Axonal mitochondrial mobility and its impact on synaptic transmission

轴突线粒体流动性及其对突触传递的影响

• 批准号: 7969648
• 负责人: Zu-hang Sheng
• 金额: $ 116.99万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969648
• 关键词: Aerobic; Affect; Amyotrophic Lateral Sclerosis; Axon; Binding; Binomial Distribution; Brain; Buffers; Calcium; Calcium Signaling; Cells; Complex; Cytoskeleton; Docking; Dynein ATPase; Equilibrium; Exhibits; Functional disorder; Genes; Genetic; Goals; Growth Cones; Homeostasis; Human; Image; Immobilization; Immunoprecipitation; Journals; Laos; Life; Light; Mediating; Microtubules; Mitochondria; Molecular; Motor; Movement; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurosciences; Pan Genus; Paper; Pathogenesis; Pathologic Processes; Pattern; Phase; Phenotype; Physiological; Physiology; Plastics; Play; Presynaptic Terminals; Process; Production; Proteins; Proteomics; Publishing; Ranvier' s Nodes; Recruitment Activity; Research; Role; Spectrum Analysis; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Time; base; cell motility; cellular imaging; density; dynein light chain; improved; inhibitor/antagonist; insight; mouse model; mutant; neuron development; novel; presynaptic; response; synaptic function; syntaphilin; trafficking

Specific Aims: Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein initially identified in our lab as a candidate inhibitor of presynaptic function (1). Our effort in generating SNPH KO mice has led to the discovery of a novel role for SNPH in the control of axonal mitochondrial motility (2). Our study reveals that SNPH is required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule (MT)-based cytoskeleton. Three lines of evidence support this view. First, the mitochondria that associated with exogenously expressed GFP-SNPH were almost entirely immobile. This occurs through interactions with the cytoskeleton because SNPH contains a MT-binding domain, which is necessary and sufficient for SNPH-mediated immobilization of axonal mitochondria. Second, by recording mitochondrial movement in living neurons followed by retrospective immunostaining for endogenous SNPH, we demonstrate that the immobility of axonal mitochondria depends on their association with endogenous SNPH, and further reveal a binomial distribution with a strong correlation between the endogenous SNPH-tagged mitochondria (62%) and stationary mitochondria (65%). Finally, deletion of the snph gene in mice resulted in a substantially higher proportion of axonal mitochondria in the mobile state than that found in wild-type neurons, and reduced the densities of total and inter-bouton mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. Thus, SNPH acts as a static anchor for docking/retaining mitochondria in axons and at synapses. These findings reveal for the first time a neuron-specific protein capable of docking axonal mitochondria and regulating their densities within axons. Mitochondrial balance between the motile and stationary phases is a target for regulating mitochondrial redistribution. How are motile mitochondria recruited to the stationary pool? In particular, the mechanisms regulating SNPH-mediated mitochondrial anchoring at axons remain elusive. By applying a proteomic approach combined with time-lapse imaging in live snph (+/+) and (-/-) neurons, we revealed that dynein light chain LC8 enhances the docking efficiency by binding to SNPH (3). Four lines of evidence support this view. First, SNPH interacts with LC8 via its 7-residue motif (ERAIQTD); the SNPH-LC8 complex is detected by immunoprecipitation of brain homogenates; the interaction is independent of the dynein motor complex. Second, SNPH recruits LC8 to axonal mitochondria via its 7-residue LC8-binding motif. Deleting this motif reduces the SNPH capacity in docking axonal mitochondria. Third, elevated LC8 expression in snph (+/+) neurons inhibits the mobility of axonal mitochondria. In contrast, this effect is not observed in snph null neurons, suggesting that the role of LC8 is depending on its interaction with SNPH. Forthermore, CD spectrum analysis revealed that LC8 enhances docking by stabilizing an α−helical coiled-coil within the MT-binding domain of SNPH against thermal unfolding. Altogether, our studies provide new mechanistic insights into how SNPH and LC8 coordinately immobilize mitochondria through enhanced interaction of SNPH and MTs. In summary, using genetic mouse models combined with time-lapse imaging in live neurons, we elucidate molecular mechanism underlying the complex mobility patterns of axonal mitochondria. Such a mechanism enables neurons to maintain proper mitochondrial densities within axons and near synapses. We further provide the physiological evidence that the mobility and density of axonal mitochondria play a critical role in short-term synaptic plasticity. It is expected that defective mitochondrial docking/anchoring could affect neuronal functions. Dysfunction and defective trafficking of axonal mitochondria have been implicated in the pathologic processes of neurodegenerative diseases such as Alzheimers and Huntingtons and amyotrophic lateral sclerosis. The continued application of live cell imaging in combination with a multi-disciplinary analysis of genetically crossed mouse models will improve our understanding as how the changes in mitochondrial mobility affect axonal neurodegeneration. Papers published from the lab related to the project: 1. Guifang Lao, Volker Scheuss, Claudia M. Gerwin, Qingning Su, Sumiko Mochida, Jens Rettig, and Zu-Hang Sheng (2000). Neuron 25, 191-201. 2. Jian-Sheng Kang,Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng. (2008). Cell 132, 137-148. 3. Yan-Min Chen, Claudia Gerwin, and Zu-Hang Sheng. (2009). Journal of Neuroscience 29, 9428-9437.

