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Pathophysiology of rare diseases due to ciliary dysfunction: nephronophthisis, Oral-facial-digital type 1 and Bardet-Biedl syndromes

Final Report Summary - EUCILIA (Pathophysiology of rare diseases due to ciliary dysfunction: nephronophthisis, Oral-facial-digital type 1 and Bardet-Biedl syndromes)

Primary cilia have been recognised on nearly all-mammalian cells. As such, defects in cilia formation or function have profound effects on the development of body pattern and on the physiology of multiple organ systems. Ciliary defects underlie a wide range of human disorders, including the rare and heritable Bardet-Biedl (BBS), oral-facial-digital type I (OFDI) and nephronophthisis (NPHP) syndromes. Each of these disorders present with defining features but all are characterised by polycystic renal disease. The proteins encoded by the genes responsible for these disorders, as well as those implicated in heritable forms of cystic kidneys, are all located to the cilium / basal body / centrosome complex, suggesting that ciliary dysfunction might be the unifying pathogenic concept underlying cystic kidney disease EUCILIA has used animal models and cell culture systems to unravel the role of cilia in basic cellular mechanisms, in order to gain insight into the pathogenetic mechanism of ciliary dysfuntion. EUCILIA has put together a consortium that brings together scientists working on three different animals models (Xenopus, zebrafish and mouse) and methodologies (cell biology, molecular biology, biochemistry) to answer the same biological question, i.e the role cilia play in rare diseases such as BBS, OFDI and NPHP. The EUCILIA project has:

1) generated in vivo models for the rare genetic conditions under study to establish a comprehensive list of phenotypic changes caused by abnormal BBS / OFD1 / NPHP functions;
2) developed in vitro models for BBS / OFD1 / NPHP proteins to identify signalling pathways involved in these rare genetic disorders;
3) uncovered the dynamics of BBS / OFD1 / NPHP trafficking and recruitment to the cilium to determine the basis of the pathophysiology of BBS / OFD1 / NPHP mutations;
4) determined the importance of BBS / OFD1 / NPHP function for renal homeostasis, to comprehend other renal disorders, such as polycystic kidney disease (PKD);
5) shed light on the role of BBS / OFD1 / NPHP proteins in Wnt signalling and tubulogenesis to identify, test and validate potential therapeutic agents.

A better understanding of ciliary function and structure is likely to have an enormous impact both at the basic research level, by gaining more insight into the function of this organelle, and more significantly in the translation of this knowledge into understanding the clinical consequences of lack or dysfunction of cilia. The wide distribution of cilia in all cell types suggests that ciliary defects could have a broader role in modern human epidemics such as hypertension, obesity, and diabetes. We believe that the biological relevance and significance of the results obtained by this consortium will have implications that go far beyond the rare OFDI, BBS and NPHP patients and may shed light on the mechanisms underlying the role of primary cilia in polycystic kidney disease paving the way to possible new therapeutic approaches.

Project context and objectives:

Cilia are evolutionarily conserved eukaryotic organelles that extend from, and are continuous with, the cell membrane. Nearly all-mammalian cells form cilia, and the ciliary apparatus is connected to cell cycle progression and proliferation (Khodjakov and Rieder, 2001). Cilia comprise a microtubular backbone (the ciliary axoneme), which is surrounded by the plasma membrane and is nucleated and organised by the basal body, located at the base of cilia (Badano et al., 2005). Cilia can be broadly subdivided into motile, characterised by a typical '9+2' architecture with nine outer microtubule doublets and a central pair of microtubules (e.g. bronchi) and primary, which appear typically as single appendages on the apical surface of cells and lack the central pair of microtubules (e.g. epithelium of kidney tubules). Ciliary proteins are synthesised in the cell body and must be transported to the tip of the axoneme. This is achieved by intraflagellar transport (IFT), an ordered and highly regulated anterograde and retrograde translocation of polypeptide complexes (IFT particles) along the length of the ciliary axoneme (Gerdes and Katsanis, 2005).

Emerging data suggest that cilia act as cellular antennae with diverse motility and sensory functions, detecting a wide variety of signals. As such, defects in cilia formation or function have profound effects on the development of body pattern and the physiology of multiple organ systems. Cilia dysfunction has been implicated in a wide spectrum of genetic disorders such as BBS, Joubert, Meckel, Alstrom, OFD1 and NPHP syndromes. Each of these disorders present with defining features but all are characterised by the presence of cystic kidneys associated with additional extrarenal symptoms. Interestingly, the protein products encoded by the genes responsible for inherited forms of cystic kidneys such as autosomal dominant (ADPKD) and, autosomal recessive polycystic kidney diseases (ARPKD), and nephronophthisis (NPHP) are all expressed in primary cilia, basal bodies, or centrosomes suggesting that ciliary dysfunction might be the unifying pathogenic concept underlying cystic kidney disease. However, the molecular mechanisms remain undetermined. The challenge and overall goal of the EUCILIA consortium has thus been to use BBS, OFD1 and NPHP syndromes as model systems to study the physiological role of primary cilia, with special emphasis on their role in the genitourinary tract and in the development of renal cysts. As proposed by the experimental plan, the consortium has been successful in generating, and making available to the scientific community, in vitro and in vivo models to study the physiological role of primary cilia and of OFD1, BBS and NPHP proteins in the formation and function of the primary cilium. These tools will allow the analysis of the ciliary protein interaction network and of the downstream pathway in the absence of BBS, OFD1, or NPHP.

