Regulation of retrograde cargo transport in axons

轴突逆行货物运输的调节

• 批准号: 10007510
• 负责人: Catherine M Drerup
• 金额: $ 86.64万
• 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/10007510
• 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; protein transport; 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) 轴突逆行线粒体运输的调节 轴突中最重要的细胞器之一是线粒体。线粒体执行许多对局部微环境重要的功能，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运作提供 ATP。此外，线粒体的位置已被证明可以调节轴突分支。线粒体不仅需要正确定位在轴突中以维持轴突健康和功能，线粒体还需要移动以保持自身健康：线粒体经历裂变融合动力学，允许蛋白质、脂质和线粒体的交换脱氧核糖核酸。如果这些动力学被破坏，线粒体就会迅速降解。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逆行运动蛋白复合物调节线粒体运输的因素。 在我的博士后培训即将结束时，我发现了一种几乎缺乏所有逆行线粒体运动的突变体。该品系中的致病突变导致 Actr10（肌动蛋白相关蛋白 10）的耗尽，Actr10 是动力蛋白相关复合物动力蛋白的已知成分。 Actr10 突变体线粒体运动的体内分析显示，线粒体缺乏逆行运动，但顺行（非动力蛋白相关）运输正常。 actr10 突变体中其他检测的货物（包括溶酶体和动力蛋白马达本身）的运输没有改变。为了确定 Actr10 是否确实是连接线粒体与逆行马达所必需的，我们对 actr10 突变体和野生型兄弟姐妹进行了线粒体分离实验。这些实验证实 Actr10 对于动力蛋白马达与线粒体相互作用是必需的。然而，这种联系可能不是直接的，因为 Actr10 不具有已知的膜相关结构域。为了鉴定构成 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物，我们目前正在测试它们在轴突逆行线粒体运输中的作用。我们的工作将共同确定轴突中细胞器逆行运动的动力蛋白-线粒体附着机制。 2）轴突逆行线粒体运动的功能 虽然我们对短期内（大约几分钟）线粒体运动的动力学了解很多，但我们对较长时间内神经元中线粒体的生命史几乎一无所知。此外，我们不了解线粒体运动在神经元中的作用，尽管很明显这种细胞器的主动运输对于神经元的健康和功能至关重要。为了开始解决该领域这些长期存在的问题，我们开发了分析斑马鱼线粒体定位、运输、健康和体内功能的工具。此外，我们还建立了合作来分析线粒体转运受到抑制时的神经回路功能。总之，该分析揭示了线粒体逆行运输在维持神经元中线粒体稳态分布中的关键作用。抑制线粒体逆行通量导致细胞体线粒体耗尽，细胞器在远端轴突中积累。积累的线粒体显示出健康状况不佳的迹象，并且无法再缓冲钙。这会导致神经回路活动受损。虽然信息丰富，但这留下了一个明显的问题：为什么线粒体需要移回细胞体。我们推断线粒体可以移回这个隔室以补充蛋白质。线粒体有超过 1200 种蛋白质，其中一些蛋白质的周转时间约为数小时。 99%的这些蛋白质是由核DNA编码的。也许将细胞器移回蛋白质合成区域比将蛋白质带到细胞器更有意义。通过对线粒体蛋白质组的质谱分析，我们揭示了逆行线粒体运输对于维持线粒体蛋白质组的重要作用。破坏这一过程会导致近 100 种线粒体蛋白损失 50% 以上。我们的工作共同定义了神经元中线粒体运输的动力学，并首次表明逆行运动是神经元中线粒体蛋白质补充和细胞器稳态所必需的。 2）识别轴突中逆行货物运输的新调节因子 正向遗传学是识别在细胞过程中具有关键功能的蛋白质的理想且公正的方法。我们在斑马鱼中启动了一项正向遗传筛选，以鉴定对轴突中特定货物逆行运输重要的蛋白质。在本次筛选中，我们使用转基因品系，用细胞质 GFP（绿色荧光蛋白）标记斑马鱼的感觉神经元轴突和运动神经元轴突。由于未能经历逆行运输的货物会随着时间的推移在轴突末端累积，因此我们可以使用 GFP 荧光指示剂筛选突变家族的轴突末端大小，以识别逆行轴突运输中断的菌株。除了是一种有效的筛选程序之外，在不同发育阶段进行活体筛选的能力还为我们提供了研究同一动物在不同时间点发育的多种类型轴突所需的灵活性。此外，我们的转基因系包含第二个转基因，用红色荧光蛋白 TagRFP 标记线粒体。因此，我们的筛选还将使我们能够识别线粒体定位缺陷的突变株以及逆行转运破坏的更一般的标记。总之，该屏幕将识别逆行货物运输的新型调节剂以及轴突中线粒体定位和运动的调节剂。这将推进我们定义轴突中货物特异性逆行运输机制的目标。