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

描述(申请人提供)：线粒体在细胞生物能量学中发挥核心作用，并控制神经元生死的多个方面，包括调节钙信号。线粒体通过在兴奋过程中缓冲钙，然后将钙释放回胞浆，塑造[钙]i信号，并调节神经元中许多钙依赖的功能，如兴奋性、突触可塑性和基因表达。然而，在中风以及阿尔茨海默氏症、帕金森氏症和亨廷顿病中，过量的线粒体和钙离子会触发神经毒性过程。尽管神经元线粒体钙转运很重要，但目前尚不清楚介导神经元线粒体钙摄取和释放的分子。这一知识差距是我们在理解和治疗上开发线粒体功能方面取得进展的主要障碍。因此，这项建议的第一个目标是确定神经元线粒体钙摄取的分子成分。线粒体是高度动态的细胞器，能迅速发生分裂和融合，影响线粒体的运输、突触的可塑性和神经元的存活。值得注意的是，线粒体分裂是中风的早期事件，碎片化的线粒体在阿尔茨海默病和亨廷顿病中很常见。鉴于线粒体钙转运在神经元生死中的核心作用，MFF对神经元存活的影响可能部分是通过线粒体钙转运的变化来调节的，尽管这一观点尚未得到验证。因此，我们的第二个目标是确定MFF状态如何影响神经元中线粒体的钙转运和钙稳态。根据我们的初步数据和已发表的文献，我们假设CCDC109A、CCDC109B和MICU1-3是神经元线粒体钙摄取的重要分子成分，MFF过程对神经毒性条件下神经元的CCDC109A和/或CCDC109B活性、线粒体钙转运和钙稳态起着重要的控制作用。我们将使用创新的方法，包括遗传编码的线粒体钙传感器、电子探针X射线显微分析和新的遗传小鼠品系，以验证我们的假设在三个特定的目标。目的1鉴定新蛋白CCDC109A、CCDC109B和MICU1、2和3在神经元线粒体钙摄取中的作用。目的2研究线粒体重组对神经元线粒体钙转运的调节作用，以及CCDC109A和CCDC109B磷酸化在这一过程中的具体作用。目的3研究MFF在神经毒性条件下维持神经元钙稳态的作用，如谷氨酸过量暴露和脑缺血。该项目将深入了解神经元线粒体钙转运的分子组织，并将在线粒体动力学、钙信号和神经元钙稳态之间建立机械联系。我们预计这些研究将具有变革性，因为它们将发现新的分子和遗传工具，用于探索神经元中线粒体钙摄取的许多功能，并可能导致针对线粒体钙转运和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.