The project specific objectives have been to:

1) generate in vivo models for BBS, OFDI and NPHP syndromes. These models will be utilised to establish a comprehensive list of phenotypic changes caused by abnormal BBS / OFD1 / NPHP functions, delineating the commonalities between these three types of ciliopathies to predict the pathways involved in these diseases;
2) generate in vitro models for BBS / OFD1 / NPHP proteins to analyse and validate predicted signalling pathways involved in these rare genetic disorders at the molecular, biochemical, and cellular level;
3) determine the dynamics of BBS / OFD1 / NPHP trafficking and recruitment to the cilium, providing a framework for the understanding of the pathophysiology of BBS / OFD1 / NPHP mutations.
4) determine the importance of BBS / OFD1 / NPHP function for renal homeostasis, and address the role of BBS / OFD1 / NPHP proteins in Wnt signalling and tubulogenesis;
5) study the downstream pathway dysregulation events consequent upon the absence of BBS, OFD1 and NPHP proteins;
6) identify, test and validate potential therapeutic agents for the amelioration and prevention of renal cyst - a major cause of morbidity and mortality in this group of patients and patients with related diseases.

The biological significance and relevance of the results obtained by the EUCILIA project have thus implications that go far beyond the rare OFD1, BBS and NPHP patients and may shed light on the mechanisms underlying the role of primary cilia in polycystic kidney disease paving the way to possible new therapeutic approaches.

References to section

1. Badano, J. L., Teslovich, T. M., and Katsanis, N. (2005). The centrosome in human genetic disease. Nat Rev Genet 6, 194-205.
2. Gerdes, J. M. and Katsanis, N. (2005). Microtubule transport defects in neurological and ciliary disease. Cell Mol Life Sci 62, 1556-1570.
3. Khodjakov, A. and Rieder, C. L. (2001). Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression. J Cell Biol 153, 237-242.

Project results:

Cilia are hair-like structures located on the membrane of almost all mammalian cells. Studies generated from different laboratories have demonstrated that these structures have important roles in transferring information from the space surrounding the cell to the inside of the cell. Genetic studies have established that mutations in genes important for formation and function of cilia are associated with a number of genetic diseases, such as BBS, OFDI and NPHP, which are characteried by the presence of common clinical features including renal cystic disease, and brain and skeletal abnormalities. Ciliary dysfunction is thus associated to this group of disorders, which is why they are commonly named 'ciliopathies'.

The EUCILIA consortium has used animal models and cell culture systems to unravel the role of cilia in basic cellular mechanisms, in order to gain insight into the pathogenetic mechanism of ciliary dysfuntion. EUCILIA has put together a consortium that brings together scientists working on three different animals models (Xenopus, zebrafish and mouse) and methodologies (cell biology, molecular biology, biochemistry) to answer the same biological question, i.e. the role cilia play in rare diseases such as BBS, OFDI and NPHP.

The main science and technology (S&T) results obtained by the EUCILIA consortium are outlined below and in the following pages.

1) New murine and zebrafish models recapitulating the disease syndromes have been developed.

In mouse we generated the following models mimicking the OFDI phenotype observed in the human disease:

a) A mouse model with ubiquitous inactivation from the early phases of development. This model had a very severe phenotype, which was not compatible with life. In fact, male mutants died in early phases of pregnancy and female mutants died at birth.
b) A subsequent mutant with kidney-specific inactivation allowed us to study the role of the Ofd1 transcript specifically in renal cystic disease. This model allowed us to overcome the embryonic male lethality and female perinatal lethality observed in the previous model. In this model, we observed that primary cilia initially form and then disappear after the development of cysts, suggesting that the absence of primary cilia is a consequence rather than the primary cause of renal cystic disease. In addition, we also provided experimental evidence that indicated abnormalities in an important signalling pathway, mTOR, and the amelioration of the renal cystic phenotype upon administration of an mTOR inhibitor. This result might have an important implication since different clinical trials are now evaluating the possible use of molecules interfering with the mTOR pathway in the treatment of patients with renal cystic disease.
c) We also generated an inducible model in which Ofd1 inactivation could be achieved upon administration of a drug, which allowed switching off the gene at specific time points. This model will allow studying the effect of Ofd1 inactivation at different stages and in adult life. We first decided to study the effect of Ofd1 inactivation in the adult stage by inducing Ofd1 inactivation at birth. The renal phenotype of this model as well as other phenotypes affecting additional organs, including the liver and the brain are currently under investigation.
d) We also established a model in which the Ofd1 transcript was specifically inactivated in the limb and skeletal structures. This model allowed us to study the role of the Ofd1 transcript and primary cilia in the transmission of the biological signal important for limb and skeletal development. Our results clearly established that Ofd1 plays an important role in the correct determination of the antero-posterior axis in limb development. Moreover, we demonstrated that Ofd1 has a role also in the formation of the long bones.

