Bilateral BBSRC-SFI: Structure-function relationships in the ciliary transition zone

Lead Research Organisation: University of Leeds
Department Name: School of Medicine

Abstract

Cilia are small 'antennae-like' structures which protrude from the surface of most animal cells. Like antennae, they receive and transduce signals from other cells and their surroundings in order to coordinate appropriate cell behaviours. This is especially important in development, and defects in cilia lead to a range of human developmental diseases called "ciliopathies". These conditions range from severe, lethal conditions that involve complex defects in multiple organs including the brain, to relatively mild and organ-specific conditions such as retinitis pigmentosa, which is a form of hereditary, progressive sight loss. Scientists still do not fully understand how cilia help to control brain and retina development. Although these conditions are individually rare, collectively they are a common cause of morbidity and mortality in babies and young children but remain difficult to diagnose and treat.

This research proposes to identify genes that contribute individually, or within groups (pathways), to the formation of a sub-structure of the cilium called the transition zone. The transition zone at the base of the cilium is thought to function as a type of 'gate', controlling which signal transduction molecules are allowed to enter and exit the cilium. Many of the genes that cause ciliopathies are thought to function in the transition zone and regulate its 'gating' function. To achieve our aims, we will take advantage of recent exciting advances in genetic technology that allow us to evaluate the contribution of every gene to cilia and transition zone formation ("reverse genetics screen"). We are uniquely placed to do this work and the team of investigators have a proven track record of success in this field: we have formed excellent research partnerships with other workers in the field to participate in gene identification studies; we have the appropriate state-of-the-art technology, image analysis tools and experience; and we have already produced and validated large data-sets from existing work that we now wish to exploit more extensively in the present research proposal. We will study key genes ("screen hits") and their contributions to cilia and transition zone formation, and, in particular we will use specialised cell model systems in combination with a versatile animal model (a nematode roundworm).

The identification and characterisation of new genes required for the structure and function of the cilium and the transition zone 'gate' provides a number of major benefits. Firstly, important and often unexpected scientific insights are made into disease processes and into the normal function of the disease gene that can lead to new treatments. Secondly, new ciliary genes often enable accurate genetic testing for patients and families with ciliopathies, which improve diagnosis and genetic counselling. Thirdly, our proposed work has a wider biological relevance because cilia have recently been shown to control metabolism, which may link to neurodegenerative diseases and metabolic disorders such as insulin resistance. Thus, a better understanding of cilium biology may provide opportunities for developing drugs or new treatments to prevent the progression of more common ailments (e.g., diabetes. obesity) that present in some ciliopathy patients. Finally, we expect that our work will provide new insights into how cilia disease proteins are arranged relative to one another within the transition zone, and how this molecular organisation facilitates the gating function of this discrete region of the cilium. This new knowledge will have important implications for molecular 'gates' that exist elsewhere in the cell, and serve to expand the impact of our work to scientists working on related questions.

Technical Summary

This research programme builds on the applicants' combined expertise in reverse genetics screening and in vivo modelling of transition zone (TZ) and ciliary function in cell and C. elegans animal models. The proposal impetus, and an example of our collaboration, was our 2015 study on TMEM107. Using high-throughput siRNA technology in mammalian cells (Workpackage 1; WP1) and C. elegans mutant alleles (WP2), we will identify novel candidate TZ genes. The resulting functional genomics data-sets will be integrated and common 'hits' validated and functionally interrogated in both systems (including knockout cell lines) to define new TZ proteins (WP3). Specifically, we will dissect their roles in cilium-based signalling, transport, composition, 'gating', and relationship to known or novel functional modules. In WP4, both models will integrate new and known TZ proteins into the emerging architectural framework of the ciliary gate, and provide new insights into structure-function relationships within the ciliary apparatus such as the TZ permeability barrier. These experiments will involve advanced confocal imaging to visualise protein-protein interactions in living cells (FRET; BioID; BiDC) and worms (BiFC), and complementary super-resolution microscopy approaches (STED, dSTORM), combined with multiplexed imaging using artificial binding proteins ("Adhirons"), to enable unprecedented spatial and temporal resolution of TZ proteins. We provide preliminary data on exemplar projects to show the exciting discoveries that rapidly arise from functional genomics to guide hypothesis generation. In vitro high content imaging and in vivo genetic complementation assays is a cost and time effective approach to complex combinatorial and multiplexed experimentation, compared to more conventional approaches. The research will significantly improve the functional annotation of genes contributing to TZ formation and ciliogenesis, and will enhance our understanding of basic cilium biology.

