Nudc as a new molecular target to investigate the pathogenesis and treatment of skeletal ciliopathies.

Cilia are hair-like protrusions found on the surface of almost every cell in the body. Research over the last two decades has shown that cilia play crucial roles in diverse biological processes, and that mutations that disrupt cilia lead to a number of important human diseases including birth defects, blindness, obesity, heart disease and also cancer. These many essential roles for cilia in disease stem from the concentration of many key proteins within the cilium. This is achieved by key 'molecular motors' which transport specific proteins into cilia, including a motor protein known as dynein.

The proposed research will investigate how dynein is trasnported into the cilium, and will also investigate how manipulation of dynein transport may be used to treat human disease. We have identified a new protein that forms part of this molecular motor complex, known as NUDC. Specific aims of the proposal are:

1) To understand how NUDC interacts with dynein and related proteins.
2) To identify other proteins that NUDC interacts with to regulate dynein.
3) To investigate how disrupting Nudc affects cilia in vertebrates.
4) To create a mouse which lacks Nudc, to investigate which human disease this protein is relevant to.
5) To test whether loss of Nudc in mice carrying other ciliary mutations can rescue disease - this will therefore determine whether or not it might be possible to manipulate dynein trasnport for treatment of this category of disease.

Overall, the aim is to investigate the basic biology of dynein and cilia, and to investigate whether this process might be targeted for therapy.

Key aims of this proposal are:
1) To characterise the protein-protein interaction between IFT80 and NUDC: Having identified a novel interaction between IFT80 and NUDC using tandem affinity purification (TAP), we will now confirm this interaction using co-immunoprecipitation (co-IP) experiments in various ciliated and non-ciliated cells lines, using full length and also deletion constructs for both proteins, to characterise the particular domains mediating this interaction.

2) To systematically identify NUDC-interacting proteins: We will now use further TAP experiments, this time using NUDC as bait, to identify the full repertoire of NUDC-interacting proteins. We will then confirm any novel interactions identified in this screen using further co-IP experiments.

3) To analyse the function of IFT80- and NUDC-interacting proteins in zebrafish: In order to evaluate the significance of these protein-protein interactions in a vertebrate model of ciliopathies, we will use morpholinos to knock-down genes encoding novel interactors in zebrafish and assess ciliopathies and other phenotypes, as well as cilia formation and function. Genetic interactions between these and other intraflagellar transport proteins will also be evaluated using pairwise morpholino injections.

4) To generate and characterise a Nudc mouse mutant: Using ES cells that have been successfully targeted for Nudc, we will generate mice carrying a mutation in this gene. These mice will be fully characterised for ciliopathy phenotypes and cilia formation. We will also generate primary mouse embryonic fibroblasts for cell biological and immuonflourescence studies.

5) To test for rescue of ciliopathies following knockdown of Nudc: We will cross Nudc or Ift80 mutant mice with Dync2h1 mutant mice to test for genetic rescue of ciliopathies. This will provide proof-of-concept in terms of targeting dynein trafficking for therapy. MEFs from these mice will also be analysed for ciliary trafficking defects.

I have previously identified RAB23 and MEGF8 mutations in Carpenter syndrome, a skeletal ciliopathy. Causative mutations have now been identified in over 40 families with affected individuals. My discovery has therefore provided such families with important information for genetic counselling and a firm diagnosis for their affected children. As a consequence, screening for RAB23 mutations has now been transferred to the NHS as a clinical diagnostic test. This work on RAB23 was recently highlighted as an 'Achievement' in the MRC Annual Review 2010/11 (http://perspectives.mrc.ac.uk/chapters/cells-genes-and-molecules#achievement-6). Therefore, the human genetics aspect of my work, including an ongoing collaborations with leading clinicians in the field of ciliopathies, including Prof. Philip Beales and Prof. Andrew Wilkie, to identify/investigate new disease genes based on results from my proposed protein interaction mouse studies will have direct impact on human subjects, and this is likely to happen within the next few years.

Patients with ciliopathies suffer from a range of conditions including skull malformations (craniosynostosis), heart malformations, blindness, renal cystic disease and postnatal obesity. Ciliary dysfunction has also been linked to cancer, particularly peadiatric brain tumours and adult skin cancers, related to abnormal hedgehog signalling. Therefore, the studies on ciliary trafficking that I propose are likely to illuminate the pathogenesis of several important medical problems. However, there are currently no treatment options for ciliopathies. The current proposal will serve as proof-of-concept to evaluate whether manipulation of a specific component within cilia - namely the dynein molecular motor - may be used for therapy. This will be tested directly by genetic rescue experiments in mutant mice, together with other functional studies to monitor dynein transport. In future, it is anticipated that experiments such as this will lead to new therapies for patients in the clinic.