CAREER: Identifying the roles of mitochondria at the neuronal presynaptic terminal

这个项目的目标是确定线粒体对哺乳动物大脑神经元之间突触传递的贡献。线粒体为所有细胞提供能量，对支持大脑功能非常重要。然而，神经元的大尺寸和延伸的形状代表了这些细胞在远距离活动部位维持能量的主要挑战，特别是在脑细胞相互交流的突触上。在突触处，线粒体被认为产生能量来支持神经递质的释放，但这一点尚未得到证实。了解维持神经元细胞能量的机制对于理解脑功能和理解这些机制在各种疾病状态下是如何分解的至关重要。该项目旨在为健康神经元突触的能量供应途径提供新的、关键的和基础的知识，并说明突触如何补偿线粒体功能的丧失。这些结果将为实验提供信息，以纠正线粒体功能受损的系统，例如在神经退行性疾病和衰老中，为社会提供重大利益。这一信息对于更好地理解日益老龄化的人口中神经疾病状态的影响至关重要。此外，本科生和研究生将接受最先进的细胞和分子生物学技术的培训，重点是神经科学技术，为下一代研究人员提供培训机会。此外，这个项目将产生一个新的基于纸牌游戏的学习工具，以帮助向小学和大学水平的学生教授突触功能的基本机制。最近的报道表明，糖酵解和ATP缓冲可以补偿突触前末端线粒体功能的丧失，甚至在活动期间也是如此。因此，关于突触前末端线粒体功能的主流观点是不准确的，需要从根本上重新评估突触前线粒体的作用。研究小组将利用hold突触花萼的特殊实验可及性作为模型来评估突触前线粒体的作用。在第一个目标中，线粒体ATP合酶将被急性抑制，并且将使用电生理学和基于荧光的ATP成像方法测量突触传递和ATP维持。第二个目标将确定如何突触前线粒体Ca2+缓冲形状短期突触可塑性，通过消除线粒体Ca2+单转运体（MCU）从突触前终端使用体内病毒介导的基因缺失小鼠萼。MCU消除对线粒体定位、突触前胞质Ca2+缓冲和突触可塑性的影响将通过共聚焦显微镜、电生理学和荧光Ca2+成像来测量。第三个目标将确定突触前定位线粒体如何微调突触传递。病毒表达的显性阴性TRAK2肽破坏线粒体和运动蛋白之间的相互作用，并将被用于特异性地损害线粒体的轴突运输，消耗它们形成选定神经元群体的突触前末端。将使用共聚焦显微镜观察突触线粒体，并使用电生理学评估突触传递的短期可塑性。ATP和Ca2+的荧光成像将用于评估错误定位的突触前线粒体对传递和突触成熟的影响。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The goal of this project is to determine the contribution of mitochondria to synaptic transmission between neurons in the mammalian brain. Mitochondria provide energy in all cells, and are important to support brain function. However, the large size and extended shapes of neurons represents a major challenge for these cells to maintain energy at distant sites of activity, especially at synapses where brain cells communicate with each other. At synapses, mitochondria are assumed to produce energy to support the release of neurotransmitter, but this remains untested. Knowing the mechanisms used to maintain cellular energy in neurons is essential to comprehend brain function and understand how these mechanisms break down in various disease states. This project aims to provide new, critical, and fundamental knowledge on the routes of energy supply in healthy neuronal synapses, and illustrate how synapses compensate for loss of mitochondrial function. These results will inform experiments to correct systems where mitochondrial function is impaired, such as in neurodegenerative disease and aging, providing a significant benefit to society. This information will be essential to better comprehend the impact of neurological disease states in an increasingly aging population. Additionally, undergraduates and graduate students will be trained in state-of-the-art techniques for cell and molecular biology, with a focus on neuroscience techniques, providing training opportunities for the next generation of researchers. In addition, this project will generate a novel card game-based learning tool to help teach fundamental mechanisms of synaptic function to primary and college-level students. Recent reports suggest that glycolysis and ATP buffering can compensate for loss of mitochondrial function at the presynaptic terminal, even during bouts of activity. Thus, the prevailing view of mitochondrial function at the presynaptic terminal is inaccurate, requiring a fundamental re-evaluation of the role of presynaptic mitochondria. The research team will utilize the exceptional experimental accessibility of the calyx of Held synapse as a model to evaluate the role of presynaptic mitochondria. In the first objective, mitochondrial ATP synthase will be acutely inhibited, and synaptic transmission and ATP maintenance will be measured using electrophysiology and fluorescence-based ATP-imaging methods. The second objective will determine how presynaptic mitochondrial Ca2+-buffering shapes short-term synaptic plasticity, by eliminating the mitochondrial Ca2+ uniporter (MCU) from the presynaptic terminal using in vivo viral-mediated genetic deletion at the mouse calyx of Held. The effect of MCU elimination on mitochondrial localization, presynaptic cytosolic Ca2+ buffering, and synaptic plasticity will be measured using confocal microscopy, electrophysiology, and fluorescent Ca2+ imaging. The third objective will determine how presynaptically localized mitochondria fine-tune synaptic transmission. Viral expression of a dominant-negative TRAK2 peptide sabotages the interaction between mitochondria and kinesin motor proteins, and will be used to specifically impair axonal trafficking of mitochondria, depleting them form the presynaptic terminal of selected neuronal populations. Synaptic mitochondria will be visualized using confocal microscopy, and short-term plasticity of synaptic transmission will be evaluated using electrophysiology. Fluorescent imaging of ATP and Ca2+ will be used to evaluate the effect of mislocalized presynaptic mitochondria on transmission and synaptic maturation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.