Utilising zebrafish and mice we performed a thorough characterisation of BBS protein expression and observed that BBS4, 6, 8, and 9 are highly expressed in centrosomes and basal bodies. By contrast, Ift80 and Nphp2 are found in the basal body and ciliary axoneme.

Furthermore, the EUCILIA consortium performed an extensive analysis of NPHP deficient zebrafish. Depletion of NPHP proteins produced a surprisingly uniform set of phenotypes, including a dorsally bend body, the formation of cystic structures within the proximal pronephros, pericardial edema, hydrocephalus, and an abnormal left-right asymmetry. While these developmental defects emphasise the role of nephrocystins in embryogenesis and implicate their requirement for motile cilia, they did not provide molecular insight into the function of nephrocystins. One curious exception was the cloaca malformation associated with the knockdown of zebrafish NPHP4. The zebrafish cloaca, a joint opening of the embryonic pronephric ducts and the gut, is largely regulated by BMP signalling, which specifies the ectodermal cells that form this opening through a well orchestrated series of cell migration, apoptosis and reorganisation. Knockdown of zebrafish NPHP4 revealed that this gene product is required to direct the migration of distal pronephric duct cells towards the ectodermal cells. While the ectodermal cells, defined to form the cloaca are specified by BMP signals, contact with pronephric cells is required to induce programmed death in those ectodermal cells that finally open the joint gut / pronephros fusion to the environment. NPHP4-deficient pronephros cells fail to migrate towards the ectodermal cells, resulting in defective apoptosis. These observations suggest that NPHP4 may play a distinct role in directed cell migration. Since NPHP4 also interacts with focal adhesion proteins, this function may involve a localisation outside of the ciliary compartment.

Finally, we also demonstrated that a tight balance of nephrocystins is required for normal embryonal development in Xenopus. Depletion of nephrocystins in Xenopus did not cause cyst formation. However, there was an almost uniform simplification of the proximal pronephros convolute after knockdown of Xenopus NPHP gene products. We showed that two antagonistic cell movements form the proximal pronephros convolute. Similar to the collective cell migration first described in zebrafish (Vasilyev et al., 2009), migrations of tubular epithelial cells from the posterior to the anterior pronephros contribute to the convolution of the proximal segments; NPHP proteins are not required for this particular cell movement. A second cell movement originates in the dorsal renal primordial and pushes the developing pronephros in a ventral direction (Lienkamp et al., 2010). This dorsal-toventral directed cell migration requires the presence of nephrocystins; without nephrocystin, the proximal convolution is drastically reduced. The consequence of this simplified proximal convolute appears to be defective handling of water. Edema formation, which characterises NPHP-deficient Xenopus embryos, may be the functional correlate of polyuria observed in patients with Nephronophthisis.

In conclusion, the consortium has generated a number of mutant phenotypes in mice, zebrafish and Xenopus, which will be made available to the scientific community via a public website, once the data is published, useful to study the role of cilia-related protein in health and disease.

2) The consortium has generated cell lines with selective depletion and selective overexpression of BBS / OFDI / NPHP allowing to study several aspects of cilia formation and function.

We used cell biology-based approaches to investigate a series of key molecular mechanisms at the basis of the formation and function of the cilium. First, we focused on the study of the functional interactions among selected important ciliopathy genes by generating a dataset of protein-protein interactions This dataset was integrated with data obtained by mining published information from a wide range of sources, such as databases for co-expression profiles and protein-protein interactions. This work has had two main outcomes:

a) the discovery of a novel protein involved in cilium formation; and
b) the disclosure of a large number of unappreciated physical / functional associations among various key components of intracellular membrane trafficking, including the Golgi complex, the exocyst complex (involved in the targeting of post-Golgi vesicles to the plasma membrane), the BBSome (a multisubunit protein complex involved in transporting cargoes to the cilium) and components of the centrosome, an intracellular organelle that organises the cellular cytoskeleton and function as the basal body of the cilium.

To study the function of the newly-identified proteins, the consortium focused on generating in vitro cellular models for cilia biogenesis and phenotypic cell-based assay that are suitable for microscope-based high-content screening. To this end, we set up the experimental condition for induction of cilium formation in different cells lines, such as the murine inner medullary collecting duct (IMCD3), retinal pigmented epithelial cells (RPE) and HeLa cells. We selected IMCD3 and RPE cells, as these cells are well-characterised in vitro models for studies related to the formation of the primary cilium related to cystic kidneys and retinal degeneration, respectively. HeLA cells form only short cilia but they are wellsuited for various experimental manipulations and they are an ideal model for studies of intracellular transport, as most of their membrane transport routes have been extensively characterised both at the morphological and molecular level. The experimental manipulation of the intracellular levels of the newly identified protein established its essential role in ciliogenesis. Moreover, by using electron- and lightmicroscopy approaches, we found that Ofd1 is located on the Golgi complex and it nicely colocalises with TGN46. Moreover, Ofd1 is sensitive to the action of brefeldin A, a formal proof that even if Ofd1 is not an integral Golgi protein, it is strongly associated to this compartment. Ofd1 knock-down cells appear to have a problem in intra-Golgi transport or in exiting the Golgi complex of VSV-G cargo proteins. All these data point to a role of Ofd1 at the Golgi complex. The consortium has also been able to demonstrate that knockdown of Inversin (NPHP2) as well as of other nephrocystins nephrocystin-1 and -4) in MDCK cells results in almost complete loss of ciliated cells, suggesting that cultured epithelial cells cannot replace the loss of NPHP proteins to form normal cilia. Thus, depletion of ciliary proteins disrupts ciliogenesis.

