Mitochondria fission and fusion (MFF)-dependent mechanisms in neuronal toxicity

描述（由申请人提供）：线粒体在细胞生物能量学中发挥核心作用，并控制神经元生命和死亡的多个方面，包括Ca 2+信号传导的调节。通过在兴奋过程中缓冲Ca 2+，随后将Ca 2+释放回胞质溶胶，线粒体形成[Ca 2 +]i信号并调节神经元中的许多Ca 2+依赖性功能，如兴奋性、突触可塑性和基因表达。然而，在中风和阿尔茨海默氏病、帕金森氏病和亨廷顿病中，线粒体与Ca 2+的过度负荷触发神经毒性过程。尽管神经元线粒体Ca 2+转运的重要性，但神经元中介导线粒体Ca 2+摄取和释放的分子尚不清楚。这种知识差距是我们理解和利用线粒体功能进行治疗的主要障碍。因此，该建议的第一个目标是确定神经元中线粒体Ca 2+摄取的分子组分。线粒体是高度动态的细胞器，其快速地进行裂变和融合（MFF），这影响线粒体的运输、突触可塑性和神经元存活。值得注意的是，线粒体分裂是中风的早期事件，而线粒体碎片在阿尔茨海默病和亨廷顿病中普遍存在。鉴于线粒体Ca 2+转运在神经元生命和死亡中的中心作用，MFF对神经元存活的影响可能部分通过线粒体Ca 2+处理的变化介导，尽管这一想法尚未得到验证。因此，我们的第二个目标是确定MFF状态如何影响神经元线粒体Ca 2+转运和Ca 2+稳态。基于我们的初步数据和已发表的文献，我们假设CCDC 109 A、CCDC 109 B和MICU 1 -3是神经元中线粒体Ca 2+摄取的重要分子组分，并且MFF过程提供了对暴露于神经毒性条件的神经元中的CCDC 109 A和/或CCDC 109 B活性、线粒体Ca 2+转运和Ca 2+稳态的重要控制。我们将使用创新的方法，包括遗传编码的线粒体Ca 2+传感器，电子探针X射线微量分析和新的遗传小鼠品系，以测试我们的假设在三个特定的目标。目的1研究新蛋白CCDC 109 A、CCDC 109 B和MICU 1、2、3在神经元线粒体Ca ~（2+）摄取中的作用。目的2将确定线粒体重组如何调节神经元中线粒体Ca 2+转运，并研究CCDC 109 A和CCDC 109 B磷酸化在此过程中的特定作用。目的3将研究MFF在神经毒性条件下（如过量暴露于谷氨酸和缺血）维持神经元Ca 2+稳态的功能。该项目将深入了解神经元中线粒体Ca 2+转运的分子组织，并将建立线粒体动力学，Ca 2+信号传导和神经元Ca 2+稳态之间的机制联系。我们预计这些研究将是变革性的，因为它们将确定新的分子和遗传工具，用于探索神经元中线粒体Ca 2+摄取的许多功能，并可能导致针对线粒体Ca 2+转运和MFF的新疗法，用于治疗中风和神经退行性变。

DESCRIPTION (provided by applicant): Mitochondria play a central role in cell bioenergetics and control multiple aspects of neuronal life and death, including regulation of Ca2+ signaling. By buffering Ca2+ during excitation, and subsequently releasing Ca2+ back to the cytosol, mitochondria shape [Ca2+]i signals and regulate numerous Ca2+-dependent functions in neurons, such as excitability, synaptic plasticity and gene expression. However, excessive load of mitochondria with Ca2+ trigger neurotoxic processes in stroke and in Alzheimer's, Parkinson's and Huntington's diseases. In spite of the importance of neuronal mitochondrial Ca2+ transport, the molecules mediating mitochondrial Ca2+ uptake and release in neurons are not known. This knowledge gap presents a major obstacle in our progress toward understanding and therapeutically exploiting mitochondrial functions. Thus, the first objective of this proposal is to identify the molecular components of mitochondrial Ca2+ uptake in neurons. Mitochondria are highly dynamic organelles that rapidly undergo fission and fusion (MFF), which affects transport of mitochondria, synaptic plasticity and neuronal survival. Notably, mitochondrial fission is an early event in stroke, and fragmented mitochondria are prevalent in Alzheimer's and Huntington's disease. Given the central role of mitochondrial Ca2+ transport in neuronal life and death, it is possible that the effects of MFF on neuronal survival are mediated in part through changes in mitochondrial Ca2+ handling, although this idea has not been tested. Thus, our second objective is to determine how MFF status affects mitochondrial Ca2+ transport and Ca2+ homeostasis in neurons. Based on our preliminary data and published literature, we hypothesize that CCDC109A, CCDC109B and MICU1-3 are essential molecular components of mitochondrial Ca2+ uptake in neurons, and that the MFF process provides important control of CCDC109A and/or CCDC109B activities, mitochondrial Ca2+ transport and Ca2+ homeostasis in neurons exposed to neurotoxic conditions. We will use innovative approaches, including genetically encoded mitochondrial Ca2+ sensors, electron probe X-ray microanalysis and novel genetic mouse strains, to test our hypothesis in three specific aims. Aim 1 will identify the roles of novel proteins CCDC109A, CCDC109B and MICU1, 2 and 3 in mitochondrial Ca2+ uptake in neurons. Aim 2 will determine how mitochondrial restructuring regulates mitochondrial Ca2+ transport in neurons and examine specific roles of CCDC109A and CCDC109B phosphorylation in this process. Aim 3 will examine the function of MFF in maintaining neuronal Ca2+ homeostasis under neurotoxic conditions, such as excessive exposure to glutamate and ischemia. This project will provide insight into the molecular organization of mitochondrial Ca2+ transport in neurons and will establish mechanistic links between mitochondrial dynamics, Ca2+ signaling and neuronal Ca2+ homeostasis. We anticipate that these studies will be transformative because they will identify new molecular and genetic tools for exploring many functions of mitochondrial Ca2+ uptake in neurons and may lead to new therapeutics targeting mitochondrial Ca2+ transport and MFF for treating stroke and neurodegeneration.