Progressive liver, kidney, and heart degeneration in children and adults affected by TULP3 mutations.

Organ fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-ß signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.

The ciliary transition zone protein TMEM218 synergistically interacts with the NPHP module and its reduced dosage leads to a wide range of syndromic ciliopathies.

Mutations in genes that lead to dysfunctional cilia can cause a broad spectrum of human disease phenotypes referred to as ciliopathies. Many ciliopathy-associated proteins are localized to the evolutionary conserved ciliary transition zone (TZ) subdomain. We identified biallelic missense and nonsense mutations in the gene encoding the transmembrane protein TMEM218 in unrelated patients with features related to Bardet-Biedl, Joubert and Meckel-Gruber syndrome (MKS) and characterized TMEM218 as a major component of the ciliary TZ module. Co-immunoprecipitation assays resulted in the physical interaction of TMEM218 with the MKS module member TMEM67/Meckelin that was significantly reduced by the TMEM218 missense change harboured by one of our patients. We could further validate its pathogenicity by functional in vivo analysis in zebrafish (Danio rerio) as a well-established vertebrate model for ciliopathies. Notably, ciliopathy-related phenotypes were most prominent by genetic interactions with the NPHP module component Nphp4. Conclusively, we describe TMEM218 as a new disease gene for patients with a wide spectrum of syndromic ciliopathy phenotypes and provide evidence for a synergistic interaction of TMEM218 and the NPHP module crucial for proper ciliary function.

PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity.

The conserved multiple PDZ-domain containing protein PATJ stabilizes the Crumbs-Pals1 complex to regulate apical-basal polarity and tight junction formation in epithelial cells. However, the molecular mechanism of PATJ's function in these processes is still unclear. In this study, we demonstrate that knockout of PATJ in epithelial cells results in tight junction defects as well as in a disturbed apical-basal polarity and impaired lumen formation in three-dimensional cyst assays. Mechanistically, we found PATJ to associate with and inhibit histone deacetylase 7 (HDAC7). Inhibition or downregulation of HDAC7 restores polarity and lumen formation. Gene expression analysis of PATJ-deficient cells revealed an impaired expression of genes involved in cell junction assembly and membrane organization, which is rescued by the downregulation of HDAC7. Notably, the function of PATJ regulating HDAC7-dependent cilia formation does not depend on its canonical interaction partner, Pals1, indicating a new role of PATJ, which is distinct from its function in the Crumbs complex. By contrast, polarity and lumen phenotypes observed in Pals1- and PATJ-deficient epithelial cells can be rescued by inhibition of HDAC7, suggesting that the main function of this polarity complex in this process is to modulate the transcriptional profile of epithelial cells by inhibiting HDAC7.

Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment.

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

Loss of CBY1 results in a ciliopathy characterized by features of Joubert syndrome.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.

The nucleoside-diphosphate kinase NME3 associates with nephronophthisis proteins and is required for ciliary function during renal development.

Nephronophthisis (NPH) is an autosomal recessive renal disease leading to kidney failure in children and young adults. The protein products of the corresponding genes (NPHPs) are localized in primary cilia or their appendages. Only about 70% of affected individuals have a mutation in one of 100 renal ciliopathy genes, and no unifying pathogenic mechanism has been identified. Recently, some NPHPs, including NIMA-related kinase 8 (NEK8) and centrosomal protein 164 (CEP164), have been found to act in the DNA-damage response pathway and to contribute to genome stability. Here, we show that NME/NM23 nucleoside-diphosphate kinase 3 (NME3) that has recently been found to facilitate DNA-repair mechanisms binds to several NPHPs, including NEK8, CEP164, and ankyrin repeat and sterile α motif domain-containing 6 (ANKS6). Depletion of nme3 in zebrafish and Xenopus resulted in typical ciliopathy-associated phenotypes, such as renal malformations and left-right asymmetry defects. We further found that endogenous NME3 localizes to the basal body and that it associates also with centrosomal proteins, such as NEK6, which regulates cell cycle arrest after DNA damage. The ciliopathy-typical manifestations of NME3 depletion in two vertebrate in vivo models, the biochemical association of NME3 with validated NPHPs, and its localization to the basal body reveal a role for NME3 in ciliary function. We conclude that mutations in the NME3 gene may aggravate the ciliopathy phenotypes observed in humans.

The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin.

Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.

Genetic loci associated with renal function measures and chronic kidney disease in children: the Pediatric Investigation for Genetic Factors Linked with Renal Progression Consortium.

