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

在正常和 ALS 相关小鼠模型中，通过 RNA TU 标记新技术研究神经元亚细胞转录组。

• 批准号: MR/K018523/1
• 负责人: Elizabeth Fisher
• 金额: $ 48.22万
• 依托单位: University College London
• 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%2FK018523%2F1
• 关键词: Investigating; neuronal; subcellular; transcriptome; novel

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.

肌萎缩性侧索硬化症（ALS，“运动神经元疾病”）是一种毁灭性的神经退行性疾病，会导致肌肉功能的逐渐丧失和瘫痪。ALS导致死亡，通常由呼吸困难引起，平均仅在诊断后3年死亡，到85岁时的终生风险为1 / 250。这种疾病的主要受累细胞是运动神经元（MNs）。神经网络将大脑与肌肉连接起来，从而使运动成为可能。肌萎缩侧索硬化症患者在病程中逐渐死亡。MNs是人体最大的细胞之一。它们的主体位于脊髓中，包含许多被称为树突的细分支过程。它们也有一个很细的突起，叫做轴突，它从脊髓延伸到我们的每一块肌肉。单个轴突的长度可以超过一米，从脊髓延伸到手指或脚趾的末端。MN和肌肉之间的连接称为神经肌肉连接处（NMJ）。一个人体内的所有细胞，尽管彼此差异很大，但都含有相同的DNA，即向每个细胞发出指令的遗传物质。因此，每种细胞类型（例如，细胞是神经细胞还是心脏细胞）的身份是DNA的哪个区域活跃并产生另一种称为RNA的化学物质的结果。RNA携带着细胞功能所需的所有信息。细胞中所有RNA的总和，称为转录组，是表征每种细胞类型的特征。了解一个细胞转录组可以深入了解其生物学，并有助于确定疾病的原因。这与ALS中的MNs特别相关，因为有充分的证据表明，与RNA“代谢”相关的生物过程主要在ALS中受到影响。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发挥作用；在MNs中，它通过轴突和NMJs转运，发挥特定的作用。NMJs和轴突被认为是肌萎缩侧索硬化症中最先受到影响的MNs部分。因此，了解哪些rna存在于细胞体和树突、轴突和MNs的神经肌肉连接处是很重要的，以了解它们如何正常运作，以及在ALS中出现了什么问题。当神经元及其轴突在培养皿中人工培养或在显微镜下从固定组织中分离出细胞体时，就有可能从神经元及其轴突中分离和鉴定RNA。这些发现表明，数千种不同的RNA物种被积极地转运到轴突，但从这些实验中很难提取出与体内自然环境下成熟神经元相关的信息，因此也很难提取出与疾病相关的信息。我们将使用一种新技术，“tu标记”，这种技术已经在老鼠身上成功应用，它允许我们在活体动物的背景下“标记”特定细胞中的RNA。然后可以通过高通量测序分离和鉴定标记的RNA。我们将把这项技术应用于MNs，因此我们首次可以从MNs细胞体和树突，以及它们的轴突和NMJs中分离和鉴定RNA。我们将用正常小鼠和我们开发的一种新的小鼠模型进行研究，这种模型在导致ALS的基因Tardbp（也称为Tdp-43）中存在缺陷。从我们目前的工作中，我们知道该基因的缺陷在该小鼠的MN细胞体中产生了异常的RNA谱。目前还没有Tdp-43突变小鼠完全模拟人类ALS，但它们告诉我们很多关于Tdp-43在正常和异常状态下的功能。这些结果将极大地有助于我们进一步了解MN生物学，以及是什么原因导致这些细胞在ALS中如此特别脆弱。这个项目将有助于研究许多其他疾病，在这些疾病中，不同细胞区域的RNA代谢是重要的，在神经损伤的反应中，它也起着关键作用。

项目成果

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

• DOI: 10.15252/embj.201798684
• 发表时间: 2018-06-01
• 期刊: The EMBO journal
• 作者: Fratta P;Sivakumar P;Humphrey J;Lo K;Ricketts T;Oliveira H;Brito-Armas JM;Kalmar B;Ule A;Yu Y;Birsa N;Bodo C;Collins T;Conicella AE;Mejia Maza A;Marrero-Gagliardi A;Stewart M;Mianne J;Corrochano S;Emmett W;Codner G;Groves M;Fukumura R;Gondo Y;Lythgoe M;Pauws E;Peskett E;Stanier P;Teboul L;Hallegger M;Calvo A;Chiò A;Isaacs AM;Fawzi NL;Wang E;Housman DE;Baralle F;Greensmith L;Buratti E;Plagnol V;Fisher EM;Acevedo-Arozena A
• 通讯作者: Acevedo-Arozena A

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

• DOI: 10.1038/s41586-022-04436-3
• 发表时间: 2022-03
• 期刊: Nature
• 影响因子: 64.8
• 作者: Brown AL;Wilkins OG;Keuss MJ;Hill SE;Zanovello M;Lee WC;Bampton A;Lee FCY;Masino L;Qi YA;Bryce-Smith S;Gatt A;Hallegger M;Fagegaltier D;Phatnani H;NYGC ALS Consortium;Newcombe J;Gustavsson EK;Seddighi S;Reyes JF;Coon SL;Ramos D;Schiavo G;Fisher EMC;Raj T;Secrier M;Lashley T;Ule J;Buratti E;Humphrey J;Ward ME;Fratta P
• 通讯作者: Fratta P

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation.

• DOI: 10.1126/sciadv.abf8660
• 发表时间: 2021-07
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Birsa N;Ule AM;Garone MG;Tsang B;Mattedi F;Chong PA;Humphrey J;Jarvis S;Pisiren M;Wilkins OG;Nosella ML;Devoy A;Bodo C;de la Fuente RF;Fisher EMC;Rosa A;Viero G;Forman-Kay JD;Schiavo G;Fratta P
• 通讯作者: Fratta P

Widespread RNA metabolism impairment in sporadic inclusion body myositis TDP43-proteinopathy.

• DOI: 10.1016/j.neurobiolaging.2013.12.029
• 发表时间: 2014-06
• 期刊: Neurobiology of aging
• 影响因子: 4.2
• 作者: Cortese A;Plagnol V;Brady S;Simone R;Lashley T;Acevedo-Arozena A;de Silva R;Greensmith L;Holton J;Hanna MG;Fisher EM;Fratta P
• 通讯作者: Fratta P

Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice.

• DOI: 10.1093/brain/awx248
• 发表时间: 2017-11-01
• 期刊: Brain : a journal of neurology
• 作者: Devoy A;Kalmar B;Stewart M;Park H;Burke B;Noy SJ;Redhead Y;Humphrey J;Lo K;Jaeger J;Mejia Maza A;Sivakumar P;Bertolin C;Soraru G;Plagnol V;Greensmith L;Acevedo Arozena A;Isaacs AM;Davies B;Fratta P;Fisher EMC
• 通讯作者: Fisher EMC