Establishing the role of OCRL as a novel ciliary gene in weight regulation in human and murine models

项目总结 了解体重调节背后的分子生物学对于解决日益流行的 肥胖症和迫切需要治疗。初级纤毛被认为是 真核细胞的信号和功能，包括参与能量稳态的细胞。睫状体功能障碍 导致几种人类综合征，统称为纤毛病，与不同的 几乎影响到每个组织和器官系统的表型。一些纤毛疾病，如Bardet-Biedl综合征 以及阿尔斯特伦综合征，表现为儿童期肥胖。已知初级纤毛表达在 下丘脑的体重调节中心。这些初级纤毛的功能丧失会导致 神经内分泌信号通路参与能量动态平衡，导致肥胖。动物模型秀 分泌前阿片黑素皮质素的神经元中纤毛基因(S)的破坏，产生饱腹感的神经肽， 会导致吞噬过多和肥胖。已确定的与初级基因功能有关的基因数量 纤毛随着时间的推移而增加。最近的证据表明，肌醇磷酸酶OCRL是一种已知的 Lowe眼脑综合征(LS)，表达于纤毛，可能在调节 肌醇-4-磷酸的水平和纤毛的贩运。OCRL也高表达于 下丘脑，特别是表达饱腹感神经肽的细胞，前阿片黑素皮质素(POMC)，以及 生长激素释放激素。我们已经确认了一个有两个男性兄弟姐妹的家庭， OCRL C末端的未知突变，仅在大脑特定的亚型中表达。先证者 有严重肥胖和其他不同的临床特征，不同于LS，但与 纤毛病。我们假设，这种仅限于大脑特定异构体的功能变异的丧失导致了 表型多样，包括严重肥胖，但不影响其他体细胞组织。因此，它提供了一个 这是研究OCRL功能丧失对大脑有限影响的独特机会。这项提案试图利用 这种表型确定OCRL作为一种纤毛基因在人类和 老鼠模型。我们将使用我们的患者特异性诱导多能干细胞系，它们的同基因对照和 等位基因序列诱导表达POMC对下丘脑弓状神经元分化的影响 神经肽的突变。我们还将使用AAV介导的RNA有条件地敲除该基因 LS特异性人源化小鼠模型对弓状核和室旁核的干扰 评估表型。这些研究将为越来越多的与体重有关的基因名单贡献一个新的基因 调节和睫毛功能。它还将扩展LS的表型，并可能提供 探索未来的治疗方法。

PROJECT SUMMARY Understanding the molecular biology behind weight regulation is imperative to address the growing epidemic of obesity and the urgent need for therapies. The primary cilium has been identified as an important organelle for signaling and function of eukaryotic cells including the cells involved in energy homeostasis. Ciliary dysfunction results in several human syndromes, collectively called ciliopathies, that are associated with diverse phenotypes affecting nearly every tissue and organ system. Some ciliopathies, such as Bardet-Biedl syndrome and Alström syndrome, present with obesity in childhood. Primary cilia are known to be expressed in the weight regulation centers of the hypothalamus. Loss of function in these primary cilia causes disruption in the neuroendocrine signaling pathways involved in energy homeostasis, resulting in obesity. Animal models show that disruption of ciliary gene(s) in neurons secreting pro-opiomelanocortin, the satiety producing neuropeptide, causes hyperphagia and obesity. The number of genes identified to be involved in the function of the primary cilium have increased over time. Recent evidence shows that the inositol phosphatase OCRL, a gene known to cause Oculocerebral syndrome of Lowe (LS), is expressed in the cilia, and may have a role in regulating the levels of phosphoinositol-4-phosphate and trafficking in the cilia. OCRL is also highly expressed in the hypothalamus, especially the cells expressing the satiety neuropeptide, pro-opiomelanocortin (POMC), and growth hormone releasing hormone. We have identified a family of two male siblings with a previously unknown mutation in the C-terminus of OCRL that is only expressed in the brain specific isoform. The proband has severe obesity and other diverse clinical features, different from those seen in LS, but overlapping with ciliopathy. We hypothesize that this loss of function variant limited to the brain-specific isoform causes a diverse phenotype including severe obesity, but does not affect the other somatic tissues. Thus, it provides a unique opportunity to study the brain limited impact of the loss of function of OCRL. This proposal seeks to use this phenotype to establish the role of OCRL as a ciliary gene involved in weight regulation in human and mouse models. We will use our patient-specific induced pluripotent stem cell line, their isogenic control and allelic series to differentiate into arcuate-like hypothalamic neurons expressing POMC to assess the impact of the mutation on the neuropeptide. We will also use conditional knockout of the gene by AAV-mediated RNA interference in the arcuate and paraventricular nuclei of the brain in LS-specific humanized mouse model to assess the phenotype. These studies will contribute a new gene to the growing list of genes involved in weight regulation and ciliary function. It will also expand the phenotype for LS and potentially provide avenues to explore therapies in future.