Investigating a neuronal subcellular transcriptome by the novel technique of RNA TU-tagging, in a normal and ALS-related mouse model.

肌萎缩侧索硬化症(ALS，运动神经元病)是一种破坏性的神经退行性疾病，会导致肌肉功能进行性丧失和瘫痪。肌萎缩侧索硬化症通常会导致死亡，通常是由于无法呼吸所致，平均在确诊后3年内，85岁到250岁之间的终生风险为1。这种疾病的主要细胞是被称为运动神经元(MNS)的神经细胞。MNS将大脑与肌肉联系起来，从而使运动成为可能。在肌萎缩侧索硬化症过程中，MN逐渐死亡。MN是人体内最大的细胞之一。它们的主体位于脊髓内，含有许多称为树突的细小分支突起。它们还有一个细小的突起，叫做轴突，从脊髓延伸到我们的每一块肌肉。一个轴突可以超过一米长，从脊髓延伸到我们的手指或脚趾的末端。MN和肌肉之间的连接被称为神经肌肉连接(NMJ)。一个人身体中的所有细胞，尽管彼此非常不同，但都含有相同的DNA，这种遗传物质向每个细胞发出指令。因此，每种细胞类型(例如，细胞是神经细胞还是心脏细胞)的特性取决于DNA的哪些区域是活跃的，并产生另一种称为RNA的化学物质。RNA携带细胞运作所需的所有信息。细胞中所有RNA的总和称为转录组，是每种细胞类型的特征。了解一个细胞转录组有助于深入了解其生物学特性，并有助于确定疾病的原因。这与肌萎缩侧索硬化症的MNS尤其相关，因为有很好的证据表明，与RNA‘新陈代谢’相关的生物过程在肌萎缩侧索硬化症中主要受到影响。重要的是，我们现在知道RNA在单个细胞内的不同区域运输和功能；在MNS中，它在轴突中运输，并为特定的角色而到达NMJ。NMJ和轴突被认为是肌萎缩侧索硬化症中最早受到影响的MNS部分。因此，重要的是要知道哪些RNA存在于细胞体和树突中，以及轴突和MN的神经肌肉接头处，以了解它们的正常功能以及ALS中的问题。当神经元及其轴突在培养皿中人工生长时，或者当细胞体在显微镜下从固定组织中解剖时，从神经元及其轴突中分离和鉴定RNA是可能的。这些发现表明，成千上万种不同的RNA物种被活跃地运输到轴突，但从这些实验中很难提取与体内自然环境中的成熟神经元相关的信息，从而与疾病相关。我们将使用一种新技术-TU-Tagging，该技术已经在小鼠身上成功使用，并允许我们在活动物的环境中对特定细胞的RNA加标签。然后，可以通过高通量测序来分离和鉴定标记的RNA。我们将把这项技术应用于MNS，这样我们就可以第一次从MNS细胞体和树突以及活着的成年小鼠的轴突和NMJ中分离和鉴定RNA。我们将使用正常小鼠，以及我们开发的一种新的小鼠模型，该模型在导致ALS的TardBP(也称为TDP-43)基因中存在缺陷。从我们目前的工作中，我们知道该基因的缺陷使这种小鼠的MN细胞体中的RNA图谱发生了异常。目前还没有TDP-43突变小鼠准确地模拟人类ALS，但它们教会了我们大量关于TDP-43在正常和异常状态下如何发挥作用的知识。这些结果将非常有助于我们进一步了解MN的生物学以及是什么原因导致这些细胞在ALS中如此特异地脆弱。该项目将有助于对许多其他疾病的研究，在这些疾病中，不同细胞区域的RNA新陈代谢是重要的，以及在应对神经损伤方面，它再次发挥关键作用。

Amyotrophic Lateral Sclerosis (ALS, 'motor neuron disease') is a devastating neurodegenerative disorder which causes progressive loss of muscle function and paralysis. ALS leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of ~1 in 250 by 85 years old.The principal cells affected in this disease are nerve cells called motor neurons (MNs). MNs connect the brain to the muscles therefore making movement possible. MNs progressively die during the course of ALS. MNs are amongst the largest cells of the body. Their main body lies in the spinal cord and contains numerous thin branching processes called dendrites. They also have one thin process, named the axon, which extends from the spinal cord out to each of our muscles. A single axon can measure over a meter, running from the spinal cord to ends of our fingers or toes. The connection between the MN and muscles is called the neuromuscular junction (NMJ). All cells in an individual's body, although very diverse from each other, contain the same DNA, the genetic material that gives instructions to each cell. So the identity of each cell type (whether the cell is a nerve cell or a heart cell, for example) is the result of which regions of DNA are active and produce another type of chemical called RNA. RNA carries all the necessary information for the cell to function. The sum of all the RNA in a cell, named the transcriptome, is the signature that characterizes each cell type.Knowing one cell transcriptome provides insights into its biology and helps determine the causes of disease. This is particularly relevant with MNs in ALS since there is good evidence showing that the biological processes linked to RNA 'metabolism' are primarily affected in ALS. Importantly, we now know that RNA is transported and functions in different regions within an individual cell; in MNs it is transported in axons and to NMJs for specific roles. NMJs and axons are thought to be the first parts of the MNs to be affected in ALS. Therefore it is important to know which RNAs are present in cell bodies and dendrites, and in axons, and at neuromuscular junctions of MNs, to understand how they function normally and what goes wrong in ALS.It is possible to isolate and identify RNA from neurons and their axons when these are artificially grown in a culture dish or when cell bodies are dissected out under a microscope from fixed tissues. These findings have shown that thousands of different RNA species are actively transported to the axons, but it is difficult from these experiments to extract information that is relevant to mature neurons in their natural context in vivo and therefore to disease.We will work with a new technique, 'TU-tagging', which has already been successfully used in mouse and which allows us to 'tag' RNA in specific cells in the context of a living animal. The tagged RNA can then be isolated and identified through high-throughput sequencing. We will apply this technique to MNs so for the first time we can isolate and identify RNA from MNs cell bodies and dendrites, and from their axons and NMJs in the living adult mouse. We will work with normal mice, and with a new mouse model that we have developed that has a defect in a gene, Tardbp (also known as Tdp-43) that causes ALS. From our current work we know defects in this gene give an aberrant RNA profile in the MN cell bodies of this mouse. Currently no Tdp-43 mutant mice exactly model human ALS, but they teach us a great amount about how Tdp-43 functions in the normal and abnormal state.These results will be extremely helpful in furthering our understanding of MN biology and of what causes these cells to be so specifically vulnerable in ALS. This project will help research in the many other diseases in which RNA metabolism in different cell regions is important, and in response to nerve injury where again it plays a key role.