Roles of ER in distal axon pathologies

我们的运动依赖于神经细胞沿着被称为轴突的狭窄突起沿着传递信号的能力，在人类中，轴突可以从细胞中心（细胞体）延伸一米。维持较长轴突的挑战表现在它们出错时的症状-轴突变性，瘫痪或缺乏感觉。这些发生在遗传性条件下，如遗传性痉挛性截瘫（HSP），其中较长的脊髓运动轴突的退化选择性地麻痹下半身，或获得性条件下，如糖尿病或化疗引起的神经病变，其中周围神经元疾病可导致身体四肢疼痛或麻木。然而，使远端轴突在这些条件下脆弱的生理过程尚不清楚;我们的目标是回答这个问题。我们专注于遗传条件，因为基因为我们提供了有关受影响机制的线索，而遗传学的见解通常适用于后天条件。我们研究了一种称为内质网（ER）的结构，它由纵向穿过轴突的中空小管组成，并相互融合和分裂形成网络。由于它们的长度和连续性，以及它们携带长距离信号的潜力，它们被称为“神经元中的神经元”。许多HSP突变破坏了帮助ER建模的蛋白质，例如通过插入ER膜的一面并弯曲它以形成小管：这表明ER网络在维持轴突功能和存活中的重要作用。轴突ER是一种未被充分研究的结构，我们只是最近才开发出可视化和观察其缺陷的工具。我们发现，在果蝇突变体中，破坏运动轴突中的ER网络，例如ER小管水平改变、小管变大或连续性中断; ER连续性的丧失是远端轴突状况的有吸引力的模型，因为沿着轴突沿着某处的间隙的概率随着与细胞体的距离而增加。在果蝇中取得这些进展比在人类或哺乳动物神经元中更容易，而且果蝇还允许我们在完整的生物体中研究轴突。ER小管储存钙离子，并且可以将钙释放到周围的细胞质中作为局部瞬时信号，或者从那里去除多余的钙，从而维持细胞质中的低水平钙。因此，我们主要集中在ER结构变化的影响，在飞HSP突变体，为本地和远程信号钙流。我们有四个主要目标，共同应该揭示改变ER的数量或存在，其连续性或其小管直径的后果;通过主要（不完全）分析远端轴突或突触前末梢，我们希望了解更多影响离细胞体最远区域的机制。首先，我们研究了如何在苍蝇同源物的一个常见的突变HSP基因，atlastin突变，影响轴突ER的连续性。第二，我们研究的后果，ER消耗局部钙处理，突触传递，和ER和质膜之间的脂质转移，使用另一种HSP突变体，缺乏ER在一些突触分支。这项工作将测试这些缺陷是否可能是病理学中的合理因素。第三，我们将测试ER损失或不连续对线粒体性质的影响-线粒体是为细胞提供可用能量的细胞器，其活性由ER释放的钙控制，如果不受控制，可能是有害的。最后，我们将测试ER异常对轴突中钙信号传播的影响，因为较长的轴突可能比较短的轴突更容易受到信号传播受损的影响。这些分析将为HSP的疾病机制提供模型，可以在哺乳动物或人类神经元中进行测试，就像我们以前的果蝇发现一样。它们可以为热休克蛋白或其他远端轴突疾病的治疗提供合理的方法。

Our movement depends on the ability of nerve cells to carry signals along narrow projections known as axons, which in humans can extend a metre from the centre of the cell, the cell body. The challenge of maintaining longer axons is shown by symptoms when they go wrong - axon degeneration, paralysis, or lack of sensation. These occurs in genetic conditions like Hereditary Spastic Paraplegia (HSP), in which degeneration of longer spinal cord motor axons selectively paralyses the lower body, or acquired conditions like diabetic or chemotherapy-induced neuropathy, in which peripheral neuron disease can cause pain or numbness of body extremities. However, the physiological processes that make distal axons vulnerable in these conditions are not known; our goal is to answer this question. We focus on genetic conditions, since genes give us clues about the affected mechanisms, and insights from genetics often apply to acquired conditions. We study a structure called endoplasmic reticulum (ER), consisting of hollow tubules that run lengthwise through the axon, and fuse and split from each other to form a network. Due to their length and continuity, and their potential to carry signals for long distances, they have been termed a "neuron within a neuron". Many HSP mutations that disrupt proteins that help model ER, for example by inserting in one face of the ER membrane and curving it to make tubules: this suggests an important role for the ER network in maintaining axon function and survival.Axonal ER is an underexplored structure and we have only recently developed tools to visualize and see defects in it. We see that removing some HSP membrane-curving proteins, in fruitfly mutants, disurupts the ER network in motor axons, e.g. altered levels of ER tubules, larger tubules, or interrupted continuity; loss of ER continuity is an attractive model for distal axon conditions, since the probability of a gap somewhere along the axon increases with distance from the cell body. It has been easier to make these advances in fruitflies than with human or mammalian neurons, and fruitflies also allow us to work with axons in an intact organism. ER tubules store calcium ions, and can either release calcium into the surrounding cytoplasm as a local transient signal, or remove excess calcium from there, thus maintaining low levels of calcium in cytoplasm. We therefore focus mainly on the consequences of ER structural changes in fly HSP mutants, for local and long-range signalling by calcium flows. We have four main aims, that together should uncover the consequences of altering the amount or presence of ER, its continuity, or its tubule diameter; by analysing mainly (not exclusively) distal axons or presynaptic terminals, we hope to learn more of the mechanisms that affect the regions furthest from the cell body. First we examine how mutations in the fly homolog of a commonly mutated HSP gene, atlastin, affect continuity of axonal ER. Second we examine the consequences of ER depletion for local calcium handling, synaptic transmission, and on lipid transfer between ER and plasma membrane, using another HSP mutant that lacks ER in some synaptic branches. This work will test whether these defects could be plausible factors in pathology. Third, we will test the consequences of ER loss or discontinuity on the properties of mitochondria - organelles that provide cells with usable energy, whose activity is controlled by release of calcium from ER, and which can be harmful if uncontrolled. Finally, we will test the consequences of ER abnormalities for the spread of calcium signals in axons, since longer axons could be more vulnerable to impaired spread of a signal than shorter ones. Together these analyses will provide models for the disease mechanisms of HSP, that can be tested in mammalian or human neurons as our previous fly findings were. They can underpin rational approaches to therapy of HSPs or other diseases of distal axons.