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

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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.