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

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.

Eye movement disorders are relatively common in humans (~1%). Such disorders include Duane Retraction Syndrome (DRS) a form of squint or strabismus, in which horizontal eye movements are impaired. If the condition is severe, it can lead to weakness of vision and partial blindness, or if less severe, it can be debilitating in daily life. We have previously demonstrated that mutations in the alpha2-chimaerin RacGAP cytoplasmic signalling protein are causal in a congenital form of DRS. Patients with these mutations manifest wiring defects of the ocular motor system - the system of three cranial nerves and six muscles which control eye movements. Our previous work has demonstrated that alpha2-chimaerin plays a key role in integrating axon guidance information and transducing it to remodel the cytoskeleton, allowing precise axon navigation. Manipulation of alpha2-chimaerin signalling in the ocular motor system in vivo produces striking axon guidance defects, akin to DRS in humans. In order to decipher the cellular mechanisms that cause DRS, we require a detailed understanding of the signalling pathways within neurons that involve alpha2-chimaerin. In this project, we will use state-of-the-art unbiassed proteomics approaches to identify novel proteins which interact with alpha2-chimaerin as components of a signalling 'module'. In parallel we will explore the role of previously identified alpha2-chimaerin-interacting proteins. Promising candidates will be validated using subcellular localisation assays and reversed co-immunoprecipitation, and ranked lists of candidates will be refined using bioinformatic tools and databases. Once several key candidates have been identified, their functional role will be tested using in vivo assays for nerve wiring and eye movements in the zebrafish. Our data will be used to generate maps of the protein networks that comprise the alpha2-chimaerin signalling module, and will allow us to determine mechanisms which lead to DRS.

As we have stated in the 'Academic Beneficiaries' section, researchers in the fields of neural development, cell biology, bioinformatics and ' interactomics', and clinical neurologists will benefit directly from our research.

In order to ensure appropriate impact of our research for the wider community we intend to pursue a number of avenues. Firstly we will explore the possibility of exploitation of our discoveries through discussion with the Technology Transfer team at King's College Business, who can help us to identify Intellectual Property (IP) and to liaise with appropriate IP law firms, pharmaceutical and biotechnology companies. Relevant existing partnerships include those with Pfizer and GSK. In the longer term, the commercial private sector is likely to be interested in alpha2-chimaerin interacting proteins identified by us due to their involvement in axon growth and potentially regeneration.

Research that follows on from the current project will involve screening small molecule libraries on zebrafish embryos to identify compounds which rescue axon guidance phenotypes resulting from morpholino knockdown of candidate genes, and restore normal axon guidance and ocular motility. This screening procedure is available at modest cost over a short timescale and will be used to identify drugs which may then be promising targets for therapies for axon guidance defects in humans. We will also consult with Dr Nick Gutowski, a clinical neurologist in Exeter who has done extensive work on cohorts of DRS patients including characterisation of patient mutations. He can search for mutations in novel proteins which we identify within his patient DNA samples to uncover novel disease associations.

The alpha2-chimaerin interacting proteins which we identify in this project and any ensuing commercial development will benefit the wider public by feeding into clinical practice and improving human health. Knowledge about the signalling pathways that underlie nerve connectivity in the ocular motor system will inform decisions made by clinicians about the likely course of eye movement disorders, and whether/when to perform squint surgery. The third (charitable) sector will also benefit from our research via our existing relationship with Fight for Sight, a blindness charity. We visit Fight for Sight to present and discuss our work, and to gain constructive insights into about how our system can be applied to tackling vision problems in humans.

We will also have impact in the training of staff involved in this project. Dr Ivana Poparic, who is already a talented molecular biologist and neuroscientist will develop her skill set extensively in a marketable research area which is at the boundary between basic and clinical science. She will learn about the conduct of mass spectrometry experiments and various validation and functional assays in vitro and in vivo. She will also benefit from the opportunity to train and mentor MSc and BSc students who each year perform a practical project in our lab. The unnamed research technician will similarly acquire extensive skills, focussing on the mass spectrometry, bioinformatics and in vitro aspects. SG's lab will also provide training over the summer months for school students from a socially-deprived area (Southwark, London) who will come to acquire work experience as part of the 'In2ScienceUK' organisation.