3) A bioinformatics analysis has revealed potential gene networks linking the BBS / OFD1 / NPHP proteins.

The interaction network among various components of intracellular membrane traffic created a rational framework for the investigation of the molecular mechanisms behind the formation of transport carriers from the Golgi complex, and to study how vesicles are targeted for fusion specifically at the periciliary membrane. Based on these findings, we investigated the role in ciliogenesis of a selected number of Golgi-associated proteins that are present in the interaction network. This functional study led to suggest that a series of Golgi-associated proteins that are known for their role in maintaining a 'ribbon' structure of the Golgi complex, have also an essential role for directional protein transport, which, in turn, is required for cell polarisation during migration or for cilium formation. Our results suggest that this polarised delivery is achieved through the Golgi-based nucleation of a specific subclass of microtubules that are oriented toward the direction of cargo delivery. This complex mechanism of directional delivery of cargos to the basal body is of fundamental importance for cilium biogenesis, as many signalling proteins, including receptors and protein kinases, need to be transported from the Golgi complex to the cilium to carry out their correct function in signal transduction. Moreover, the most represented cellular compartments for cilia-associated proteins are the cilium, cytoskeleton and cell projection. These ciliary associated transcripts appear to be co-expressed with transcripts associated to cytoskeletal functions (11 %), Golgi and trafficking (11 %), chaperons (10 %), and protein translation (10 %). Altogether, we have determined that a complex and coordinated interplay among multiple intracellular systems, which include the Golgi complex, the basal bodies and the cytoskeleton, is fundamental for the formation and functional / structural homeostasis of cilia. The delivery of cargoes to their target membranes is a multistep process that involves their sorting into carriers and the subsequent transport, tethering and fusion of these carriers with a specific membrane target. In particular, we have identified novel proteins that are probably involved at two levels:

a) directional delivery of cargo;
b) cargo sorting, which is mediated by the decoding of the targeting sequence during the process of transport carriers formation.

Furthermore, nephrocystins function as gatekeepers that control ciliary cargo transport. Several nephrocystins localise to the transition zone or a segment immediately adjacent to the transition zone. Although the function of the transition zone has remained unclear, it has been proposed to act as a 'pore complex' that restricts the access to the flagellar compartment. Recently, NPHP6 was found to localise to the Y-shaped connectors that link the plasma membrane enclosing the flagella with the microtubules (Craige et al., 2010), suggesting that NPHP6 might regulate the cargo transport in and out of the ciliary compartment. The EUCILIA consortium found that NPHP5 interacts with NPHP6, potentially changing the conformation of this large coiled-coil protein (Schafer et al., 2008). It is therefore conceivable that NPHP5 regulates the gate-keeping function of NPHP6, potentially through the recruitment of additional adaptor and effector proteins such as RPGR and RPGRIP1. Finally, we were able to show that nephrocystins are required for ciliary polarisation. Motile cilia display a coordinated beating pattern to move fluid or particles. To achieve this synchronisation, motile cilia are precisely positioned in relationship to the apical cell surface. For example, to generate a coordinated left-to-right directed fluid flow at the ventral node, the motile cilium is moved to the posterior side of the cell (translational polarity) and tilted before it starts a rotating movement (rotational polarity); failure to achieve this polarisation results in an abnormal body axis (situs inversus), a manifestation observed in Nephronophthisis. Using the epidermis of the Xenopus embryo as a model system, the EUCILIA consortium gained novel insight in the role of nephrocystins in cilia formation and polarisation. The Xenopus epidermis contains single isolated cells with multiple motile cilia that generate a directed fluid flow from the anterior to the posterior end of the embryo. The cilia are formed during early embryogenesis, when basal bodies formed in the interior of the epidermal cell start nucleating the typical 9+2 microtubular axoneme while they are migrating towards the apical cell surface. After docking to the apical membrane, the basal bodies are oriented in an anterior-to-posterior direction defined by the orientation of the basal foot and rootlet (Mitchell et al., 2007). The multifunctional adaptor protein Dishevelled associates with the budding basal bodies before they attach to the apical membrane, and finally assumes an asymmetric position opposite of the basal foot. Nephrocystins can target Dishevelled for ubiquitin-dependent degradation (APC/C) (Simons et al., 2005). The EUCILIA consortium has now discovered that ANAPC2, a component of the anaphase promoting complex that interacts with nephrocystins, plays a crucial role during the polarisation of motile cilia: without an intact APC/C, the motile cilia on the Xenopus epidermis remain disoriented (Ganner et al., 2009). Since depletion of nephrocystins (e.g. NPHP1, NPHP2, NPHP5) cause identical defects, these findings suggest that nephrocystins in combination with the APC/C help to polarise motile cilia, and help to explain why patients with Nephronophthisis not only experience laterality defects (e.g. situs inversus), but also an increased frequency of chronic bronchitis and sinusitis.

