neuronal progenitor cells derived from human induced pluripotent stem cells with and without functional MKRN3. The successful completion of these aims will help us to understand the actions of MKRN3, a novel regulator of GnRH re-activation, in the neuroendocrine control of pubertal timing and may also have a broader role in neuronal differentiation, development and maturation. A complete understanding of the role of MRKN3 in GnRH regulation may also identify novel factors involved in the neuroendocrine control of reproduction, and lead to the development of new tools for the management of reproductive disorders.