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

这项提案关注的是一种名为spastin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，i)影响spastin的遗传基因突变会导致一种称为遗传性痉挛截瘫的遗传性运动神经元疾病，ii)spastin与阿尔茨海默氏症的病理有关，以及iii)spastin执行控制在许多不同细胞类型中发现的细胞通路的功能，因此了解它的工作方式对于了解细胞的基本生物学很重要。在这项工作中，我们的目标是尽可能全面地确定spastin蛋白的正常功能是什么，并了解这些功能的异常如何导致遗传性痉挛性截瘫。我们将在现有的痉挛知识基础上再接再厉。人类细胞被一层脂膜包围，并含有许多不同的膜结合的细胞内间隔(称为细胞器)，这些间隔通过运输途径连接在一起。货物通过被称为微管的轨道从一个细胞器到另一个细胞器，在膜结合的囊泡或小管中运输。我们知道，痉挛蛋白的一个主要功能是切断微管，当痉挛蛋白异常时，这种情况就不会发生。这种切割微管的失败有几个后果--它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spastin控制了膜运输小管从母细胞中释放的程度。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会导致运输小管的异常释放，但我们不知道微管切断是如何促进它们的释放的。我们将使用高倍显微镜方法来检查这一点，以可视化活细胞中的这一过程。Ii)我们将研究这些运输和成形过程的失败如何影响受影响细胞器的已知功能。例如，其中一个细胞器被称为内小体，它可以被认为是细胞内的分拣中心。在这里，决定是像膜受体这样的货运蛋白被送去降解，还是被回收到细胞表面，在那里它们可以感觉到细胞环境。因此，我们将找出在痉挛异常的细胞中哪些细胞表面受体受到影响。iii)我们还将确定我们在ii)中发现的哪些功能异常可能与遗传性痉挛截瘫疾病有关。我们将通过检测培养皿中培养的神经元来实现这一点。当神经元出现痉挛蛋白异常时，它们会出现肿胀的部分，我们将测试是否可以通过治疗来防止这些异常，以使我们发现的特定功能异常正常化。例如，一些膜受体控制着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，我们将检查是否通过适当的药物抑制或激活它控制的信号通路在防止神经元异常方面有效。这将是寻找治疗由痉挛异常引起的遗传性痉挛截瘫的第一步。总体而言，这些实验将确定痉挛蛋白在细胞中起什么作用，它是如何发挥作用的，以及这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截瘫以及其他类似的神经疾病提供合理的治疗方法。

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.