4) We have consistently demonstrated a link between primary cilia and PCP/noncanonical Wnt signalling.

In fact, we observed the following:

a) Ofd1 inactivation in zebrafish is important for convergent extension during gastrulation.
b) The actin cytoskeletal organisation, adhesion properties and the PCP / non-canonical Wnt pathway are perturbed in Ofd14-5/+ female mutant mouse embryos.
c) Several nephrocystins (NPHP4 > NPHP9 > NPHP2 > NPHP3) interfere with the canonical Wnt signalling pathways, apparently through down-regulation of Dishevelled steady-state levels. At least some nephrocystins can interact with subunits of the Anaphase Promoting Complex, and perhaps use this E3 ubiquitin ligase to target Dishevelled for degradation. However, the functional implications remain unknown; for example, Dishevelled degradation could be limited to the basal body to achieve polarisation of motile cilia. In this case, nephrocystins may not interfere with canonical Wnt signalling outside of this confined compartment.
d) Similar findings to those cited above have been reported for BBS proteins (Gerdes et al., 2007), suggesting a general inhibitory function of ciliary / centrosomal proteins on canonical Wnt signalling.
e) A novel molecular function for nephrocystin-2 (Inversin) has been identified by using the Xenopus animal cap assay system. This protein is necessary to allow translocation of Dishevelled to the plasma membrane in response to Frizzled-8 (Lienkamp et al., 2010). Although Frizzled-8 has been implicated in several branches of the Wnt signalling cascades, recruitment of Dishevelled to the plasma membrane is a key event in non-canonical, planar cell polarity signalling. Our observation confirms the prevailing hypothesis that at least a subset of nephrocystins participates in planar cell polarity signaling during kidney morphogenesis.
f) Cilia defects arise, at least in part, from defective Wnt signalling (planar cell polarity (PCP)) resulting in deficient basal body migration to the apical surface of the cell.

5) We have generated data indicating an abnormal activation of the mTOR pathway in mutant Ofd1 and Bbs mice kidneys.

To understand what the consequences are of the loss or deficiency of key ciliopathy genes / proteins on whole organisms and at the cellular level, the consortium focused its attention on determining the molecular pathway by which kidney disease (cysts) arise in both mouse and zebrafish models. Based on existing knowledge of the contribution of three main pathways in kidney development and/or cyst formation we focused on the following:

a) The mTOR pathway - mTOR functions to sense cellular nutrient and energy levels. The pathway is dysregulated in human diseases, especially certain cancers. It is believed to control cell proliferation a key component of renal cyst formation.
b) The autophagy pathway - autophagy is a key component of cell biology involving the degradation of a cell's own components destined for destruction. It plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It may play a role in the prevention of neurodegeneration and cancer and is linked to the mTOR pathway.
c) The Sonic Hedgehog (HH) pathway - HH is a key regulator of animal development, important in later stages of embryogenesis and body planning in some organisms. It has been shown to be important in development of the brain, skeleton, musculature,
GI tract, lungs and kidneys. It also has a role in regeneration. HH has been implicated in some forms of cancer.

We began by compiling a list of the effects of loss of 11 different ciliopathy related genes in zebrafish morphants (gene deficient young fish). We found that perturbation of the function of genes associated with Bardet-Biedl syndrome, Meckel syndrome, OFD1, Jeune syndrome and NPHP gave rise to renal cysts in five day-old fish embryos. These provided us with the resources to further investigate the pathways described above. We showed that BBS proteins are required for different levels of HH signalling but not nphp2 / inversin. Next we sought to investigate these pathways in mouse models, specifically kidneys taken from OFD1, BBS and NPHP deficient mice. We showed that mTOR activity is increased in Ofd1 and Bbs4 and 6 mutant mouse renal tissue but especially in cysts linking this pathway to disease pathogenesis. SHH pathway is also perturbed in Ofd1 and Bbs4, 6 and 8 deficient mammalian cells (but not in Nphp2/Inv) and is likely to account for the development of polydactyly in both OFD1 and BBS patients but not in NPHP patient. SHH did not seems to affect renal development. We did not see major perturbations of the autophagy pathways in BBS, NPHP and OFD1 model systems. Cilia-related proteins are thus important for the structure and function of cilia at the cellular level but their loss can lead to devastating clinical consequences ranging from perinatal death to childhood onset blindness, kidney failure, severe brain defects and skeletal anomalies including extra digits. We can now conclude that these arise from multiple and differing actions of key proteins on cellular, tissue and organ development and function and can in a major part be ascribed to at least three important pathways; mTOR, SHH and WNT (previously published data) signalling.

