The mechanistic basis and potential disease relevance of microtubule disorganisation in axons

在这里，我们将研究神经元轴突微管（MT）细胞骨架的特性，以更好地了解MT在神经元形成、维持和退化过程中所起的重要作用。轴突是细长的、像电缆一样的、长达几米的神经元突起，它们构成了连接我们身体的神经。它们通常无法被替换，因此需要在人体中维持长达一个世纪。不出所料，随着年龄的增长，我们逐渐失去了约50%的轴突，而在神经退行性疾病（ND）中，这一比例要高得多。尽管它们非常重要，但我们对长期维持这些微妙结构或导致它们在神经变性中过早衰退的机制知之甚少。轴突的形成和维持主要取决于微管（MT）细胞骨架。MTs由丝状蛋白聚合物组成，排列成25nm厚的小管。在轴突中，mt形成连续的平行束，作为维持生命的货物/细胞器运输的结构骨干和高速公路。在衰老和ND中，轴突经常形成肿胀，其中MT束被打乱成纵横交错的卷曲，从而捕获细胞器，并被认为引发轴突损失。为了研究轴突紊乱，我们基于果蝇神经元的实验数据建立了轴突稳态的工作模型；该模型包括三个步骤（详见CfS第1页）：(1)mt必须经历不断的聚合/解聚合以自我更新。(2)每个MT聚合事件都有MT破坏的风险，特别是在轴突中，高密度MT和分子马达产生剪切力，可以诱导MT卷曲。(3)由mt结合蛋白介导的一系列不同机制（例如，通过引导mt成平行束，或消除偏离轨道的mt）施加秩序。我们认为，单个（或多个）这些强制机制的缺失增加了轴突紊乱导致轴突肿胀的风险，从而为与各种轴突调节因子遗传相关的晚发性轴突变性提供了潜在的解释。到目前为止，我们主要通过培养果蝇和小鼠神经元的实验工作来测试和完善该模型，重点关注我们模型的第3步机制（即强制顺序MT调节器）。在这里，我们将重点关注步骤2中涉及的机制（即引起mt卷曲），并将我们对培养神经元的了解与体内神经系统的情况进行比较。这项工作很重要，原因有几个：首先，这里获得的数据将揭示在培养神经元的高效模型中观察到的程度，反映体内轴突肿胀的机制。这将为旨在揭示轴突肿胀如何形成和如何预防的实验工作提供重要方向。其次，我们将生成关于MT动力学及其在轴突中的空间排列的重要数据。这些数据将为MT行为的数学模型提供重要信息（见支持信），我们正在并行项目中开发这些模型，旨在最终进行长期的计算机实验，以测试MT在迟发性神经变性中的病理作用。第三，我们的数据将提供重要的理解、描述和轴突MT的概念，这将有助于全球范围内对轴突运输、细胞器动力学和MT调节的研究，从而促进我们对轴突生物学在发育、衰老、再生和退化过程中的理解。

Here we will study the properties of the microtubule (MT) cytoskeleton of neuronal axons to gain a better understanding of the important roles that MTs play during the formation, maintenance and degeneration of neurons. Axons are the slender, cable-like, up to several meter long protrusions of neurons which form the nerves that electrically wire our bodies. They can usually not be replaced, hence need to be maintained for up to a century in humans. Unsurprisingly, we gradually lose ~50% of our axons towards old age - and far more in neurodegenerative diseases (ND). In spite of their enormous importance, we know far too little about the mechanisms that maintain these delicate structures long-term or lead to their premature decay in neurodegeneration. Axon formation and maintenance essentially depends on the microtubule (MT) cytoskeleton. MTs consist of filamentous protein polymers arranged into 25nm thick tubules. In axons, MTs form continuous parallel bundles, serving as structural backbones and highways for life sustaining cargo/organelle transport. In ageing and ND, axons often form swellings where MT bundles become disorganised into criss-crossing curls, which trap organelles and are believed to trigger axonal loss. For the study of MT disorganisation, we developed the working model of axonal homeostasis based on our experimental data obtained in Drosophila neurons; this model involves three steps (details in CfS pt. 1): (1) MTs have to undergo constant polymerisation/depolymerisation to self-renew. (2) Each MT polymerisation event poses a risk of MT disorganisation, particularly in axons where high densities of MTs and molecular motors generate shear forces which can induce MT curling. (3) Order is imposed by a range of different mechanisms mediated by MT-binding proteins (e.g. by guiding MTs into parallel bundles, or eliminating MTs that have gone off-track). We propose that loss of single (or multiple) of these order-imposing mechanisms increases the risk of MT disorganisation leading to axon swellings - thereby providing potential explanations for late-onset axon degeneration linked genetically to various MT regulators. So far we have tested and refined this model primarily through experimental work in cultured fly and mouse neurons, by focusing on mechanisms regarding step 3 of our model (i.e. order-imposing MT regulators). Here we will focus on the mechanisms involved in step 2 (i.e. causing the curling of MTs), and compare our knowledge in cultured neurons to the situation in the nervous system in vivo. This work is important for several reasons: First, data obtained here will reveal the degree to which observations made in the highly efficient model of cultured neurons, reflect mechanisms underlying axon swellings in vivo. This will give important direction for experimental work aiming to unravel how axon swellings form and can be prevented. Second, we will generate important data concerning MT dynamics and their spatial arrangements in axons. These data will provide important information for the mathematical models of MT behaviours (see support letters) which we are developing in parallel projects - aiming to eventually perform long-term in silico experiments that can test pathological roles of MTs in late-onset neurodegeneration.Third, our data will provide important understanding, descriptions and concepts of axonal MTs that will aid worldwide research into axonal transport, organelle dynamics and MT regulation, thus promoting general advances in our understanding of axon biology during development, ageing, regeneration and degeneration.