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

项目摘要 了解体重调节背后的分子生物学对于解决日益流行的 肥胖和治疗的迫切需要。初级纤毛是一种重要的细胞器， 真核细胞的信号传导和功能，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，与多种 影响几乎所有组织和器官系统的表型。某些纤毛病变，如Bardet-Biedl综合征 和Alström综合征，在儿童时期表现为肥胖。已知初级纤毛表达于 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稳态，导致肥胖。动物模型显示 在分泌阿黑皮素原（产生饱腹感的神经肽）的神经元中， 会导致暴食和肥胖。被鉴定为参与主要功能的基因的数量 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，一个已知的基因， 导致劳氏眼脑综合征（LS）的基因在纤毛中表达，可能在调节 磷酸肌醇-4-磷酸水平和纤毛中的运输。OCRL也高度表达于 下丘脑，特别是表达饱腹感神经肽、阿黑皮素原（POMC）的细胞，以及 生长激素释放激素我们发现一个有两个男性兄弟姐妹的家庭， OCRL的C-末端的未知突变，仅在脑特异性同种型中表达。先证者 具有严重肥胖和其他不同的临床特征，与LS中观察到的不同，但与 纤毛病变我们假设，这种功能丧失的变异局限于脑特异性亚型，导致了脑功能的丧失。 多种表型，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 独特的机会来研究大脑有限的影响功能丧失的OCRL。该提案旨在利用 这种表型，以建立OCRL作为纤毛基因参与体重调节的作用， 小鼠模型。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， 等位基因系列分化成表达POMC的下丘脑神经元，以评估 神经肽的突变我们还将使用AAV介导的RNA条件性敲除该基因 在LS特异性人源化小鼠模型中， 评估表型。这些研究将为不断增长的与体重有关的基因列表贡献一个新的基因 调节和纤毛功能。它还将扩大LS的表型，并可能提供途径， 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.