6) The results testing the efficacy of available drugs, evaluated in both fish and mice (preliminary) by morphological and functional analysis, suggest that the mTOR inhibitor, rapamycin, and to a lesser extent the cell cycle inhibitor, roscovitine, rescue renal cysts and function, while other drugs do not.

To discover therapeutic interventions in the form of drugs that are already approved for use in man that might alleviate renal cyst formation and even prevent renal failure, we again chose to use well-defined model organisms, namely zebrafish and mouse.

Here we adopted a dual approach:

a) a pathway targeted evaluation of compounds previously shown to have an effect on cyst formation in kidneys; and
b) a blind screen for compounds that could improve cyst formation.

For the first targeted approach, we chose to evaluate rapamycin, an mTOR inhibitor first discovered to have a positive effect on shrinking cyst size in autosomal dominant polycystic kidney disease, which is relatively common. Rapamycin is used as part of the anti-rejection therapy for renal transplants and it was first noted in PKD transplant patients that the retained disease kidney cysts had reduced in volume. Given our result from this study, we tested rapamycin in Bbs4, Nphp and Ofd1 mice. For the second approach, we needed to develop a high-throughput system not possible in mice and thus we adopted zebrafish for their key properties of easy gene suppression (morpholinos) and their transparent embryos enabling us to follow developmental progress. We administered a drug library consisting of 880 compounds, 90 % of which are already approved for use in man. This would hopefully allow us two key benefits:

a) reduced lead time to patient trials;
b) revelation of novel pharmacological targets.

Concerning the targeted approach, we established that rapamycin was able to reduce renal cyst size and even recover excretory function in several zebrafish morphants. However, we were unable to demonstrate any efficacy in Bbs4 or Nphp2 mice of rapamycin on cyst formation. The results were better for Ofd1 conditional mice and might be explained by some limitations of the system for delivery and imaging. For example Nphp2 null mice die early precluding a longer period of administration. In Bbs4 mice, the cysts were variable in number, size and onset (always late - 6-9 months). Our imaging modalities (MRI or USS) were not as good as we had hoped for monitoring progress in live mice.

Regarding the drug library screen, we completed screening of 880 compounds and discovered 40 potentially useful drugs that could halt or prevent cyst formation in morphant fish. These drugs have been retested with fresh batches at several different doses and a handful remain of interest. We were able to group the compounds into categories based on their pharmacological actions. Surprisingly, several drugs belonging to the quinolone group were shown to be the most active in preventing cysts. The anti-malarial compound primaquine proved to be the top most consistent hit.

We can now begin to think about therapies in ciliopathy patients particularly in conditions in which gene therapy would not be amenable (most of these have multi-organ involvement). We would expect that, based on the pathway studies, different phenotypes (clinical problems) may arise from different impacts on tissues / organs of loss of protein function. This would likely indicate that different drugs will be sued for separate clinical features eventually leading to a polypharmacy approach to treating ciliopathy patients.

Beyond the scope of this successful programme, we are now continuing to further evaluate other drug hits, embark on screens to rescue other disease components (e.g. retinal degeneration) and to further develop mammalian assays (renal explants) in preparation for human clinical trials in the not too distant future.

In conclusion the EUCILIA consortium has:

1) generated in vivo models for the rare genetic conditions under study to establish a comprehensive list of phenotypic changes caused by abnormal BBS / OFD1 / NPHP functions;
2) developed in vitro models for BBS / OFD1 / NPHP proteins to identify signalling pathways involved in these rare genetic disorders;
3) uncovered the dynamics of BBS / OFD1 / NPHP trafficking and recruitment to the cilium to determine the basis of the pathophysiology of BBS / OFD1 / NPHP mutations;
4) determined the importance of BBS / OFD1 / NPHP function for renal homeostasis, to comprehend other renal disorders, such as PKD;
5) shed light on the role of BBS / OFD1 / NPHP proteins in Wnt signalling and tubulogenesis to identify, test and validate potential therapeutic agents.

A better understanding of ciliary function and structure is likely to have an enormous impact both at the basic research level, by gaining more insight into the function of this organelle, and more significantly in the translation of this knowledge into understanding the clinical consequences of lack or dysfunction of cilia. The wide distribution of cilia in all cell types suggests that ciliary defects could have a broader role in modern human epidemics such as hypertension, obesity, and diabetes. We believe that the biological relevance and significance of the results obtained by this consortium will have implications that go far beyond the rare OFDI, BBS and NPHP patients and may shed light on the mechanisms underlying the role of primary cilia in polycystic kidney disease paving the way to possible new therapeutic approaches.

