Understanding axonopathy by defining physiological and pathological functions of the microtubule severing protein spastin at membrane traffic pathways

通过定义膜交通途径中微管切断蛋白 spastin 的生理和病理功能来了解轴突病

• 批准号: MR/M00046X/1
• 负责人: Evan Reid
• 金额: $ 43.87万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FM00046X%2F1
• 关键词: Understanding; axonopathy; defining; physiological; pathological

This proposal focuses on a protein called spastin. Spastin is present in all of the cells of the body and is important for three main reasons, i) inherited gene mutations affecting spastin cause a type of hereditary motor neuron disease called hereditary spastic paraplegia, ii) spastin has been implicated in the pathology of Alzheimer's dementia, and iii) spastin carries out functions that control cellular pathways found in many different cell types and so knowing how it works is of importance in understanding the basic biology of cells.In this work we aim to determine as fully as possible what the normal functions of the spastin protein are, and to understand how abnormality of these functions leads to hereditary spastic paraplegia. We will build on our existing knowledge of spastin. Human cells are bounded by a lipid membrane and contain many different membrane-bound intracellular compartments (called organelles) that are connected by transport pathways. Cargoes are transported in membrane-bound vesicles or tubules from one organelle to another over tracks called microtubules. We know that a main function of spastin is to cut microtubules, and when spastin is abnormal this does not happen. This failure to cut microtubules has several consequences- it results in some cellular organelles having an abnormal shape, and in the inefficient transport of some cargoes. We believe that this may be because spastin controls how well membrane transport tubules are released from the parent organelle.To achieve our aims we will carry out the following experiments:i) Although we know that failure of microtubule severing results in abnormal release of transport tubules, we do not know how microtubule severing promotes their release. We will examine this using high powered microscopy approaches to visualise the process in living cells. ii) We will examine how failure of these transport and shaping processes affects the known functions of the organelles affected. For example, one of the organelles involved is called the endosome, which can be thought of as a sorting centre within the cell. Here, decisions are made as to whether cargo proteins like membrane receptors are sent for degradation, or are recycled back to the cell surface, where they can sense the cellular environment. We will therefore find out which cell surface receptors are affected in cells with abnormal spastin.iii) We will also determine which of the functional abnormalities that we discover in ii) are likely to be involved in causing the hereditary spastic paraplegia disease. We will do this by examining neurons cultured in Petri dishes. When neurons have abnormality of spastin, they develop swollen sections, and we will test whether these can be prevented by treatments to normalise the particular functional abnormalities that we find. For example, some membrane receptors control signaling pathways that influence how neurons grow. If we find that such a receptor is controlled by spastin, we will examine whether inhibiting or activating, with appropriate drugs, the signaling pathway that it controls is effective at preventing the neuronal abnormalities. This will be a first step in finding treatments for hereditary spastic paraplegia caused by abnormality of spastin.Overall, these experiments will identify what spastin does in cells, how it does it, and how this translates to pathology in neurons. This level of detailed understanding will likely give rise to rational treatment approaches for hereditary spastic paraplegia, and perhaps other similar neurological condiditons.

这项提议的重点是一种叫做痉挛蛋白的蛋白质。Spastin存在于身体的所有细胞中，其重要性主要有三个原因，1)影响Spastin的遗传基因突变导致一种称为遗传性痉挛性截瘫的遗传性运动神经元疾病，2)Spastin与阿尔茨海默氏痴呆症的病理有关，3)Spastin执行控制许多不同细胞类型中发现的细胞通路的功能，因此了解其工作原理对于理解细胞的基本生物学非常重要。在这项工作中，我们的目标是尽可能充分地确定痉挛蛋白的正常功能是什么，并了解这些功能异常如何导致遗传性痉挛性截瘫。我们将以现有的关于痉挛的知识为基础。人体细胞由脂质膜包围，并包含许多不同的膜结合的细胞内室（称为细胞器），它们通过运输途径连接在一起。货物在膜结合的囊泡或小管中通过称为微管的轨道从一个细胞器运输到另一个细胞器。我们知道，spastin的一个主要功能是切断微管，当spastin异常时，这种情况不会发生。这种切割微管的失败有几个后果——它会导致一些细胞器形状异常，并导致一些货物的运输效率低下。我们认为这可能是因为痉挛素控制着膜运输小管从母体细胞器中释放的程度。为了达到我们的目的，我们将进行以下实验：i)虽然我们知道微管切断失败会导致运输管的异常释放，但我们不知道微管切断如何促进运输管的释放。我们将使用高倍显微镜方法来观察活细胞中的过程。ii)我们将研究这些运输和成型过程的失败如何影响受影响细胞器的已知功能。例如，其中一种细胞器被称为核内体，它可以被认为是细胞内的分选中心。在这里，决定是否像膜受体这样的货物蛋白被送去降解，或者被回收到细胞表面，在那里它们可以感知细胞环境。因此，我们将发现哪些细胞表面受体受到异常痉挛素细胞的影响。iii)我们还将确定我们在ii)中发现的哪些功能异常可能涉及导致遗传性痉挛性截瘫病。我们将通过检查培养皿中培养的神经元来做到这一点。当神经元出现痉挛素异常时，它们会出现肿胀部分，我们将测试是否可以通过治疗来防止这些异常，使我们发现的特定功能异常正常化。例如，一些膜受体控制影响神经元生长的信号通路。如果我们发现这种受体是由spastin控制的，我们将研究是否通过适当的药物抑制或激活它控制的信号通路，从而有效地预防神经元异常。这将是寻找治疗由痉挛蛋白异常引起的遗传性痉挛性截瘫的第一步。总的来说，这些实验将确定spastin在细胞中起什么作用，它是如何起作用的，以及它如何转化为神经元的病理。这种程度的详细了解可能会为遗传性痉挛性截瘫以及其他类似的神经系统疾病提供合理的治疗方法。

项目成果

Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.

• DOI: 10.1083/jcb.201609033
• 发表时间: 2017-05-01
• 期刊: The Journal of cell biology
• 作者: Allison R;Edgar JR;Pearson G;Rizo T;Newton T;Günther S;Berner F;Hague J;Connell JW;Winkler J;Lippincott-Schwartz J;Beetz C;Winner B;Reid E
• 通讯作者: Reid E

Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia

ER-内体接触缺陷影响遗传性痉挛性截瘫的溶酶体功能

• DOI: 10.17863/cam.9693
• 发表时间: 2017
• 作者: Allison R

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

• DOI: 10.1371/journal.pone.0152413
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Connell JW;Allison R;Reid E
• 通讯作者: Reid E

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin

使用电动跑步机系统进行定量步态分析可灵敏地检测表达 ATP 酶缺陷型 Spastin 的小鼠的运动异常

• DOI: 10.17863/cam.543
• 发表时间: 2016
• 作者: Connell J