Planned Impact

Although ciliopathies are a heterogenous group of rare inherited conditions, collectively they are a common cause of perinatal morbidity and mortality (1 in 500 births) and remain difficult to diagnose and treat. In the UK, about 4000 patients require renal replacement therapy (dialysis and transplantation) as a result of cystic kidney disease leading to kidney failure. The identification of new disease gene functions and pathways related to the ciliary transition zone (TZ) may enable the future rational design of preventative treatments or new therapeutic interventions for cystic kidney disease, retinal degeneration or ciliopathy disease progression, or approaches to improve the long-term outlook of patients with these conditions. For example, by understanding how the ciliary TZ and associated ciliopathy proteins regulate signalling via diffusion barriers, our work could lead to the design of small molecules that can advantageously increase or decrease permeability at the ciliary base, and thereby alleviate some of the progressive post-natal ciliopathy pathologies. Also, since these conditions result from absence of normal TZ proteins, they could in principle be corrected by gene replacement, therapeutic approaches now undergoing Phase III clinical trials.

It is likely that new TZ genes that we discover in this project will subsequently link to ciliary disease. Patients in whom new mutations are found can therefore be given a clearer prognosis and prioritised for any emerging new treatments, making inherited disease a top priority for further characterization. Recent advances have improved the molecular diagnosis of children with a known or suspected ciliopathy, and reduced the burden and disruption of a "diagnostic odyssey" for families. For clinicians, new gene identification can also reduce misdiagnosis or late diagnosis, inform the establishment of proper care pathways for ciliopathy patients, and prioritize patients that can most benefit from future targeted therapies. In families with a ciliopathy, the identification of a novel disease gene immediately permits genetic testing to at-risk relatives. Carrier tests and prenatal diagnosis can also encourage informed reproductive choices for families.

Recently, cilia have been shown to be essential for normal autophagic processes and have a broader role in metabolic control through mTOR signalling. This has wider medical relevance because autophagy is implicated in many human pathological processes, including neurodegenerative diseases and human metabolic disorders. The understanding of these pathophysiological processes poses some of the most significant challenges to modern biomedical research, and the regulatory relationships that control cilia function could therefore provide a new appreciation of their potential medical relevance in older age. The identification of new ciliary genes and pathways provides insights into normal human physiology and development, as well as the pathophysiology of common conditions (such as such as insulin resistance, obesity and cystic kidney disease) thus benefiting the wider population.

To understand ciliary TZ structure/function regulation, we will use functional genomics strategies to evaluate the contribution of large data-sets of genes. This is a proven strategy for identifying new and unexpected disease genes, mechanisms and pathways. We will integrate functional genomics with advanced cell biology methodologies in complementary physiologically-relevant cell and animal mutant models to derive cutting edge knowledge of the TZ system. Our work also aims to demonstrate the clinical utility and validity of integrated "systems medicine" annotation and its ability to make relevant predictions about ciliopathy disease causality. The development of a strong interface between gene discovery, functional annotation and diagnostic or translational development work is therefore an indirect but important added benefit of this research.
 
