Development of a Synaptic ATP Reporter

描述（由申请人提供）：突触代表关键的转导机器，将动作电位形式的传入电信息转换为分泌的化学信息，而化学信息又转换回突触后电反应。这些事件的协调被认为是学习和记忆的关键机制的基础，突触通信功能障碍被怀疑是许多脑功能病变状态的核心。然而，突触位于离细胞体很远的地方，高度定位的细胞生物机制必须依赖于ATP的局部来源才能发挥作用。除了存在于突触前和突触后膜上的离子泵，这些离子泵在活动后恢复离子平衡，两个隔室都含有高浓度的蛋白质，这些蛋白质必须消耗ATP，以执行信号转导的作用，以及向质膜传递和提取蛋白质和脂质的关键膜运输事件。由于这些高能量需求，很大一部分神经末梢和突触后树突被赋予了局部线粒体。然而，对于这些位点的细胞内ATP浓度，这些浓度如何受到突触活性的影响，代谢需求如何与活性耦合，局部线粒体的存在如何影响局部ATP水平，或局部突触ATP生成依赖于糖酵解与氧化磷酸化的程度知之甚少。需要当地ATP水平的直接报告才能获得这些信息。鉴于线粒体功能障碍与许多神经退行性疾病有关，直接测量单个神经末梢ATP浓度动态的能力对于检查这些疾病状态下的ATP代谢也很有价值。在这里，我们建议发展成像方法来帮助填补这一信息空白。我们的方法将是开发一种结合化学发光和荧光的方法，以遗传编码和突触靶向ATP指示器的形式，提供活体神经末梢中校准的、动态的、细胞内突触ATP浓度谱。

DESCRIPTION (provided by applicant): Synapses represent key transduction machines that convert incoming electrical information in the form of action potentials into a secreted chemical message which in turn is converted back into a postsynaptic electrical response. The orchestration of these events is thought to underlie critical mechanisms of learning and memory, and dysfunction of synaptic communication is suspected to be central in a number of diseased states of brain function. Synapses however are located at significant distances from cell bodies, and the highly-localized cell biological machinery must rely on local sources of ATP for function. In addition to the ion pumps that are present on both pre and postsynaptic membranes that work to restore ionic balance following activity both compartments contain high concentrations of proteins that must consume ATP in their role carrying out signal transduction, as well as key membrane trafficking events delivering and retrieving proteins and lipids to and from the plasma membrane. Because of these high energy needs, a large fraction of nerve terminals and postsynaptic dendrites are endowed with local mitochondria. Little is known however about intracellular ATP concentrations at these sites, how these concentrations are impacted by synaptic activity, how metabolic needs are coupled to activity, how the presence of local mitochondria impacts local ATP levels, or the extent to which local synaptic ATP generation relies on glycolysis versus oxidative phosphorylation. A local direct reporter of ATP levels is required to access this information. Given that mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, the ability to directly measure ATP concentration dynamics at individual nerve terminals will be valuable for examining ATP metabolism in these diseased states as well. Here we propose to develop imaging methodology to help fill this information gap. Our approach will be to develop a combined chemo-luminescence and fluorescence approach in the form of a genetically encoded and synaptically targeted ATP indicator that will provide calibrated, dynamic, intracellular synaptic ATP concentration profiles in living nerve terminals. PUBLIC HEALTH RELEVANCE: Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. The functioning of the human brain relies on the careful orchestration of delivering neurotransmitter-laden vesicles to sites at nerve terminals where they can be used to deliver this chemical message on demand. Many known genetic mutations in diseases such as Parkinson's disease, migraine headache and schizophrenia are linked to proteins that control synapse function. Our work is aimed at understanding the machinery at a molecular level to better ensure the success of future therapies for these types of neuronal diseases. Here we are proposing to develop technologies that will allow us to examine how synapses regulate their energy supply, which is thought to be a critical area of malfunction in certain neurological diseases.