The role of tanycyte cilia in hypothalamic neurogenesis and glucose sensing

 描述（由申请人提供）：肥胖困扰着三分之一的美国人，是全球死亡和残疾的主要原因。目前的治疗方法并没有从根本上解除对能量平衡的调节，并且在很大程度上未能遏制正在进行的流行病。更好地了解下丘脑对能量平衡的控制，应该会导致更好的治疗。虽然大量的研究表明下丘脑葡萄糖反应神经元可以刺激或抑制进食，但对调节能量平衡回路的特化下丘脑神经胶质细胞（称为伸长细胞）知之甚少。延伸细胞排列在第三脑室腹侧壁，独特地位于下丘脑和脑脊液之间。它们将长的过程投射到下丘脑中，与饮食反应神经元形成广泛的接触，调节食欲神经元的活动，并且是出生后添加到该回路的新神经元的神经源性祖细胞。实验性增强或消除啮齿类动物的伸长细胞源性下丘脑神经发生影响体重增加。然而，神经源性伸长细胞的确切身份尚不清楚，调节伸长细胞增殖活性的内源性信号传导机制也不清楚。使用一种成像技术，开发了一个面向分析的细胞内衬脑室，在小鼠和人类的初步研究揭示了两个分离的亚群的tanycytes-bi-cilimated tanycytes与两个长纤毛和uni-cilimated tanycytes与一个单一的短初级纤毛。有趣的是，发现胰腺β细胞中表达的葡萄糖敏感分子定位于伸展细胞初级纤毛，这表明这些细胞用于检测能量平衡的可能反馈机制。初步实验表明，消融tanycell纤毛导致糖尿病样表型。伸展细胞亚群也显示出不同的分子标记表达，允许构建两种表达Cre的腺病毒，以特异性靶向和谱系追踪两个群体。在目的1中，本研究将采用立体定向第三脑室注射Cre腺病毒到Cre报告小鼠中以确定神经源性伸长细胞的身份。在目标2中，相同的注射将用于消融具有纤毛发生所需的Kif 3a的floxed等位基因的条件性敲除小鼠中的纤毛。然后，这些小鼠将接受一系列代谢测试，包括测量食物摄入和能量消耗，有或没有额外的代谢挑战，包括饮食诱导的肥胖和葡萄糖缺乏状态。这项工作将为深入了解伸展细胞亚型、伸展细胞源性下丘脑神经发生以及伸展细胞纤毛在葡萄糖敏感和能量平衡中的功能提供帮助。从治疗的角度来看，这些结果可能会促进未来的药理学实验，靶向tanycell纤毛与脑室内治疗，以调节能量不平衡的状态。

 DESCRIPTION (provided by applicant): Obesity afflicts one in three Americans and is a leading cause of death and disability worldwide. Current therapies do not address obesity as fundamentally deregulated energy balance and have largely failed to curtail the ongoing epidemic. Better understanding of hypothalamic control over energy balance should lead to improved therapy. While extensive work has shown that hypothalamic glucose-responsive neurons can stimulate or suppress feeding, less is known about specialized hypothalamic glial cells called tanycytes that modulate the energy balance circuit. Tanycytes line the ventral third ventricle wall and are uniquely positioned between the hypothalamus and cerebrospinal fluid. They project long processes into the hypothalamus that form extensive contacts with the diet-responsive neurons, modulate the activity of orexigenic neurons, and are neurogenic progenitors for new neurons added to this circuit postnatally. Experimentally enhancing or ablating tanycyte-derived hypothalamic neurogenesis in rodents affects weight gain. However, the precise identity of neurogenic tanycytes is unclear and endogenous signaling mechanisms that regulate tanycyte germinal activity are unknown. Using an imaging technique developed for en-face analysis of cells lining the cerebral ventricles, preliminary studies in mice and humans revealed two segregated subsets of tanycytes-bi-ciliated tanycytes with two long cilia and uni-ciliated tanycytes with a single short primary cilium. Intriguingly, glucose-sensing molecules expressed in pancreatic beta cells were found localized to tanycyte primary cilia, suggesting a possible feedback mechanism used by these cells to detect energy balance. Preliminary experiments showed that ablating tanycyte cilia resulted in a diabetic-like phenotype. Tanycyte subsets also displayed distinct molecular marker expression that permitted construction of two Cre-expressing adenoviruses to specifically target and lineage-trace the two populations. In Aim 1, this study will employ stereotactic 3rd ventricular injections of the Cre adenoviruses into Cre reporter mice to determine the identity of neurogenic tanycytes. In Aim 2, the same injections will be used to ablate cilia in conditional knockout mice with floxed alleles of Kif3a, required fo ciliogenesis. These mice will then be subject to a battery of metabolic tests including measurements of food intake and energy expenditure with or without additional metabolic challenges including diet-induced obesity and glucoprivic states. The proposed work will provide deeper understanding of tanycyte subtypes, tanycyte-derived hypothalamic neurogenesis, and the function of tanycyte cilia in glucose-sensing and energy balance. From a therapeutic perspective, these results may foster future pharmacological experiments targeting tanycyte cilia with intraventricular therapies to modulate states of energy imbalance.