具体目的： Syntaphilin（SNPH）是一种神经元特异性和轴突靶向的蛋白质，最初在我们的实验室中鉴定为突触前功能的候选抑制剂（1）。我们在产生SNPH KO小鼠方面的努力导致了SNPH在控制轴突线粒体运动中的新作用（2）。我们的研究表明，通过与基于微管（MT）基于基的细胞骨架的相互作用，将SNPH保持在固定状态下的大量轴突线粒体所必需的SNPH。三条证据支持这一观点。首先，与外源表达的GFP-SNPH相关的线粒体几乎完全不动。这是通过与细胞骨架的相互作用而发生的，因为SNPH包含一个MT结合域，这对于SNPH介导的轴突线粒体的固定化是必要且足够的。其次，通过记录活性神经元中的线粒体运动，然后对内源性SNPH进行回顾性免疫染色，我们证明，轴突线粒体的固定性取决于它们与内源性SNPH的关联，并进一步揭示了内源性SNPH snph标记的线粒体之间的二项式分布（62％）和62％）和62％的STESRAICH（62％）。最后，小鼠中SNPH基因的缺失导致移动状态下的轴突线粒体比例大大高于野生型神经元中的轴突线粒体，并降低了轴突中总和线粒体的密度。 SNPH突变神经元在长时间刺激过程中通过影响突触前胸子的钙信号传导表现出增强的短期促进作用。通过将SNPH基因重新引入突变神经元，可以完全挽救该表型。因此，SNPH充当轴突和突触中线粒体对接/保持线粒体的静态锚。这些发现首次揭示了一种神经元特异性蛋白，能够对接轴突线粒体并调节其在轴突中的密度。 运动相和固定相之间的线粒体平衡是调节线粒体重新分布的目标。如何将线粒体招募到固定池？特别是，调节SNPH介导的线粒体锚定的机制仍然难以捉摸。通过应用蛋白质组学方法与活SNPH（+/+）和（ - / - ）神经元中的延时成像相结合，我们揭示了Dynein Light Chain LC8通过与SNPH结合（3）来提高对接效率（3）。四条证据支持这一观点。首先，SNPH通过其7个残留图案（ERAIQTD）与LC8相互作用；通过对脑匀浆的免疫沉淀检测到SNPH-LC8复合物。该相互作用与动力蛋白运动复合物无关。其次，SNPH通过其7个残留的LC8结合基序将LC8募集到轴突线粒体。删除此主题可降低对接轴突线粒体的SNPH容量。第三，SNPH（+/+）神经元中的LC8表达升高会抑制轴突线粒体的迁移率。相反，在SNPH无效神经元中未观察到这种效果，这表明LC8的作用取决于其与SNPH的相互作用。 Fortermore，CD频谱分析表明，LC8通过在SNPH的MT结合域内稳定α-螺旋线圈来增强对接的影响。总的来说，我们的研究提供了新的机械洞察力，即SNPH和LC8如何通过增强SNPH和MT的相互作用将线粒体固定。 总而言之，使用与活神经元中延时成像结合的遗传小鼠模型，我们阐明了轴突线粒体复杂迁移率模式的基础的分子机制。这种机制使神经元能够在轴突和近突触中保持适当的线粒体密度。我们进一步提供了生理证据，即轴突线粒体的迁移率和密度在短期突触可塑性中起关键作用。预计有缺陷的线粒体对接/锚定可能会影响神经元功能。轴突线粒体的功能障碍和障碍的运输与神经退行性疾病的病理过程有关，例如阿尔茨海默氏症和亨廷顿氏症以及肌萎缩性侧向硬化。随着线粒体迁移率的变化如何影响轴突神经变性，将实时细胞成像与遗传交叉模型的多学科分析相结合，将持续应用。 从实验室发表的论文与该项目有关： 1。GuifangLao，Volker Scheuss，Claudia M. Gerwin，Qingning SU，Sumiko Mochida，Jens Rettig和Zu-Hang Sheng（2000）。神经元25，191-201。 2。 （2008）。牢房132，137-148。 3。Yan-Min Chen，Claudia Gerwin和Zu-Hang Sheng。 （2009）。神经科学杂志29，9428-9437。