Description Our current research has uncovered an unexpected series of genetics interactions between key proteins in the primary cilium, the sensory "antenae" of the cell. The ciliary proteins include an transmembrane receptor (TMEM67), a fundamental organizing protein of ciliary architecture (RPGRIP1L) and a protein that mediates trafficking of components within the cilium (IFT88). The genetic interactions were identified using "crispant" cells, carrying mutations engineered by CRISPR-Cas9 genome editing, in combination with high content imaging (a type of automated microscopy imaging). More recently, the crispant cells have been used to validate the effect of a repurposed drug on cilia formation. We identified this drug from a screen of known drugs and clinical development compounds using high content imaging.
Exploitation Route Our characterized crispant cell-lines will be useful to other researchers in the cilia biology field for similar high content experiments including cell-based drug screening. The repurposed drug forms the basis of new pre-clinical studies in stem cell-derived tissues (renal organoids) which are currently submitted as grant applications to LifeArc and Action Medical Research.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description development and characterization of of novel non-antibody based protein binding reagents ("Affimers") against ciliary proteins (post-translationally modified tubulin, CEP290); these reagents have potential as detection reagents in (for example in super-resolution microscopy) and for modulation of ciliary processes; identification of a potential repurposed drug that could be used in pre-clinical studies, and that could form the basis of more extensive "hit derivation" work. (There are several hundred derivatives available as clinical-development compounds). The identification of a new repurposed medical or new derivative compounds with defined mechanisms of action are very likely to result in patentable or commercially exploitable outcomes.
First Year Of Impact 2017
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Elucidating splicing factor function and retinal splicing programmes: developing new therapeutic strategies for splicing factor retinitis pigmentosa
Amount £1,456,000 (GBP)
Funding ID MR/T017503/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 12/2024
 
Description Pre-clinical testing of new therapeutic treatments for cystic kidney disease
Amount £200,000 (GBP)
Funding ID GN2628 
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description multi-user equipment grant
Amount £429,000 (GBP)
Funding ID 208395/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 08/2022
 
Description project grant
Amount £200,000 (GBP)
Funding ID GN2628 
Organisation Action Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 02/2021
 
Title Affimers against ciliary proteins 
Description Affimer artificial non-antibody affinity and binding reagents against ciliary proteins for pull-down adn localization studies 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact development of better localization precision reagents in supper resolution microscopy 
 
Title genome-edited cell-lines 
Description a panel of isogenic knock-out cell-lines for key ciliary genes (validated by sequencing, western blotting and assays of ciliary function) to be used in reverse genetics screening, drug screening and as model systems of ciliopathies. New crispant cell-lines include null alleles in CEP290 and base-edited specific class 5 (pathogenic) and class 3 (unknown significance) variants. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact The CEP290 knock-out cell-line is being used as a model system for renal cystic disease in preclinical studies that have arisen from drug screens of clinical development compounds 
 
Description functional characterization of ciliopathy proteins 
Organisation Eberhard Karls University of Tübingen
Department Institute for Ophthalmic Research
Country Germany 
Sector Academic/University 
PI Contribution completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance
Collaborator Contribution partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models
Impact Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172.
Start Year 2011
 
Description functional characterization of ciliopathy proteins 
Organisation Johannes Gutenberg University of Mainz
Department Department of Cell & Matrix Biology
Country Germany 
Sector Academic/University 
PI Contribution completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance
Collaborator Contribution partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models
Impact Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172.
Start Year 2011
 
Description functional characterization of ciliopathy proteins 
Organisation Radboud University Nijmegen Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance
Collaborator Contribution partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models
Impact Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172.
Start Year 2011
 
Description functional characterization of ciliopathy proteins 
Organisation University of California, San Diego (UCSD)
Department Institute for Genomic Medicine
Country United States 
Sector Academic/University 
PI Contribution completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance
Collaborator Contribution partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models
Impact Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172.
Start Year 2011
 
Description structural biology of ciliary proteins 
Organisation University of Leeds
Department Astbury Centre for Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution protein purification of ciliary protein domains, production and charaterization of "Affimer " artifical binding proteins and their use as novel detection reagents in super resolution microscopy; optimizartion of cellular soft X-ray tomography of primary cilia 9using teh SXt B24 beamline at Diamond)
Collaborator Contribution protein production for Affimer generation and crysallography trials; optimization of novel analysis methods for super resolution microscopy (two colour dSTORM, STED); live cell imaging
Impact collation of preliminary data into a larger UKRI grant application
Start Year 2018
 