References to section

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4. Lienkamp, S., Ganner, A., Boehlke, C., Schmidt, T., Arnold, S. J., Schafer, T., Romaker, D., Schuler, J., Hoff, S., Powelske, C., et al. (2010). Inversin relays Frizzled-8 signals to promote proximal pronephros development. Proc Natl Acad Sci U S A 107, 20388-20393.
5. Mitchell, B., Jacobs, R., Li, J., Chien, S., and Kintner, C. (2007). A positive feedback mechanism governs the polarity and motion of motile cilia. Nature 447, 97-101.
6. Schafer, T., Putz, M., Lienkamp, S., Ganner, A., Bergbreiter, A., Ramachandran, H., Gieloff, V., Gerner, M., Mattonet, C., Czarnecki, P. G., et al. (2008). Genetic and physical interaction between the NPHP5 and NPHP6 gene products. Hum Mol Genet 17, 3655-3662.
7. Simons, M., Gloy, J., Ganner, A., Bullerkotte, A., Bashkurov, M., Kronig, C., Schermer, B., Benzing, T., Cabello, O. A., Jenny, A., et al. (2005). Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat Genet 37, 537-543.
8. Vasilyev, A., Liu, Y., Mudumana, S., Mangos, S., Lam, P. Y., Majumdar, A., Zhao, J., Poon, K. L., Kondrychyn, I., Korzh, V., et al. (2009). Collective Cell Migration Drives Morphogenesis of the Kidney Nephron. PLoS Biol 7, e9.
9. Zullo, A., Iaconis, D., Barra, A., Cantone, A., Messaddeq, N., Capasso, G., Dolle, P., Igarashi, P., and Franco, B. (2010). Kidney-specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway. Hum Mol Genet 19, 2792-2803.

Potential impact:

Cilia have been observed in a number of organs including liver, pancreas, kidney as well as in numerous cell types including endothelial cell, myocardium, odondoblasts and several neuronal populations (Badano et al., 2006). Consistent with this ubiquitous presence ciliary dysfunction gives rise to a variety of human phenotypes ranging from being organ specific (such as polycystic kidney disease) and to syndromic and pleiotropic (such as BBS, OFSDI, Alstram syndromes). The role for motile cilia and flagella in cell locomotion, and fluid movement has been known for some time. However, the role for immotile cilia, associated to a larger variety of cell types, were thought to be of minor physiological importance until recently when homologs for the ciliary proteins of Chlamydomonas and C. elegans were linked to PKD in humans and mice and found to localise to primary cilia (Pazour and Rosenbaum, 2002; Hildebrandt and Otto, 2005). Since then, the role for cilia in human disease has expanded considerably beyond PKD. Recent research endeavours have led to the discovery that loss of normal ciliary function in mammals is responsible for cystic and noncystic pathology in the kidney, liver, brain, and pancreas, as well as severe developmental patterning abnormalities (Bisgrove and Yost, 2006). In addition, cilia have been implicated to have crucial roles in the signal transduction pathways that regulate Ca2+ levels as well as hedgehog and planar cell polarity pathways.

Our understanding of the association between cilia and human disease has benefited substantially from the use of lower organisms such as Chlamydomonas and Caenorhabditis elegans. An example of how basic research can translate into knowledge benefiting human health. Building on this success, the EUCILIA consortium has continued to use animal models and cell culture systems to further the role of cilia in basic cellular mechanisms, in order to gain insight into the pathogenetic mechanism of ciliary dysfuntion of three ciliary disorders, namely BBS, OFDI and nephronophthisis (NPHP). In particular, the EUCILIA consortium has studied the pathophysiology of these ciliopathies with specific emphasis on the development of renal cysts by developing and studying appropriate animal models (WP 1), by developing in vitro models (WP 2), by studying signalling pathways to understand cilia assembly and to determine protein interaction networks to establish a hierarchy of temporal and spatial interactions of BBS, OFD1, and NPHP proteins in cilium structure and function (WP 3, WP 4), to utilise alternative vertebrate models to the mouse, and zebrafish, to study novel signalling pathways that regulate cilia function (WP 5). Finally, EUCILIA has laid the foundations to identify and evaluate potential therapeutic agents to prevent or reverse renal cyst formation that might influence the life of patients with severe kidney disease due to ciliary dysfunction (WP 6). The work carried out by the EUCILIA consortium, and results achieved, fall in the 'classical' basic biomedical research in which animal models of human disease are used to recapitulate disease mechanisms and to understand the pathophysiological mechanisms. Moreover, the consortium has also significantly invested in developing in vitro models to test signalling pathways involved in cilia assembly and function in order to reduce the number of animals utilised in research.

The impact of the work and results obtained by the EUCILIA consortium on the understanding and potential treatment of rare diseases due to ciliary dysfunction is described below:

- New murine and zebrafish models recapitulating the disease syndromes have been generated, which have established a comprehensive list of phenotypic changes caused by abnormal BBS / OFD1 / NPHP functions, delineating the commonalities between these three types of ciliopathies to predict the pathways involved in these diseases.
- The consortium has used in vitro models for BBS / OFD1 / NPHP proteins that have identified signalling pathways involved in these rare genetic disorders.
- We have been successful in uncovering the dynamics of BBS / OFD1 / NPHP trafficking and recruitment to the cilium, which has determined the basis of the pathophysiology of BBS / OFD1 / NPHP mutations.
- We have determined the importance of BBS / OFD1 / NPHP function for renal homeostasis, which will have important implications in the comprehension of other renal disorders, such as polycystic kidney disease (PKD).
- By addressing the role of BBS / OFD1 / NPHP proteins in Wnt signalling and tubulogenesis, the consortium has provided insight to the role of these proteins and cilia in development.
- The consortium has set the ground knowledge in identifying, testing and validating potential therapeutic agents, which will have an enormous impact for the amelioration and prevention of renal cysts - a major cause of morbidity and mortality in this group of patients and patients with related diseases.

