Deciphering the functional role of MKRN3 in puberty and reproduction

描述（由申请方提供）：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，但在儿童期受到抑制，然后最终重新激活，首先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。脉冲性GnRH释放的重新出现导致垂体促性腺激素、促黄体生成素（LH）和卵泡分泌的增加， 刺激激素（FSH），以及随之而来的性腺功能激活。青春期的时间受到遗传、营养、环境和社会经济因素复杂相互作用的影响。HPG轴的早期激活导致促性腺激素依赖性或中枢性性早熟（CPP），其临床定义为女孩在8岁之前和男孩在9岁之前第二性征的发育。青春期的时间与随后疾病的风险有关;女孩初潮年龄较早与乳腺癌、子宫内膜癌、肥胖、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。我们最近使用全外显子组测序在CPP患者中鉴定了编码Makorin环指蛋白3的印迹MKRN 3基因的功能缺失突变。mkrn 3在幼年小鼠的弓状核中高度表达，在青春期开始之前显著降低。基于这些数据，我们假设MKRN 3在休眠的青春期前阶段作为GnRH释放的“制动器”，这是第一种被鉴定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MKRN 3控制青春期开始时间的分子，细胞和生理机制，以及MKRN 3突变导致中央生殖轴的早期激活。在第一个目标中，我们将在CPP患者中鉴定新的MKRN 3缺陷，并扩大我们对MKRN 3缺陷患者表型的理解。在第二个目标中，我们将通过在有和没有功能性Mkrn 3的小鼠中的一系列表达和功能研究来确定Mkrn 3在体内的生殖作用。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来自具有和不具有功能性MKRN 3的人诱导多能干细胞的神经元祖细胞中的基因表达谱来体外鉴定MKRN 3伴侣和/或靶标。这些目标的成功完成将有助于我们了解MKRN 3（一种新的GnRH再激活调节剂）在青春期时间神经内分泌控制中的作用，也可能在神经元分化，发育和成熟中发挥更广泛的作用。对MRKN 3在GnRH调节中的作用的全面了解也可能确定参与生殖神经内分泌控制的新因素，并导致开发用于管理生殖疾病的新工具。

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.