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

斜视在人类中很常见。如果病情严重，它可能导致视力下降和部分失明，或者如果不太严重，它可能在日常生活中，特别是在社交场合中变得虚弱。斜视是指眼睛不能正确对齐，现在已知是由于胎儿发育期间神经生长错误造成的。一个或多个控制眼肌的脑神经可能会发育异常，导致眼球运动缺乏协调。目前治疗斜视的唯一方法是手术削弱正常肌肉（“截肢手术”）或儿童眼睛修补。最近，科学证据表明，一种名为Duane Retraction Syndrome（DRS）的斜视是由α 2-chimaerin分子突变引起的。我们实验室的研究表明，α 2-chimaerin是神经系统发育过程中的关键，负责颅神经到眼肌的精确导航。α 2-嵌合蛋白存在于神经元内部，并对发育中的胎儿环境中的传入信号做出反应，使这些信号与神经元内部的机制相结合，使它们能够生长，分支并与眼肌连接。为了发挥这一作用，α 2-嵌合蛋白分子是动态的，与多个其他分子伴侣相互作用。为了详细了解α 2-嵌合蛋白突变如何以及为什么会产生斜视，我们需要更多地了解这种分子在神经元中是如何工作的，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α 2-嵌合蛋白相互作用的新分子。我们将从细胞中分离嵌合蛋白和相互作用的分子复合物，然后使用质谱法产生这种复合物的分子指纹。这组分子，我们称之为一个信号'模块'，然后将进行比较，并与分子的清单，相互作用的突变形式的α 2-嵌合蛋白在人类中发现。斜视突变的影响之一可能是改变神经元内α 2-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开始剖析导致神经连接错误和眼球运动缺陷的过程。一旦确定了一系列“候选”分子，它将通过复杂的生物信息学进行筛选，并通过分子定位技术确认单个分子与神经元中的α 2-嵌合蛋白相关。然后，将使用斑马鱼作为动物模型，进一步分析一组三到五个有希望的候选者。令人惊讶的是，这为人类斜视提供了一个很好的模型，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的眼球运动。正如我们所知，消除斑马鱼中的α 2-chimaerin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体分子表达时产生的缺陷进行比较。如果缺陷是相似的，这将表明我们的新分子在眼肌布线和DRS中发挥重要作用。建立我们鉴定的分子的重要性的进一步步骤将是将它们引入携带人类α 2-嵌合蛋白突变的斑马鱼中。由于这些突变鱼的神经线路有缺陷，眼睛运动也有缺陷，我们将通过拍摄鱼的眼睛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，它可以确定一种或多种分子，这些分子可能在未来为人类斜视提供治疗。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带到临床。

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.