Neurochemistry/Physiology of Proopiomelanocortin Neurons

描述(申请人提供)：肥胖及其相关的代谢性疾病是世界范围内的主要健康问题。尽管肥胖率快速上升的原因是多方面的，但很明显，大脑中的神经回路在感知能量储存和调节能量平衡方面发挥着重要作用。这些神经通路是开发旨在逆转肥胖流行的新药物疗法的候选靶点，因此，我们有必要详细了解它们的功能，包括与其他神经系统的相互联系的完整图谱，以及它们对多种神经递质和细胞内信号通路的利用。这个项目的重点是大脑能量平衡回路的一个关键组成部分，即位于下丘脑和脑干的前阿片黑素皮质素(POMC)神经元。大脑POMC功能的基因缺失会导致严重的肥胖和代谢综合征，其特征是极度吞噬和基础代谢率降低。然而，POMC神经元在许多方面是不同的，越来越多的证据表明，不同的神经元亚群调节不同的神经过程，共同导致正常或病理的热量平衡控制。这个项目的总体目标是确定这些神经元亚群的特定功能以及它们所利用的神经解剖学和分子通路。具体目标1包括一系列行为和药理学研究，以探索POMC缺陷小鼠模型中导致过度吞噬的潜在成分、过程和神经底物。这些实验利用了一种新开发的进食模式分析方法，并将测试这一假设，即黑素皮质素信号协调调节摄食行为的刻板印象的运动、奖励和享乐方面；在肥胖的临床发病机制中，这些过程与围绕食物选择和食物大小的人类问题特别相关。具体目标2将进一步定义连接弓状POMC神经元和边缘前脑核团的神经回路。一种新的逆行示踪方法，包括将表达Cre重组酶的犬腺病毒载体定点微量注射到POMC基因等位基因可逆沉默的突变小鼠中，将用于将轴突侧支映射到特定的靶点组合。由POMC终末支配的边缘区域的二级神经元将通过跨突触标记和小麦胚凝集素在POMC细胞体中表达和顺行运输来识别。我们还将使用遗传技术和多标记免疫组织化学来研究从远端部位接受突触输入的POMC神经元出人意料的复杂树突。最后，在特定的目标3中，我们将使用互补的遗传方法来研究空间上不同的POMC神经元亚群或发育变化的POMC基因表达在预防或减轻肥胖中的独特功能作用。涉及的技术包括聚集嵌合体形成和Cre重组酶介导的POMC表达的重新激活，这些表达来自神经元特异性的可逆地沉默的POMC等位基因。公共卫生相关性：当前对公共健康的最大威胁之一是超重、肥胖、糖尿病和代谢综合征的比率持续上升。一套复杂的神经回路将卡路里需求和利用之间的平衡与与进食相关的行为和心理过程结合在一起。该项目以大脑喂养回路的关键组成部分--丙黑素皮质素神经元为中心，解释这些神经元如何协调调节食欲、进餐开始和结束以及代谢率，以正常将体重维持在严格控制的范围内。

DESCRIPTION (provided by applicant): Obesity and its associated metabolic diseases are major health problems world-wide. Although the reasons for the rapid increase in rates of obesity are multifactorial, it is clear that neural circuits in the brain play a major role in sensing energy stores and regulating energy balance. These neural pathways are candidate targets for the development of new pharmacotherapies aimed at reversing the obesity epidemic and therefore it is essential that we understand their function in detail, including the complete map of interconnections with other neural systems and their utilization of multiple neurotransmitters and intracellular signaling pathways. This project focuses on a key component of the brain's energy balance circuitry, the proopiomelanocortin (POMC) neurons located in the hypothalamus and brainstem. Genetic deletion of POMC function from the brain results in profound obesity and a metabolic syndrome characterized by extreme hyperphagia and reduced basal metabolic rate. However, POMC neurons are heterogeneous in many aspects and accumulating evidence suggests that different subpopulations of the neurons regulate separate neurological processes that together result in normal or pathological control of caloric balance. The overall goals of this project are to identify specific functions of these neuronal subpopulations and the neuroanatomic and molecular pathways that they utilize. Specific aim 1 includes a series of behavioral and pharmacological studies to probe the underlying component processes and neural substrates contributing to hyperphagia in POMC-deficient mouse models. These experiments utilize a newly developed method for meal pattern analysis and will test the hypothesis that melanocortin signaling coordinately modulates stereotyped motor, reward, and hedonic aspects of feeding behavior; processes which are particularly relevant to human issues surrounding food choice and meal size in the clinical pathogenesis of obesity. Specific aim 2 will further define the neurocircuitry connecting arcuate POMC neurons to limbic forebrain nuclei. A novel retrograde tracing method involving site-specific microinjections of a canine adenoviral vector expressing Cre recombinase into mutant mice with a reversibly silenced POMC gene allele will be used to map axon collaterals to specific combinations of target sites. Second order neurons in limbic areas innervated by POMC terminals will be identified by trans-synaptic labeling with wheat germ agglutinin expressed in, and transported anterogradely from, POMC cell bodies. We will also use genetic techniques and multilabel immunohistochemistry to study the unexpectedly complex dendrites of POMC neurons that receive synaptic inputs from distal sites. Finally, in Specific aim 3 we will use complementary genetic approaches to study the unique functional role of spatially distinct POMC neuron subpopulations or developmentally altered POMC gene expression in the prevention or mitigation of obesity. The techniques involved are aggregation chimera formation and Cre recombinase-mediated reactivation of POMC expression from a neuron-specific and reversibly silenced POMC allele. PUBLIC HEALTH RELEVANCE: Among the greatest current threats to public health are the continually increasing rates of overweight, obesity, diabetes, and the metabolic syndrome. A complex set of neural circuits integrates the balance between caloric demand and utilization with the behavioral and psychological processes related to feeding. This project centers on a key component of the brain's feeding circuits, propiomelanocortin neurons, to explain how these neurons coordinately regulate appetite, meal initiation and termination, and metabolic rate to normally maintain body weight within tightly controlled limits.