Regulation of retrograde cargo transport in axons

轴突逆行货物运输的调节

• 批准号: 10266546
• 负责人: Catherine M Drerup
• 金额: $ 64.44万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/10266546
• 关键词: Actins; Active Biological Transport; Address; Afferent Neurons; Animals; Axon; Axonal Transport; Back; Biological Assay; Buffers; Calcium; Calcium ion; Carrier Proteins; Cell physiology; Cells; Collaborations; Complex; DNA; Defect; Development; Disease; Distal; Dynein ATPase; Family; Fractionation; Genetic Screening; Goals; Green Fluorescent Proteins; Health; Homeostasis; Hour; Immunoprecipitation; Impairment; Interruption; Ions; Kinesin; Label; Lead; Left; Link; Lipids; Location; Lysosomes; Maintenance; Mass Spectrum Analysis; Membrane; Metabolic; Metabolism; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular Motors; Motor; Motor Neurons; Movement; Neurons; Nuclear; Organelles; Positioning Attribute; Presynaptic Terminals; Process; Protein Biosynthesis; Protein Region; Proteins; Proteome; Regulation; Role; Screening procedure; Siblings; System; Testing; Time; Transgenes; Transgenic Organisms; Work; Zebrafish; causal variant; cell motility; dynactin; experimental study; flexibility; forward genetics; in vivo; life history; mutant; neural circuit; neuronal cell body; novel; organelle movement; post-doctoral training; protein complex; protein protein interaction; red fluorescent protein; relating to nervous system; retrograde transport; tool

1) Regulation of retrograde mitochondrial transport in axons One of the most crucial organelles in axons are mitochondria. Mitochondria perform many functions important for local microenvironments including: 1) generate the energy necessary for cellular metabolism; 2) buffer calcium ion levels; and 3) supply ATP for the proper functioning of ion transporters that regulate neural excitability. In addition, the location of mitochondria has been shown to regulate axon branching. Not only do mitochondria need to be properly localized in axons to maintain axon health and function, mitochondria also need to move in order for them to maintain their own health: Mitochondria undergo fission-fusion dynamics which allow the exchange of proteins, lipids, and mitochondrial DNA. If these dynamics are disrupted, mitochondria rapidly undergo degradation. Consequently, mitochondrial transport is of the utmost importance for axon function and health. Our lab is working to identify the factors that regulate mitochondrial transport by the retrograde motor protein complex. Towards the end of my post-doctoral training, I discovered a mutant which lacks almost all retrograde mitochondrial movement. The causative mutation in this line results in depletion of Actr10 (actin related protein 10) a known component of the dynein-associated complex, dynactin. In vivo analyses of mitochondrial movement in actr10 mutants revealed a lack of retrograde mitochondrial movement but normal anterograde (non-dynein related) transport. Transport of other cargos assayed, including lysosomes and the dynein motor itself, was not altered in actr10 mutants. To determine if Actr10 was in fact necessary to link mitochondria to the retrograde motor, we performed mitochondrial fractionation experiments from actr10 mutants and wildtype siblings. These experiments confirmed that Actr10 is necessary for the dynein motor to interact with mitochondria. This linkage is likely not direct, however, as Actr10 does not have known membrane-associated domains. To identify the proteins which make up this link between Actr10 and mitochondria, we performed an immunoprecipitation experiment followed by mass spectrometry analysis. These experiments yielded a number of interesting candidates which we are currently testing for their role in retrograde mitochondrial transport in axons. Together, our work will define the mechanism of dynein-mitochondrial attachment for retrograde movement of this organelle in axons. 2) The function of retrograde mitochondrial motility in axons While we know quite a bit about the dynamics of mitochondrial motility in the short term (on the order of minutes), we know almost nothing about the life history of mitochondria in neurons over longer periods of time. Additionally, we do not understand the role of mitochondrial movement in neurons though it is clear that active transport of this organelle is critical to the health and function of neurons. To begin to address these long-standing questions in the field, we have generated tools to analyze mitochondrial localization, transport, health, and function in vivo in zebrafish. Additionally, we have established collaborations to analyze neural circuit function when mitochondrial transport is inhibited. Together, this analysis revealed a critical role for mitochondrial retrograde transport in the maintenance of a homeostatic mitochondrial distribution in neurons. Inhibiting mitochondrial retrograde flux resulted in a depletion of cell body mitochondria with organelle accumulation in the distal axon. Accumulated mitochondria show signs of failed health and can no longer buffer calcium. This leads to impaired neural circuit activity. While informative, this left an obvious question of why mitochondria need to move back to the cell body. We reasoned that mitochondria could be moved back to this compartment for protein replenishment. Mitochondria have >1200 proteins,some of which turnover on the order of hours. 99% of these proteins are encoded by nuclear DNA. Perhaps it would make more sense to move the organelle back to the region of protein synthesis rather than bring the proteins to the organelle. Using mass spec analysis of the mitochondrial proteome, we revealed an essential role for retrograde mitochondrial transport for maintenance of the mitochondrial proteome. Disrupting this process leads to a >50% loss of almost a hundred mitochondrial proteins. Together, our work defines the dynamics of mitochondrial transport in neurons and has shown for the first time that retrograde movement specifically is required for mitochondrial protein replenishment and organelle homeostasis in neurons. 2) Identifying novel regulators of retrograde cargo transport in axons Forward genetics is an ideal and unbiased way to identify proteins with critical functions in cellular processes. We have initiated a forward genetic screen in zebrafish to identify proteins important for the retrograde transport of specific cargos in axons. For this screen, we are using a transgenic line that marks both the sensory and motor neuron axons in zebrafish with cytoplasmic GFP (Green Fluorescent Protein). Because cargos that fail to undergo retrograde transport accumulate over time in axon terminals, we can screen our mutagenized families for axon terminal size using the GFP fluorescent indicator to identify strains with disruptions in retrograde axonal transport. In addition to being an efficient screening procedure, the ability to screen live at various developmental stages also gives us the flexibility necessary to study multiple types of axons that develop at different time-points in the same animals. Additionally, our transgenic line contains a second transgene to label mitochondria with the red fluorescent protein TagRFP. Consequently, our screen will also allow us to identify mutant strains with defect in mitochondrial positioning as well as more general markers of retrograde transport disruption. Together, this screen will identify novel regulators of retrograde cargo transport and regulators of mitochondrial localization and motility in axons. This will advance our goal of defining the mechanisms of cargo-specific retrograde transport in axons.

