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代谢和神经元兴奋性之间的关系，并研究如何在疾病状态（如癫痫）中改变这种关系。我们的近期目标是了解关键代谢物ATP的区室化如何调节神经元的兴奋性。 能量代谢和ATP依赖过程对所有哺乳动物细胞都至关重要。一个长期存在且尚未解决的假设是ATP隔室存在于细胞质内。有证据表明，ATP区室化可能对许多细胞类型中的化学和电信号的调节至关重要，但这一假设存在争议。这一争议的解决将为我们对细胞内信号传导的基本理解提供重大进展，并对我们对糖尿病，缺血性损伤和癫痫等健康问题的理解产生影响。 特别是在神经元中，ATP的区室化可能是影响可塑性和膜兴奋性的关键因素。ATP区室化的发生可能是因为神经元的特殊几何结构，其神经突延伸远离细胞体。例如，高代谢要求和局部ATP消耗树突区室可能会影响突触可塑性。在另一种情况下，ATP在大量细胞质和质膜附近（“亚膜”区室）之间的受限扩散可能会影响兴奋性。Na，K-ATP酶是神经元中的主要能量消耗者，并且神经元活动后的泵激活可耗尽膜下ATP。神经元ATP敏感性钾通道（KATP channels）对膜下ATP和ADP敏感，通过负反馈回路控制兴奋性。虽然使用电生理学，生物化学和细胞生物学的实验支持ATP区室化的重要作用，但缺乏直接证据。为了直接研究ATP区室化，需要更好的光学工具来成像细胞内ATP。因此，在这次研究期间，我将研究神经元中的ATP区室化，有三个具体目标：（1）我将开发使用改进的遗传编码的比率荧光传感器成像神经元中ATP与ADP比率的方法，该传感器针对亚细胞位置。(2)我将研究ATP水平如何对神经元激活作出反应，以及ATP是否在树突内或在大量细胞质和膜下空间之间局部分隔。(3)我将研究ATP水平如何对燃料源的变化作出反应，以及燃料的选择是否会影响细胞体、膜下室和树突之间的ATP区室化。使用荧光显微镜来研究这些特定的目标，我将能够研究ATP区室化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.