Compartmentation of Neuronal ATP and Metabolic Regulation of Excitability

描述(由申请人提供)：我们的长期目标是了解新陈代谢和神经元兴奋性之间的关系，并研究这种关系在疾病状态下如何改变，如癫痫。我们的直接目标是了解关键代谢物三磷酸腺苷的区隔如何调节神经元的兴奋性。能量代谢和ATP依赖的过程对所有哺乳动物细胞都是至关重要的。一个长期存在但仍未解决的假说是，细胞质中存在三磷酸腺苷隔室。有证据表明，在许多类型的细胞中，ATP区隔对化学和电信号的调节可能是关键的，但这一假说是有争议的。这一争议的解决将为我们对细胞内信号的基本理解提供重大进展，并对我们理解糖尿病、缺血性损伤和癫痫等健康问题具有重要意义。在神经元中，三磷酸腺苷的区隔作用可能是影响可塑性和膜兴奋性的关键因素。ATP区隔的发生可能是因为神经元的特殊几何形状，其神经突延伸到远离细胞体的地方。例如，高代谢需求和树突间的局部ATP消耗可能会影响突触的可塑性。在另一种情况下，ATP在大块细胞质和靠近质膜(“亚膜”隔室)之间的有限扩散可能会影响兴奋性。Na，K-ATPase是神经元的主要能量消耗，神经元活动后泵的激活可能会耗尽细胞膜下的ATP。神经元型ATP敏感性钾通道(KATP通道)对膜下ATP和ADP敏感，可通过负反馈环控制兴奋性。尽管使用电生理学、生物化学和细胞生物学的实验支持了ATP区隔的重要作用，但缺乏直接的证据。为了直接研究三磷酸腺苷的分区，需要更好的光学工具来成像细胞内的三磷酸腺苷。因此，在这次研究中，我将以三个具体目标来研究神经元中的ATP区隔：(1)我将开发一种方法，使用一种针对亚细胞位置的改进的遗传编码的比率荧光传感器来成像神经元中的ATP/ADP比率。(2)我将研究ATP水平如何响应神经元的激活，以及ATP是在树突内局部划分，还是在胞浆和膜下间隙之间局部划分。(3)我将研究ATP水平如何响应燃料来源的变化，以及燃料的选择是否会影响细胞体、亚膜室和树突之间的ATP区隔。使用荧光显微镜来研究这些特定的目标，我将能够研究ATP区隔是如何作为调节神经元兴奋性的关键参数。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the relationship between metabolism and neuronal excitability and to investigate how this relationship can be altered in diseased states such as in epilepsy. Our immediate goal is to understand how compartmentation of the key metabolite ATP can modulate neuronal excitability. Energy metabolism and ATP-dependent processes are vital to all mammalian cells. A long-standing and still unresolved hypothesis is that ATP compartments exist within the cytoplasm. Evidence suggests that ATP compartmentation could be critical to the regulation of chemical and electrical signaling in many cell types, but this hypothesis is controversial. Resolution of this controversy would provide a significant advance in our basic understanding of intracellular signaling, and it has implications for our understanding of health problems such as diabetes, ischemic injuries, and epilepsy. In neurons specifically, compartmentation of ATP could be a critical factor affecting plasticity and membrane excitability. ATP compartmentation may occur because of the specialized geometry of neurons whose neurites extend far from the cell body. For example, high metabolic requirements and local ATP consumption in dendritic compartments may affect synaptic plasticity. In another scenario, restricted diffusion of ATP between the bulk cytoplasm and near the plasma membrane (the "submembrane" compartment) may impact excitability. The Na,K-ATPase is a major energy consumer in neurons, and pump activation following neuronal activity may deplete submembrane ATP. Neuronal ATP-sensitive potassium channels (KATP channels) are sensitive to submembrane ATP and ADP and could control excitability through a negative feedback loop. Although experiments using electrophysiology, biochemistry, and cell biology support an important role for ATP compartmentation, there is a lack of direct evidence. To directly investigate ATP compartmentation, better optical tools are needed for imaging intracellular ATP. Therefore, during this fellowship I will investigate ATP compartmentation in neurons with three specific aims: (1) I will develop methods for imaging the ATP-to-ADP ratio in neurons using an improved genetically-encoded, ratiometric fluorescent sensor that is targeted to subcellular locations. (2) I will investigate how ATP levels respond to neuronal activation and whether ATP is compartmented locally within dendrites or between the bulk cytoplasm and a submembrane space. (3) I will investigate how ATP levels respond to a change in fuel source and whether choice of fuel affects ATP compartmentation between the cell body, submembrane compartment, and dendrites. Using fluorescence microscopy to investigate these specific aims, I will be able to study how ATP compartmentation acts as a critical parameter in modulating neuronal excitability.