1）调节轴突中逆行线粒体转运 轴突中最关键的细胞器之一是线粒体。线粒体对局部微环境的许多功能包括：1）产生细胞代谢所需的能量； 2）缓冲钙离子水平； 3）为调节神经兴奋性的离子转运蛋白的正常功能提供ATP。另外，线粒体的位置已显示用于调节轴突分支。线粒体不仅需要在轴突中正确定位以维持轴突的健康和功能，还需要移动，以使它们保持自己的健康：线粒体经历裂变融合动力学，允许蛋白质，脂质，脂质和线粒体DNA交换。如果这些动态被破坏，线粒体会迅速降解。因此，线粒体运输对于轴突功能和健康至关重要。我们的实验室正在努力确定通过逆行运动蛋白复合物调节线粒体转运的因素。 在我的博士后训练结束时，我发现了一个突变体，几乎缺少所有逆行线粒体运动。该系中的致病突变导致ACTR10耗竭（肌动蛋白相关蛋白10）是动力蛋白相关复合物Dynactin的已知成分。 ACTR10突变体中线粒体运动的体内分析表明，线粒体运动缺乏，但正常的顺序（非二烯素相关）转运。在ACTR10突变体中，其他碳糖分的运输，包括溶酶体和动力蛋白运动本身，并未改变。为了确定将线粒体与逆行电动机联系起来是否确实需要ACTR10，我们从ACTR10突变体和野生型兄弟姐妹进行了线粒体分级实验。这些实验证实，Dynein运动与线粒体相互作用是必需的。但是，由于ACTR10没有已知的膜相关域，因此这种连接可能不是直接的。为了确定构成ACTR10和线粒体之间这种联系的蛋白质，我们进行了免疫沉淀实验，然后进行了质谱分析。这些实验产生了许多有趣的候选者，我们目前正在测试它们在轴突中逆行线粒体转运中的作用。总之，我们的工作将定义轴突中该细胞器的逆行运动的动力蛋白 - 位体附着的机制。 2）轴突中逆行线粒体运动的功能 虽然我们对短期内线粒体运动的动态（按几分钟的顺序）了解了很多，但我们几乎对长时间神经元中线粒体的生活史一无所知。此外，我们不了解线粒体运动在神经元中的作用，尽管显然该细胞器的主动运输对神经元的健康和功能至关重要。为了开始解决该领域的这些长期存在的问题，我们生成了工具来分析Zebrafish在体内的线粒体定位，运输，健康和功能。此外，当抑制线粒体传输时，我们还建立了合作来分析神经回路功能。总之，该分析揭示了线粒体逆行转运在维持神经元中稳态线粒体分布中的关键作用。抑制线粒体逆行通量会导致细胞体线粒体的耗竭，并在远端轴突中积聚细胞器。累积的线粒体表现出健康状况失败的迹象，无法再缓冲钙。这导致神经回路活性受损。在提供信息的同时，这留下了一个明显的问题，即为什么线粒体需要移回细胞体。我们认为线粒体可以转回此隔室进行蛋白质补充。线粒体具有> 1200个蛋白质，其中一些蛋白质在小时的顺序上。这些蛋白质中的99％由核DNA编码。也许将细胞器重新回到蛋白质合成区域而不是将蛋白质带到细胞器会更有意义。使用线粒体蛋白质组的质量分析，我们揭示了逆行线粒体传输在维持线粒体蛋白质组方面的重要作用。破坏此过程会导致近100个线粒体蛋白的损失> 50％。总之，我们的工作定义了神经元中线粒体转运的动力学，并首次表明了逆行运动专门用于线粒体蛋白补充和神经元中细胞器稳态。 2）识别轴突中逆行货物运输的新型调节剂 正向遗传学是一种理想且公正的方法，可以鉴定在细胞过程中具有关键功能的蛋白质。我们已经在斑马鱼中启动了一个正向遗传筛选，以鉴定蛋白质对轴突中特定千圈逆行转运重要的蛋白质。对于此屏幕，我们使用的是具有细胞质GFP（绿色荧光蛋白）的斑马鱼中的感觉和运动神经元轴突的转基因线。由于无法在轴突终端中随着时间的推移积累的逆转录逆转录的货物，因此我们可以使用GFP荧光指示器筛选诱变家族的轴突末端大小，以识别逆行轴突运输中有干扰的菌株。除了是有效的筛选程序外，在各种发育阶段进行筛查的能力还使我们具有研究多种类型的轴突的灵活性，这些轴突在同一动物的不同时间点发展。此外，我们的转基因系列与红色荧光蛋白TAGRFP标记线粒体的第二个转基因。因此，我们的屏幕还将使我们能够鉴定线粒体定位缺陷的突变菌株以及逆行转运破坏的更一般标记。该屏幕将共同确定逆行货物运输的新型调节因子以及轴突中线粒体定位和运动性的调节剂。这将促进我们定义轴突中货物特异性逆行运输机制的目标。