Description super-resolution microscopy of ciliary proteins 
Organisation University College Dublin
Department UCD Conway Institute of Biomedical annd Biomolecular Research
Country Ireland 
Sector Academic/University 
PI Contribution We have developed a panel of antibodies specific for ciliary proteins that have been used for both PALM and STORM imaging. We are developing non-antibody binding proteins (Adhirons) for this application.
Collaborator Contribution Provided the facilities and technical expertise for both STORM and PALM image capture, processing and analysis.
Impact The collaboration has led to the publication: Lambacher NJ, et al. (2015). TMEM107 recruits ciliopathy proteins to anchored periodic subdomains of the ciliary transition zone membrane and is mutated in Joubert syndrome. Nat. Cell Biol. doi: 10.1038/ncb3273 New funding application have been made to the MRC and BBSRC/SFI joint funding stream
Start Year 2015
 
Description super-resolution microscopy of ciliary proteins 
Organisation University of Leeds
Department Faculty of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed a panel of antibodies specific for ciliary proteins that have been used for both PALM and STORM imaging. We are developing non-antibody binding proteins (Adhirons) for this application.
Collaborator Contribution Provided the facilities and technical expertise for both STORM and PALM image capture, processing and analysis.
Impact The collaboration has led to the publication: Lambacher NJ, et al. (2015). TMEM107 recruits ciliopathy proteins to anchored periodic subdomains of the ciliary transition zone membrane and is mutated in Joubert syndrome. Nat. Cell Biol. doi: 10.1038/ncb3273 New funding application have been made to the MRC and BBSRC/SFI joint funding stream
Start Year 2015
 
Title Pre-clinical testing of ROCK2 inhibition as a new therapeutic treatments for cystic kidney disease 
Description Cilia are small structures that protrude from the surface of most animal cells like antennae. Like antennae, they receive signals from other cells and their surroundings to ensure that cells form tissues correctly. This is important in development, and defects in cilia cause inherited developmental disorders called "ciliopathies". These invariably have cystic kidney disease as a feature, and are a major cause of childhood morbidity and mortality, with about 5000 patients in the UK needing either dialysis or transplantation every year as a result of kidney failure. The lack of preventative or therapeutic treatments has created the opportunity to "repurpose" existing approved drugs as potential treatments. We have identified and validated one approved drug that improves cilia formation and function, determined the drug's target protein (ROCK2) and molecular mechanism of action, and have tested efficacy in animal models of ciliopathies. We now want to test the lead drug and related derivatives in cell-based disease models of ciliopathies and kidney cysts. We will use kidney organoids that we can artificially produce from special "induced pluripotent stem cells" (iPSCs) that can be differentiated into embryonic organ systems. This is the continuation of an existing project into a more extensive validation programme ("pre-clinical testing"). The overall aim is to re-purpose and translate a known drug into Phase II clinical trials in less than 5 years. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2019
Development Status Actively seeking support
Impact Initial studies have leveraged further internal funding for development work, and we are activity seeking external funding for pre-clinical studies in disease models. Funding bodies include LifeArc, Action Medical Research, and Harrington UK Rare Disease scholarship. 
 
Description BioJapan2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues
Year(s) Of Engagement Activity 2017
 
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact About 50 attendees to the Cafe Scientifique meeting in Feb 2020; I presented on recent advances in the diagnosis and treatment of inherited blindness, and was able to answer questions and speak to patients and supporters after the formal presentation.
Year(s) Of Engagement Activity 2020
 
Description FASEB "Polycystic Kidney Disease" meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact to facillitate and establish research collabirations, with critique of current research
Year(s) Of Engagement Activity 2017
 
Description Johnson Lab Twitter feed 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Twitter feed for the lab group which reports on our professional activities and events
Year(s) Of Engagement Activity 2020
URL https://twitter.com/johnsoncilialab
 
Description PEGS Boston 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues
Year(s) Of Engagement Activity 2018
 
Description UK Cilia Club 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact informal research club for reseachers in the north of England and Scotland; first meeting was in Sheffield Sept 2014, with on-going meetings planned for every 6 months in Leeds, Newcastle adn Edinburgh

new collaboration with University of Newcastle
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
 
Description school out-reach activities 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact discussed careers in medical research with about 60 Year 12 and 13 students on a visit and presentation at their school; 10 had a follow-on visit to the lab to discuss research methods
Year(s) Of Engagement Activity 2015,2016,2017,2018