The Intracellular Dynamics of AGRP Neurons under Different Metabolic Conditions

 描述（由申请人提供）：能量代谢的维持是从人类到简单细胞的所有生物体中发现的基本稳态功能。能量代谢的中断可导致危及生命的状况，包括慢性代谢紊乱，如肥胖和糖尿病。了解控制能量代谢的调节原则对于帮助设计更好的代谢紊乱治疗方法至关重要。下丘脑中的AGRP神经元参与能量代谢的调节，并且在食物剥夺期间被激活。奇怪的是，我们发现AGRP神经元活性在饮食诱导的肥胖小鼠中也升高。我们最近发现，当小鼠从负能量平衡切换到正能量平衡时，AGRP神经元中的线粒体发生融合（即，从食物剥夺到高脂肪喂养）。当我们在喂食高脂饮食的小鼠中阻断AGRP神经元中的线粒体融合（通过敲低Mfn 2）时，由于细胞内ATP水平降低，AGRP神经元活性降低，小鼠对饮食诱导的肥胖具有抵抗力。因为在食物剥夺和高脂肪饮食喂养的小鼠中，AGRP神经元的活性都很高，我们假设AGRP神经元的活性在这两种情况下受到不同机制的支持。这是由AGRP神经元中的线粒体在食物剥夺期间的分裂状态和在高脂肪喂养的小鼠中的融合状态所说明的。本申请的目的是提供机械洞察的复杂性，生物学参与AGRP神经元的适应不同的代谢条件。在目标1中，我们将使用AGRP神经元的细胞特异性核糖体分析结合RNA测序来确定AGRP神经元翻译景观的变化。在子目标1.1中，我们将描述在食物剥夺、进食和高脂饮食喂养的小鼠中参与AGRP神经元功能的核糖体相关转录组（翻译组）。在子目标1.2中，我们将描述AGRP-Mfn 2KO小鼠在饮食诱导的肥胖期间，在不存在线粒体融合的情况下，AGRP神经元的翻译组如何被修饰。这些实验将确定假定的细胞内机制，使AGRP神经元，以适应不断变化的代谢环境。在目标2中，我们将解决一个非常重要的机制问题，即AGRP神经元中的线粒体动力学是否由细胞的电活动控制。我们将使用多方面的方法来选择性地和急性激活/抑制Agrp神经元，利用转基因和AAV介导的小鼠模型，通过电子显微镜分析确定线粒体中的动态形态学变化的目标。该提案将为代谢的中枢调节提供新的见解，并为肥胖和相关代谢紊乱的药物靶点提供新的候选人。

 DESCRIPTION (provided by applicant): The maintenance of energy metabolism is a fundamental homeostatic function found in all organisms from humans to simple cells. Disruption of energy metabolism can lead to life-threatening conditions, including chronic metabolic disorders such as obesity and diabetes. Understanding the regulatory principles that control energy metabolism is of the utmost importance in helping to design better treatments for metabolic disorders. AGRP neurons in the hypothalamus participate in the regulation of energy metabolism and are activated during times of food deprivation. Paradoxically, we showed that AGRP neuronal activity is also elevated in diet- induced obese mice. We have recently found that mitochondria in AGRP neurons undergo fusion when mice switch from negative to positive energy balance (i.e., from food deprived to high-fat fed). When we blocked mitochondria fusion in AGRP neurons (by knocking down Mfn2) in mice fed a high-fat diet, AGRP neuron activity decreased due to reduced intracellular levels of ATP, and the mice became resistant to diet-induced obesity. Because in both food deprived and high-fat diet fed mice the activity of AGRP neurons is high, we hypothesize that AGRP neuron activity is supported by different mechanisms in these two conditions. This is illustrated by the fission state of mitochondria in AGRP neurons during food deprivation, and the fused state in high-fat fed mice. The goal of this application is to provide mechanistic insight into the complexity of the biology involved in the adaptations of AGRP neurons to different metabolic conditions. In Aim 1, we will use cell-specific ribosome profiling of AGRP neurons combined with RNA-sequencing to identify changes in the translational landscape of AGRP neurons. In Sub-Aim 1.1 we will characterize the ribosome-associated transcriptome (translatome) involved in AGRP neuron function in food deprived, fed and high-fat diet fed mice. In Sub-Aim 1.2 we will characterize how the translatome of AGRP neurons is modified in the absence of mitochondria fusion during diet-induced obesity in AGRP-Mfn2KO mice. These experiments will identify the putative intracellular mechanisms that allow AGRP neurons to adapt to the changing metabolic milieu. In Aim 2, we will tackle a very important mechanistic question that is whether mitochondrial dynamics in AGRP neurons is controlled by the electrical activity of the cells. We will use a multi-faceted approach to selectively and acutely activate/inhibit Agrp neurons utilizing transgenic and AAV-mediated mouse models with the goal of identifying dynamic morphological changes in mitochondria through electron microscopic analyses. This proposal will deliver novel insights into the central regulation of metabolism and offer new candidates to pursue as drug targets for obesity and related metabolic disorders.