A better understanding of ciliary function and structure is likely to have an enormous impact both at the basic research level, by gaining more insight into the function of this organelle, and more significantly it will allow translation of this knowledge into understanding the clinical consequences of lack or dysfunction of cilia. Many questions remain unanswered regarding cilia, how extracellular stimuli perceived by the cilia result in changes in cell behaviour and physiology and how functional defects in this organelle result in such a wide spectrum of pathologies, including cystic kidneys, hydrocephaly, ductal abnormalities in the liver and pancreas, embryonic and skeletal patterning abnormalities. The wide distribution of cilia in all cell types suggests that ciliary defects could have a broader role in modern human epidemics such as hypertension, obesity, and diabetes. Although we have made great advances in demonstrating the importance of cilia over the past decade, the physiological role that this organelle plays in most tissues remains elusive. Research focused on addressing this issue will be of critical importance for a further understanding of how ciliary dysfunction can lead to such severe disease and developmental pathologies.

Concluding, EUCILIA exemplifies how basic research among complementary research groups can efficiently and on a broad scale increase our knowledge of basic biological mechanisms, in this case ciliary function, with potentially significant impacts for human health and quality of life.

Main dissemination activities and exploitation of results

The partners of the EUCILIA consortium have given particular attention towards the dissemination of the results of the project throughout these three years by presenting their data in international conferences, workshops and meetings and by publishing their results in high impact, peer-reviewed international journals. The EUCILIA website has been developed and is online (please see http://www.eucilia.eu online), as planned, which has favoured the dissemination of the project to the scientific community, patient groups, industry, biotechnology, and training institutions, and has facilitated the intercommunication among partners as well as the acceleration in work progression. To this end, the site consists of a public area and a private area that is password protected (username: partners; password: europa2).

In the public area, which is easily accessible for any internet user, a first section (What is EUCILIA?) provides general information on the focus of the project, the diseases of interest to the consortium, and the aims and impact of the project in terms of EU scientific and societal objectives. A more specific description of the project work packages and their objectives is also provided together with a general overview of each partner making up the consortium and his / her role in the project, the key personnel and publications acknowledging EUCILIA. Links to other websites, relevant events and participation to meetings and symposia is also available. Finally, a separate section (internal use) with sensitive data (such as the TA, the contract and terms, etc.) is restricted to the partners of the consortium and is password protected. The EUCILIA management team has also given particular attention to keep the website up to date with the consortium annual meetings, including the PPTs presented by each partner and Minutes of each meeting, and with the publications acknowledging EUCILIA and European Union (EU) funding. The PPTs, minutes of the meetings and full articles of EUCILIA publications are again restricted to the partners and the EU and therefore are password protected.

As planned, the partners have been able to exploit the results of the project in their local research programmes, and have been successful in seeking additional major funding from national research agencies, foundations and industries to further extend the work carried out in the project towards the experimental characterisation of the most interesting findings. To this end, partners 1 (B. Franco), 2 (P. Beales) and 3 (G. Walz) are also cooperating in another two-phase EU collaborative project (SYSCILIA), which started last year (start date: 1 June 2010). The aim of the SYSCILIA consortium, consisting of 15 partners, is to identify the molecular mechanisms characterising cilium function as well as the discrete perturbations associated with dysfunction caused by mutations in inherited ciliopathies, by applying a systems biology approach. The use and dissemination of foreground to the lay public as well as contact with patient associations to disseminate results and get feedback has been planned by the EUCILIA consortium and will be formally set out as soon as all the data produced by the project is published (there are a number of papers still in preparation). Thus, whenever indicated, data is still confidential and restricted to the members of the consortium.

The exact nature of such exploitable outcome is not currently foreseeable, but definitely forms the ground knowledge on ciliopathies and ciliary dysfunction to move 'from bench to bedside' possibly in the near future.

References to section

1. Badano, J. L., Mitsuma, N., Beales, P. L., and Katsanis, N. (2006). The Ciliopathies: An Emerging Class of Human Genetic Disorders. Annu Rev Genomics Hum Genet 7, 125-148.
2. Bisgrove, B. W. and Yost, H. J. (2006). The roles of cilia in developmental disorders and disease. Development 133, 4131-4143.
3. Hildebrandt, F. and Otto, E. (2005). Cilia and centrosomes: a unifying pathogenic concept for cystic kidney disease? Nat Rev Genet 6, 928-940.
4. Pazour, G. J. and Rosenbaum, J. L. (2002). Intraflagellar transport and cilia-dependent diseases. Trends Cell Biol 12, 551-555.

Project website: http://www.eucilia.eu

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