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

肌萎缩侧索硬化症（ALS），也称为运动神经元疾病（MND），是一种破坏性的神经退行性疾病，其导致MN的进行性丧失，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，通常是由无法呼吸引起的，平均诊断后仅3年，终生风险约为1/400。ALS疾病机制中的一个关键角色是一种称为TDP-43的蛋白质。TDP-43在神经元中的错误定位发生在约97%的ALS中，无论其许多可能的原因如何。TDP-43对RNA剪接很重要，RNA剪接是一个过程，通过这个过程，来自DNA的信息RNA被切割并重新组装，为细胞提供必要的信息。当TDP-43不能执行其功能时，会发生许多剪接的改变，包括称为“隐藏外显子”的现象，其中新的随机序列插入到RNA中。在ALS大脑中，在TDP-43改变后，会发生多个隐藏外显子错误，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UNC 13 A基因中鉴定了一个新的隐蔽外显子事件。UNC 13 A编码一种对突触功能非常重要的蛋白质，突触允许神经元相互传递信息-这是大脑功能的重要步骤。出乎意料的是，UNC 13 A隐藏外显子与许多其他隐藏外显子不同的是，ALS患者的遗传学数据支持它对疾病非常重要。事实上，UNC 13 A中的一个常见的遗传变化长期以来一直被认为与ALS相关，但为什么仍然难以捉摸。在这里，随着UNC 13 A隐藏外显子的发现，我们已经能够提供缺失的一环。总之，当TDP-43基因突变发生在ALS中时，它们会诱导UNC 13 A隐性外显子，从而导致这种关键的突触蛋白的减少。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。在这项研究中，我们的目标是更好地了解ALS中UNC 13 A隐性外显子，其后果以及我们如何预防它的发生。我们将：1.确定究竟是什么神经元亚型UNC 13 A在ALS中发生了改变。这个问题对于理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病例的大脑和脊髓来回答我们的问题。我们将可视化改变的UNC 13 A RNA，并使用新的技术，使我们能够研究这些病例中多个单个神经元中的所有RNA。研究UNC 13 A隐性外显子如何影响基本神经元功能。这项工作将为ALS的疾病机制提供新的线索，并将对建立未来的治疗开发检测方法至关重要。我们常规使用人类（iPSC衍生的）ALS皮层和运动神经元进行培养，我们可以诱导UNC 13 A隐藏外显子。我们将用显微镜和电生理学研究突触在这些神经元中的功能，我们还将研究与ALS相关的其他神经元问题是否受到UNC 13 A隐藏外显子的影响。了解UNC 13 A隐性外显子的潜在机制并设计策略来预防它。这将使我们能够了解UNC 13 A隐性外显子是如何发生的，并将使我们能够设计寡核苷酸策略-目前正在临床上用于其他神经系统疾病-来干扰这种毒性机制。我们已经开发了UNC 13 A的小版本，使我们能够非常有效地研究剪接机制。我们也将使用这个系统来有效地筛选和鉴定用于预防这种事件的最佳寡核苷酸。总之，这个项目将有助于了解TDP-43诱导的UNC 13 A的变化如何影响神经元和ALS发病机制，无论潜在的原因是什么。至关重要的是，该项目还将开发一种方法来防止这些变化，并为ALS的潜在治疗策略执行初始步骤。

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.