Loss of UNC13A: how it exacerbates amyotrophic lateral sclerosis, and how to correct it

UNC13A 缺失：如何加剧肌萎缩侧索硬化症以及如何纠正

• 批准号: MR/W005190/1
• 负责人: Pietro Fratta
• 金额: $ 107.91万
• 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: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FW005190%2F1
• 关键词: Loss; UNC13A; how; exacerbates; amyotrophic

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a devastating neurodegenerative disorder that causes progressive loss of MNs, leading to impaired muscle function and paralysis. ALS is incurable and leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of about 1 in 400.A crucial player in the disease mechanism of ALS is a protein called TDP-43. TDP-43 mislocalisation in neurons occurs in ~97% of ALS, regardless of its many possible causes. TDP-43 is important for RNA splicing, a process by which RNA, the information deriving from DNA, is cut and re-assembled to provide cells with essential information. When TDP-43 cannot perform its function, numerous alterations of splicing occur, including a phenomenon called "cryptic exons", where novel random sequences are inserted into RNAs.In ALS brains, following TDP-43 alterations, multiple cryptic exon mistakes occur, and it has been difficult to understand which ones play a role in disease. We have now identified a novel cryptic exon event in the UNC13A gene. UNC13A encodes a protein that is important for the functioning of synapses, which allow neurons to transfer information between each other - the essential step for brain function.Unexpectedly, what makes the UNC13A cryptic exon different from the numerous other cryptic exons, is that genetics data from ALS patients supports it being very important for disease. In fact, a common genetic change in UNC13A has been long known to be associated with ALS, but why has remained elusive.Here, with the discovery of the UNC13A cryptic exon, we have been able to provide the missing link. In summary, when TDP-43 alterations occur in ALS, they induce the UNC13A cryptic exon, which in turn leads to a reduction of this crucial synaptic protein.Our hypothesis is that preventing this loss will have a positive impact on ALS disease course.In this research proposal we aim to better understand the UNC13A cryptic exon in ALS, its consequences and how we can prevent it from happening. We will:1. Identify in exactly what neuronal subtypes UNC13A is altered in ALS. This question is important to both understand the disease, and to know what we need to target in therapeutic interventions.We will answer our question by studying brains and spinal cords from ALS cases. We will visualise the altered UNC13A RNA and also use novel techniques that allow us to study all the RNAs in multiple single neurons from these cases.2. Investigate how the UNC13A cryptic exon impacts basic neuronal functions. This work will shed new light on disease mechanism in ALS and will be crucial to set up future assays for therapeutic development.We routinely use human (iPSC-derived) ALS cortical and motor neurons in culture and we can induce the UNC13A cryptic exon. We will study, with microscopy and electrophysiology, how the synapse functions in these neurons, and we will also study if other neuronal problems associated with ALS are impacted by the UNC13A cryptic exon.3. Understand the mechanism underlying the UNC13A cryptic exon and devise strategies to prevent it. This will allow us to understand how the UNC13A cryptic exon occurs, and will allow us to design oligonucleotide strategies - currently being used in the clinic for other neurological diseases - to interfere with this toxic mechanism. We have developed small versions of UNC13A that allow us to study the splicing mechanism very effectively. We will use this system also to efficiently screen and identify the best oligonucleotides for preventing this event.In summary, this project will contribute to understanding how changes in UNC13A, induced by TDP-43, impact on neurons and ALS pathogenesis, regardless of underlying cause. Crucially, this project will also develop an approach to prevent these changes and perform the initial steps for a potential therapeutic strategy for ALS.

