Molecular Mechanisms and Functions of Mitochondrial Ca2+ transport in Neurons

线粒体在细胞代谢中起核心作用，并控制神经元信号的多个方面。通过 有效缓冲神经元兴奋期间的Ca 2+内流并缓慢释放Ca 2+回到胞质溶胶中， 线粒体形成[Ca 2 +]i瞬变并调节Ca 2+依赖性神经元功能，如兴奋性， 突触传递和基因表达。线粒体基质中的Ca 2+升高刺激Ca 2+依赖性 增加和促进ATP的产生，以满足兴奋过程中能量需求的增加。 然而，线粒体Ca 2+超载可杀死神经元，并且线粒体Ca 2+失调与此有关 在中风和神经退行性疾病如阿尔茨海默氏症和帕金森氏症中的神经元损伤中 疾病尽管线粒体Ca 2+转运对神经元生命和死亡很重要，但 介导神经元中线粒体Ca 2+摄取和释放的机制尚不清楚。这种知识差距呈现出一种 这是我们在理解和治疗上纠正线粒体功能方面取得进展的主要障碍， 神经元本提案的主要目标是鉴定介导线粒体Ca 2+摄取的分子 在外周和中枢神经元中的作用，并确定它们在神经元Ca 2+信号传导，ATP合成， 突触传递和兴奋性毒性。我们的初步研究表明，两个新的分子，MCU MCUb（CCDC 109 A）和MCUb（CCDC 109 B）广泛表达于外周和中枢神经系统， 并且MCU是神经元中线粒体Ca 2+摄取所必需的，而MCUB抑制这种Ca 2+转运 机制此外，我们使用MCU KO小鼠的试验数据显示，MCU损失显着，但没有 完全减少线粒体Ca 2+摄取，改变Ca 2+信号传导和线粒体功能， 对谷氨酸诱导的毒性具有显著的保护作用。我们的中心假设是MCU和MCUb 在神经元线粒体Ca 2+摄取的调节中起重要但相反的作用，生物能量学，Ca 2 + 信号传导和突触传递，以及MCU的敲除，而不是MCUb的敲除，保护神经元免受 兴奋性毒性和减少缺血性中风中的神经元损伤。我们将采用多学科方法 包括遗传Ca 2+和ATP传感器，膜片钳记录，敲除小鼠和小鼠模型， 缺血性中风，以测试这一假设在三个具体目标。目标1将确立MCU和MCUb的角色 在中枢和外周神经元线粒体Ca ~（2+）转运和Ca ~（2+）信号转导中的作用。目标2将审查 MCU和MCUb对突触前Ca 2+信号传导和突触传递影响。目标3将建立 MCU和MCUb在兴奋性毒性和缺血性卒中中的作用。我们预计这项工作将是 因为它将建立遗传和药理学操纵的分子基础， 线粒体Ca 2+在神经元中的转运，并可能导致新的治疗方法的发展， 线粒体Ca 2+单向转运体，用于治疗中风和其它与兴奋性毒性相关的神经障碍。

Mitochondria play a central role in cell metabolism and control multiple aspects of neuronal signaling. By efficiently buffering Ca2+ influx during neuronal excitation and slowly releasing Ca2+ back into the cytosol, mitochondria shape [Ca2+]i transients and regulate Ca2+-dependent neuronal functions, such as excitability, synaptic transmission and gene expression. Ca2+ rise in the mitochondrial matrix stimulates Ca2+-dependent dehydrogenases and boosts ATP production to meet the increase in energy demand during excitation. However, mitochondrial overload with Ca2+ can kill neurons, and mitochondrial Ca2+ dysregulation is implicated in neuronal damage during stroke and in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Despite the importance of mitochondrial Ca2+ transport to neuronal life and death, the molecules that mediate mitochondrial Ca2+ uptake and release in neurons are not known. This knowledge gap presents a major obstacle in our progress toward understanding and therapeutically correcting mitochondrial functions in neurons. The main objectives of this proposal are to identify molecules that mediate mitochondrial Ca2+ uptake in peripheral and central neurons, and to establish their roles in neuronal Ca2+ signaling, ATP synthesis, synaptic transmission and excitotoxicity. Our preliminary studies indicate that two novel molecules, MCU (CCDC109A) and MCUb (CCDC109B), are broadly expressed in the peripheral and central nervous systems, and that MCU is required for mitochondrial Ca2+ uptake in neurons whereas MCUb inhibits this Ca2+ transport mechanism. Moreover, our pilot data using MCU KO mice showed that MCU loss dramatically, but not completely, reduced mitochondrial Ca2+ uptake, altered Ca2+ signaling and mitochondrial function and provided remarkable protection against glutamate-induced toxicity. Our central hypothesis is that MCU and MCUb play important but opposite roles in the regulation of mitochondrial Ca2+ uptake in neurons, bioenergetics, Ca2+ signaling and synaptic transmission, and that knockout of MCU, but not of MCUb, protects neurons from excitotoxicity and reduces neuronal damage in ischemic stroke. We will employ a multidisciplinary approach involving genetic Ca2+ and ATP sensors, patch-clamp recording, knockout mice and a mouse model of ischemic stroke to test this hypothesis in three specific aims. Aim 1 will establish the roles of MCU and MCUb in mitochondrial Ca2+ transport and Ca2+ signaling in central and peripheral neurons. Aim 2 will examine the impact of MCU and MCUb on presynaptic Ca2+ signaling and synaptic transmission. Aim 3 will establish the roles of MCU and MCUb in excitotoxicity and ischemic stroke. We anticipate that this work will be transformative because it will establish the molecular basis for genetic and pharmacological manipulation of mitochondrial Ca2+ transport in neurons, and may lead to the development of new therapeutics that target mitochondrial Ca2+ uniporters for treating stroke and other neurological disorders associated with excitotoxicity.