Exploration of the role of CART/GPR160 in metabolism in the setting of Magel2 deficiency: Implications for Prader Willi Syndrome

项目摘要 严重的摄食过多是Prader Willi综合征（PWS）的一个标志。多个神经回路已经被 与PWS相关的食欲亢进有关;然而，食欲神经调节的复杂性 混淆了PWS环境中摄食回路失调的确切机制。的 神经肽可卡因和安非他明调节转录物（CART）是食欲和体重的关键调节剂。 中枢输注CART可降低肥胖大鼠的摄食量和体重增加，而CART的整体缺失可降低肥胖大鼠的体重增加。 小鼠导致肥胖的易感性和对高脂肪饮食诱导的代谢功能障碍的易感性增强。 同样，CART基因的突变也与人类肥胖有关。有趣的是，下丘脑水平 在PWS动物模型中，CART肽的含量减少。尽管有大量证据表明， CART在代谢中的重要作用，CART在PWS模型中减少食物摄入的能力还没有被证实。 报道这种知识的缺乏是PWS领域的一个主要差距。重要的是，我们最近发现， CART的同源受体作为G蛋白偶联受体GPR 160，从而使化学生物学方法成为可能 设计CART激动剂用于治疗PWS相关的食欲过盛。我们假设中央 注射CART将减少父系遗传的Magel 2缺乏症的食物摄入， 缺乏将导致CART在中枢摄食中心，特别是下丘脑中的表达减少。我们 将在两个具体目标中阐述我们的假设。在目标1中，我们将CART icv注射到Magel 2缺陷大鼠中， 食物和水的摄入量和膳食模式将使用我们的BioDAQ系统进行测量。然后将大鼠置于 在旷场行为测试中，以确保CART对食物摄入的任何观察到的影响是由于主要的 CART对食欲的影响，而不是对运动行为的次要影响。虽然proCART mRNA 水平可能不会在PWS中调节，PWS是负责产生成熟激素的激素原转化酶。 CART，PSCK 1，已被证明是减少在个人与PWS，这导致减少在 成熟CART肽的产生。因此，在目标2中，我们将测量CART、GPR 160和PSCK 1水平， 与WT大鼠相比，Magel 2缺陷大鼠的脑、CSF和血浆。因为CART肽水平波动 根据代谢状态（即禁食期间减少，重新进食后恢复正常），我们将确定功能性 使用整体蛋白质组学分析，在Mage 12缺陷的情况下发生CART肽水平的变化。 最后，我们将确定是否有CART和GPR 160在大脑中的表达模式的变化， 与WT大鼠相比，Magel 2缺陷大鼠。如果我们的假设是正确的， 至少可以部分控制PWS患者的食欲。这种针对一个系统的策略， 平行于，而不是依赖于其他喂养电路可以彻底改变治疗PWS相关的 这是一种治疗食欲过盛的方法，并为PWS患者经历的严重肥胖提供了创新的解决方案。

PROJECT SUMMARY Severe hyperphagia is a hallmark of Prader Willi Syndrome (PWS). Multiple neuronal circuits have been implicated in PWS-associated hyperphagia; however the complexity of the neural regulation of appetite has obfuscated the precise mechanisms underlying the dysregulation of feeding circuits in the setting of PWS. The neuropeptide cocaine and amphetamine regulated transcript (CART) is a key regulator of appetite and weight. Central infusion of CART decreased food intake and weight gain in obese rats, and global deletion of CART in mice led to predisposition to obesity and enhanced susceptibility to high fat diet-induced metabolic dysfunction. Likewise, mutations in the CART gene have been linked to obesity in humans. Interestingly, hypothalamic levels of CART peptide were reduced in an animal model of PWS. In spite of a wealth of evidence indicating an important role of CART in metabolism, the ability of CART to reduce food intake in models of PWS has not been reported. This lack of knowledge represents a major gap in the PWS field. Importantly, we recently identified the cognate receptor of CART as the G protein coupled receptor, GPR160, thus enabling chemical biology methods to design CART agonists for the treatment of PWS-associated hyperphagia. We hypothesize that central injection of CART will reduce food intake in the setting of paternally-inherited Magel2 deficiency, and that Magel2 deficiency will lead to reduced expression of CART in central feeding centers, particularly the hypothalamus. We will address our hypothesis in two Specific Aims. In Aim 1, we will inject CART icv into Magel2-deficient rats, and food and water intakes and meal patterning will be measured using our BioDAQ system. Rats will then be placed in an open field behavioral test to ensure that any observed effect of CART on food intake is due to a primary effect of CART on appetite, rather than a secondary effect on locomotor behavior. Although proCART mRNA levels likely are not modulated in PWS, the prohormone convertase responsible for the production of mature CART, PSCK1, has been shown to be reduced in individuals with PWS, which leads to reductions in the production of mature CART peptide. In Aim 2, we therefore will measure CART, GPR160, and PSCK1 levels in brains, CSF, and plasma of Magel2-deficient rats compared to WT rats. Because CART peptide levels fluctuate with metabolic state (i.e. reduced during fasting, normalized after re-feeding), we will determine if functional changes in CART peptide levels occur in the setting of Magel2 deficiency using global proteomic analyses. Lastly, we will determine if there are changes in the expression patterns of CART and GPR160 in the brains of Magel2-deficient rats compared to WT rats. If our hypotheses are correct, then replacement with CART agonists should at least partially control appetite in individuals with PWS. This strategy of targeting a system that is parallel to, and not dependent upon, other feeding circuits could revolutionize treatment of PWS-associated hyperphagia and offer an innovative solution to the severe obesity experienced by patients with PWS.