Understanding microtubule regulation during the making and maintenance of axons

轴突是一种细长的神经元突起，在全身延伸数米，充当连接神经系统的信息高速公路。在发育过程中不能长出轴突要么是致命的，要么会导致发育性脑障碍。受伤或中风后轴突不能再生是导致终身残疾的重要原因。在老化的大脑中，轴突不能维持被认为是神经变性的一个重要原因。药理研究表明，轴突的生长和维持基本上是由高度动态的微管（MT）细胞骨架介导的。然而，mt是如何通过基因调控来促进轴突生长和维持的尚不清楚。该项目的总体目标是提供这样的理解，从而弥合我们对健康和疾病中大脑发育、再生和衰老的知识的重要差距。mt是丝状的，高度动态的微管蛋白聚合物，形成轴突的骨干。mt为轴突提供结构支持，同时也是细胞内往返于细胞体的运输通道。MTs的定向延伸驱动轴突生长，而它们的不稳定与轴突缩回或退化相关。轴突的动态持续整个轴突的生命周期（即长达数十年），这表明轴突的维持涉及到轴突的稳态翻转。轴突通过其正极的聚合/解聚合进行伸展/收缩，并且它们的正极与细胞内环境相互作用，决定轴突伸长的方向和范围。据报道，多种蛋白质可以调节MT +端动力学，包括EBs（末端结合蛋白）、+TIPs（与EBs结合的蛋白）、XMAP215（聚合MT）、DOUBLECORTIN（稳定MT +端）、STATHMIN（隔离游离微管蛋白）以及细胞皮层和细胞器中可以与MT +端相互作用的蛋白质。大多数这些MT +端调节因子的主要分子功能在体外是已知的，并且各种与大脑疾病有关，清楚地说明了它们在神经系统中的重要性。然而，这些蛋白质在不同神经元系统中的功能研究只产生了轻微的轴突表型（如果有的话），未能证明MT +端动力学在轴突生长和维持过程中预期发挥的重要作用。我假设不同的MT +端调节因子有助于一个共同的MT +端机制，并且它们的功能在该机制中重叠。因此，破译这种机制并确定驱动轴突生长和维持的关键组件集是这个项目的一个重要挑战和总体目标。这一挑战需要新颖的方法。我们使用了一种简单的遗传模式生物，果蝇。对果蝇的研究快速、廉价，而且利用了有效的遗传策略。它一直是发现支撑大脑发育和功能的机制和概念的发电站，其中许多是进化上很好的保守，为高等动物的研究奠定了重要的基础。我们在果蝇轴突生长期间的细胞骨架调节方面有8年的工作经验，并提供了大量的原理证据，证明可以产生新的理解并应用于高等动物。我们对MT +末端调控的试点研究揭示了轴突畸变的特征，并使我们能够制定详细的工作模型。在此基础上，我们将研究MT +末端调节因子的细胞机制以及它们之间的功能联系。我们的工作将证明MT +末端机制在轴突生长和维护过程中的重要性，并在理解该机制如何工作方面提供一个步骤变化。这将对大脑发育障碍、神经再生、神经退行性疾病和衰老的研究产生重要影响。

Axons are slender processes of neurons extending up to meters across the body, serving as information highways that wire the nervous system. Failure to grow axons during development is either fatal or causes developmental brain disorders. Failure to re-grow axons after injury or stroke is an essential cause for lifelong disabilities. Failure to maintain axons in the ageing brain is considered an important cause of neurodegeneration. Pharmacological studies have demonstrated that axon growth and maintenance are essentially mediated by the highly dynamic microtubule (MT) cytoskeleton. However, how MTs are genetically regulated to promote axon growth and maintenance is not understood. The overarching aim of this project is to deliver such understanding, thus bridging an important gap in our knowledge about brain development, regeneration and ageing in both health and disease. MTs are filamentous, highly dynamic tubulin polymers that form the backbone of axons. MTs provide structural support to axons as well as highways of intracellular transport from and to the cell body. The directed extension of MTs drives axon growth, whereas their destabilisation correlates with axon retraction or degeneration. MT dynamics continue throughout an axon's life (i.e. up to decades) suggesting that axonal maintenance involves steady-state turn-over of MTs. MTs extend/retract through polymerisation/depolymerisation at their plus ends, and their plus ends interact with the intracellular environment to determine the direction and extend of MT elongation. Various proteins have been reported to regulate MT plus end dynamics, and these include EBs (end binding proteins), +TIPs (proteins binding to EBs), XMAP215 (polymerising MTs), DOUBLECORTIN (stabilising MT plus ends), STATHMIN (sequestering free tubulin), and proteins of cell cortex and organelles that can interact with MT plus ends. The principal molecular functions of most of these MT plus end regulators are known in vitro, and various have been linked to brain disorders clearly illustrating their importance in the nervous system. However, functional studies of these proteins in different neuron systems have produced only mild axon phenotypes (if any), falling short of demonstrating the essential roles that MT plus end dynamics are expected to play during axon growth and maintenance. I hypothesise that the different MT plus end regulators contribute to one common MT plus end machinery and that their functions overlap within this machinery. Deciphering this machinery and identifying the key set of components that drive axon growth and maintenance is therefore an important challenge and the overarching objective of this project. This challenge requires novel approaches. We use a simple genetic model organism, the fruit fly Drosophila. Research in Drosophila is fast, cheap and capitalises on efficient genetic strategies. It has been a powerhouse for the discovery of mechanisms and concepts underpinning brain development and function, many of which are evolutionary well conserved and have laid important foundations for research in higher animals. We have 8 years of experience with work on cytoskeletal regulation during axon growth in Drosophila and have provided substantial proof of principle that novel understanding can be generated and applied to higher animals. Our pilot studies of MT plus end regulators reveal characteristic axon aberrations and allow us to formulate detailed working models. On this basis, we will study cellular mechanisms of MT plus end regulators and functional links between them. Our work will prove the importance of MT plus end machinery during axon growth and maintenance and deliver a step change in understanding of how this machinery works. This will have important implications for research on developmental brain disorders, neuroregeneration, neurodegenerative diseases and ageing.