肌萎缩性侧索硬化症（ALS），也被称为运动神经元疾病（MND），是一种毁灭性的神经退行性疾病，导致MNs逐渐丧失，导致肌肉功能受损和瘫痪。ALS是无法治愈的，通常会导致死亡，通常是由于无法呼吸，平均仅在诊断后3年就会死亡，终生风险约为400分之一。在ALS的发病机制中起关键作用的是一种叫做TDP-43的蛋白质。尽管有许多可能的原因，但约97%的ALS患者发生了神经元中TDP-43的错误定位。TDP-43对RNA剪接很重要，RNA剪接是一个过程，通过这个过程，RNA（来自DNA的信息）被切割并重新组装，为细胞提供必要的信息。当TDP-43不能发挥其功能时，就会发生许多剪接改变，包括一种称为“隐外显子”的现象，即新的随机序列插入rna中。在ALS大脑中，随着TDP-43的改变，会发生多个隐性外显子错误，并且很难理解哪些外显子在疾病中起作用。我们现在已经在UNC13A基因中发现了一个新的隐式外显子事件。UNC13A编码一种蛋白质，这种蛋白质对突触的功能很重要，突触允许神经元在彼此之间传递信息——这是大脑功能的基本步骤。出乎意料的是，使UNC13A隐外显子与许多其他隐外显子不同的是，来自ALS患者的遗传学数据支持它对疾病非常重要。事实上，UNC13A的一种常见的遗传变化早就被认为与ALS有关，但原因仍然难以捉摸。在这里，随着UNC13A神秘外显子的发现，我们已经能够提供缺失的环节。总之，当TDP-43在ALS中发生改变时，它们会诱导UNC13A隐式外显子，从而导致这种关键突触蛋白的减少。我们的假设是，防止这种损失将对ALS的病程产生积极的影响。在这项研究计划中，我们的目标是更好地了解ALS中的UNC13A隐外显子，它的后果以及我们如何预防它的发生。我们将:1。确定在肌萎缩性侧索硬化症中UNC13A究竟改变了哪些神经元亚型。这个问题对于了解这种疾病，以及了解我们在治疗干预中需要针对什么都很重要。我们将通过研究ALS患者的大脑和脊髓来回答这个问题。我们将可视化改变的UNC13A RNA，并使用新技术，使我们能够从这些病例中研究多个单个神经元中的所有RNA。研究UNC13A隐外显子如何影响基本神经元功能。这项工作将为ALS的发病机制提供新的线索，并将对建立未来治疗开发的检测方法至关重要。我们常规地使用人类（ipsc衍生的）ALS皮层和运动神经元进行培养，我们可以诱导UNC13A的隐外显子。我们将通过显微镜和电生理学研究突触如何在这些神经元中起作用，我们还将研究与ALS相关的其他神经元问题是否受到UNC13A隐式外显子的影响。了解UNC13A神秘外显子的机制，并制定预防策略。这将使我们了解UNC13A隐外显子是如何发生的，并将使我们能够设计寡核苷酸策略——目前用于临床治疗其他神经系统疾病——来干扰这种毒性机制。我们已经开发了小版本的UNC13A，使我们能够非常有效地研究剪接机制。我们还将使用该系统有效地筛选和识别预防该事件的最佳寡核苷酸。综上所述，本项目将有助于了解TDP-43诱导的UNC13A变化如何影响神经元和ALS发病机制，而不考虑其潜在原因。至关重要的是，该项目还将开发一种预防这些变化的方法，并为ALS的潜在治疗策略执行初步步骤。

项目成果

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

• DOI: 10.1038/s42003-022-03253-8
• 发表时间: 2022-04-05
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Šušnjar U;Škrabar N;Brown AL;Abbassi Y;Phatnani H;NYGC ALS Consortium;Cortese A;Cereda C;Bugiardini E;Cardani R;Meola G;Ripolone M;Moggio M;Romano M;Secrier M;Fratta P;Buratti E
• 通讯作者: Buratti E

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

错误剪接的转录本在 TDP-43 相关的 ALS/FTD 中从头生成蛋白质。

• DOI: 10.1101/2023.01.23.525149
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Seddighi,Sahba;Qi,YueA;Brown,Anna-Leigh;Wilkins,OscarG;Bereda,Colleen;Belair,Cedric;Zhang,Yongjie;Prudencio,Mercedes;Keuss,MatthewJ;Khandeshi,Aditya;Pickles,Sarah;Hill,SarahE;Hawrot,James;Ramos,DanielM;Yuan,Hebao;Roberts
• 通讯作者: Roberts

UNC13A in amyotrophic lateral sclerosis: from genetic association to therapeutic target.

• DOI: 10.1136/jnnp-2022-330504
• 发表时间: 2023-08
• 期刊: Journal of neurology, neurosurgery, and psychiatry

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