Deciphering alpha2-chimaerin signalling pathways in ocular motor development and Duane Retraction Syndrome

破译 α2-chimaerin 信号通路在眼部运动发育和杜安回缩综合征中的作用

• 批准号: MR/L020742/2
• 负责人: Sarah Guthrie
• 金额: $ 34.75万
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL020742%2F2
• 关键词: Deciphering; alpha2; chimaerin; signalling; pathways

Squint is common in humans. If the condition is severe, it can lead to weakness of vision and partial blindness, or if less severe, it can be debililitating in daily life and particularly in social situations. Squint is the failure to align the eyes correctly, and is now known to result from errors in nerve growth during development of the foetus. One or more of the cranial nerves that control the eye muscles may develop aberrantly, leading to a lack of co-ordination of eye movements. The only medical treatment currently available to treat squint is surgery to weaken a normal muscle ('mutilant surgery') or eye patching in children. Recently, scientific evidence has shown that a form of squint called Duane Retraction Syndrome (DRS) result from mutations in the molecule alpha2-chimaerin. Studies from our laboratory have shown that alpha2-chimaerin is a linchpin during development of the nervous system, being responsible for the precise navigation of cranial nerves to the eye muscles. Alpha2-chimaerin resides in the interior of neurons, and responds to incoming signals in the environment of the developing foetus, engaging these signals with the machinery inside neurons that allows them to grow, branch and connect up with the eye muscles. To perform this role, the alpha2-chimaerin molecule is dynamic, interacting with multiple other molecular partners. To understand in detail how and why alpha2-chimaerin mutations produce squint, we need to know far more about how this molecule works in the neuron, and which molecules it interacts with. Our project will use the powerful technology of mass spectrometry to identify new molecules that interact with alpha2-chimaerin. We will isolate chimaerin and the complex of interacting molecules from cells and then produce a sort of molecular fingerprint of this complex using mass spectrometry. This group of molecules which we call a signalling 'module', will then be compared and contrasted with the list of molecules that interact with mutant forms of alpha2-chimaerin found in humans. It is likely that one of the effects of squint mutations will be to change the detailed pattern of interactions of alpha2-chimaerin and its associates inside neurons. This information will start to allow us to dissect out the process that leads to faulty nerve wiring and eye movement defects.Once a list 'candidate' molecules has been identified, it will be winnowed down using sophisticated bioinformatics, and by confirming that individual molecules associate with alpha2-chimaerin in neurons using molecular localisation techniques. A group of three to five promising candidates will then be analysed further by using the zebrafish as an animal model. Surprisingly, this provides an excellent model for squint in humans, as the system of eye muscles and nerves is identical, and we can film the eye movements of the developing fish. As we know that eliminating alpha2-chimaerin function in the zebrafish produces nerve wiring defects similar to DRS in humans, we will then compare these wiring defects with those produced when we manipulate the expression of our candidate interactome molecules. If the defects are similar, this will suggest that our novel molecules play an important role in eye muscle wiring and DRS. A further step in establishing the importance of our identified molecules will be to introduce them into zebrafish which carry human alpha2-chimaerin mutations. As these mutant fish have faulty nerve wiring and defective eye movements, we will test whether the candidate molecules can restore normal development and eye function, by filming the fishes' eye movements. The key importance of this experiment is that it may pinpoint one or more molecule which might in future provide a therapy for squint in humans. Our findings will be shared with our clinical collaborators so that in time a therapy for squint may be brought to the clinic.

斜视在人类中很常见。如果病情严重，可能会导致视力减弱和部分失明，如果病情不太严重，则可能会导致日常生活尤其是社交场合的衰弱。斜视是指眼睛无法正确对齐，目前已知是由于胎儿发育过程中神经生长错误造成的。控制眼部肌肉的一根或多根脑神经可能发育异常，导致眼球运动缺乏协调。目前唯一可用于治疗斜视的药物是削弱正常肌肉的手术（“残障手术”）或儿童眼罩。最近，科学证据表明，一种称为杜安回缩综合症 (DRS) 的斜视是由 α2-嵌合蛋白分子突变引起的。我们实验室的研究表明，α2-嵌合蛋白是神经系统发育过程中的关键，负责脑神经到眼部肌肉的精确导航。 Alpha2-chimaerin 存在于神经元内部，对发育中胎儿环境中的传入信号做出反应，将这些信号与神经元内部的机制结合起来，使神经元能够生长、分支并与眼部肌肉连接。为了发挥这一作用，α2-嵌合蛋白分子是动态的，与多个其他分子伙伴相互作用。为了详细了解 α2-嵌合蛋白突变如何以及为何会导致斜视，我们需要更多地了解该分子如何在神经元中发挥作用，以及它与哪些分子相互作用。我们的项目将使用强大的质谱技术来识别与 alpha2-chimaerin 相互作用的新分子。我们将从细胞中分离嵌合蛋白和相互作用分子的复合物，然后使用质谱法产生该复合物的一种分子指纹。我们将这组分子称为信号“模块”，然后将其与与人类中发现的 α2-嵌合蛋白突变体相互作用的分子列表进行比较和对比。斜视突变的影响之一可能是改变神经元内α2-嵌合蛋白及其相关物相互作用的详细模式。这些信息将使我们能够剖析导致神经布线错误和眼球运动缺陷的过程。一旦确定了“候选”分子列表，我们将使用复杂的生物信息学对其进行筛选，并使用分子定位技术确认单个分子与神经元中的 α2-嵌合蛋白相关。然后将使用斑马鱼作为动物模型对三到五个有希望的候选者进行进一步分析。令人惊讶的是，这为人类斜视提供了一个极好的模型，因为眼部肌肉和神经系统是相同的，我们可以拍摄发育中的鱼类的眼球运动。我们知道，消除斑马鱼中的 α2-嵌合蛋白功能会产生类似于人类 DRS 的神经布线缺陷，然后我们将这些布线缺陷与我们操纵候选相互作用组分子的表达时产生的缺陷进行比较。如果缺陷相似，则表明我们的新型分子在眼肌布线和 DRS 中发挥着重要作用。确定我们所鉴定的分子的重要性的进一步步骤是将它们引入携带人类α2-嵌合蛋白突变的斑马鱼中。由于这些突变鱼的神经布线有缺陷，眼球运动也有缺陷，我们将通过拍摄鱼的眼球运动来测试候选分子是否可以恢复正常发育和眼睛功能。这项实验的关键重要性在于，它可能会查明一种或多种分子，这些分子将来可能会为人类斜视提供治疗方法。我们的研究结果将与我们的临床合作者分享，以便及时将斜视治疗方法带到诊所。

项目成果

Cadherins regulate nuclear topography and function of developing ocular motor circuitry.

• DOI: 10.7554/elife.56725
• 发表时间: 2020-10-01
• 期刊: eLife
• 影响因子: 7.7
• 作者: Knüfer A;Diana G;Walsh GS;Clarke JD;Guthrie S
• 通讯作者: Guthrie S