The Intracellular Dynamics of AGRP Neurons under Different Metabolic Conditions

 描述(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的一种基本的体内平衡功能。能量代谢的中断可能会导致危及生命的情况，包括肥胖和糖尿病等慢性代谢障碍。了解控制能量代谢的调节原理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，并在食物缺乏时被激活。矛盾的是，我们发现，在饮食诱导的肥胖小鼠中，AGRP神经元的活性也增加了。我们最近发现，当小鼠从负能量平衡转换到正能量平衡(即从食物剥夺到高脂肪喂养)时，AGRP神经元中的线粒体发生融合。当我们阻止高脂饮食小鼠AGRP神经元的线粒体融合(通过击倒Mfn2)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产生抵抗力。由于禁食和高脂喂养的小鼠AGRP神经元的活性都很高，我们推测在这两种情况下AGRP神经元的活性是由不同的机制支持的。这一点从食物剥夺期间AGRP神经元线粒体的分裂状态以及高脂肪喂养的小鼠的融合状态中得到了说明。这项应用的目的是提供对AGRP神经元适应不同代谢条件所涉及的生物学复杂性的机械性洞察。在目标1中，我们将使用AGRP神经元的细胞特异性核糖体图谱与RNA测序相结合的方法来识别AGRP神经元翻译景观的变化。在子目标1.1中，我们将描述参与AGRP神经元功能的核糖体相关转录组(翻译组)在食物剥夺、喂养和高脂饮食喂养的小鼠中的特征。在子目标1.2中，我们将描述在饮食诱导肥胖的AGRP-Mfn2KO小鼠中，在没有线粒体融合的情况下，AGRP神经元的翻译组是如何被修改的。这些实验将确定可能的细胞内机制，使AGRP神经元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受细胞的电活动控制。我们将使用多方面的方法，利用转基因和AAV介导的小鼠模型，选择性地和尖锐地激活/抑制AgRP神经元，目的是通过电子显微镜分析来识别线粒体的动态形态变化。这项提议将为新陈代谢的中央调控提供新的见解，并提供新的候选药物作为肥胖症和相关代谢紊乱的药物靶点。

 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.