BACKGROUND: Chronic kidney disease (CKD) in children is characterized by rapid progression and a high incidence of end-stage renal disease and therefore constitutes an important health problem. While unbiased genetic screens have identified common risk variants influencing renal function and CKD in adults, the presence and identity of such variants in pediatric CKD are unknown. METHODS: The international Pediatric Investigation for Genetic Factors Linked with Renal Progression (PediGFR) Consortium comprises three pediatric CKD cohorts: Chronic Kidney Disease in Children (CKiD), Effect of Strict Blood Pressure Control and ACE Inhibition on the Progression of CRF in Pediatric Patients (ESCAPE) and Cardiovascular Comorbidity in Children with CKD (4C). Clean genotype data from > 10 million genotyped or imputed single-nucleotide polymorphisms (SNPs) were available for 1136 patients with measurements of serum creatinine at study enrollment. Genome-wide association studies were conducted to relate the SNPs to creatinine-based estimated glomerular filtration rate (eGFR crea) and proteinuria (urinary albumin- or protein-to-creatinine ratio ≥ 300 and ≥ 500 mg/g, respectively). In addition, European-ancestry PediGFR patients (cases) were compared with 1347 European-ancestry children without kidney disease (controls) to identify genetic variants associated with the presence of CKD. RESULTS: SNPs with suggestive association P-values < 1 × 10(-5) were identified in 10 regions for eGFR crea, four regions for proteinuria and six regions for CKD including some plausible biological candidates. No SNP was associated at genome-wide significance (P < 5 × 10(-8)). Investigation of the candidate genes for proteinuria in adults from the general population provided support for a region on chromosome 15 near RSL24D1/UNC13C/RAB27A. Conversely, targeted investigation of genes harboring GFR-associated variants in adults from the general population did not reveal significantly associated SNPs in children with CKD. CONCLUSIONS: Our findings suggest that larger collaborative efforts will be needed to draw reliable conclusions about the presence and identity of common variants associated with eGFR, proteinuria and CKD in pediatric populations.

Kif21a deficiency leads to impaired glomerular filtration barrier function.

The renal glomerulus represents the major filtration body of the vertebrate nephron and is responsible for urine production and a number of other functions such as metabolic waste elimination and the regulation of water, electrolyte and acid-base balance. Podocytes are highly specialized epithelial cells that form a crucial part of the glomerular filtration barrier (GFB) by establishing a slit diaphragm for semipermeable plasma ultrafiltration. Defects of the GFB lead to proteinuria and impaired kidney function often resulting in end-stage renal failure. Although significant knowledge has been acquired in recent years, many aspects in podocyte biology are still incompletely understood. By using zebrafish as a vertebrate in vivo model, we report a novel role of the Kinesin-like motor protein Kif21a in glomerular filtration. Our studies demonstrate specific Kif21a localization to the podocytes. Its deficiency resulted in altered podocyte morphology leading to podocyte foot process effacement and altered slit diaphragm formation. Finally, we proved considerable functional consequences of Kif21a deficiency by demonstrating a leaky GFB resulting in severe proteinuria. Conclusively, our data identified a novel role of Kif21a for proper GFB function and adds another piece to the understanding of podocyte architecture and regulation.

A novel role for the chloride intracellular channel protein Clic5 in ciliary function.

CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left-right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation.

The Rac1 regulator ELMO1 controls vascular morphogenesis in zebrafish.

RATIONALE: Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180 complex forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. OBJECTIVE: To investigate the function of ELMO1/DOCK180 in vascular development. METHODS AND RESULTS: In situ hybridization studies for elmo1 identified a vascular and neuronal expression in zebrafish. Morpholino-based expression silencing of elmo1 severely impaired the formation of the vasculature, including intersomitic vessels, the dorsal longitudinal anastomotic vessel, the parachordal vessel, and the development of the thoracic duct in tg(fli1:EGFP) embryos. Mechanistically, we identified Netrin-1 and its receptor Unc5B as upstream activators of the ELMO1/DOCK180 complex, regulating its functional interaction and leading to Rac1 activation in endothelial cells and vessel formation in zebrafish. CONCLUSIONS: Our data have identified a novel signaling cascade regulating vasculature formation in zebrafish.

Microridge-like structures anchor motile cilia.

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.

Expression of rasgef1b in zebrafish.

The rasgef genes encode a subgroup of highly conserved Ras guanine nucleotide exchange factors. While EST projects revealed the presence of rasgef genes in organisms that range from nematodes to humans, their functions remain to be elucidated. In zebrafish two rasgef genes, rasgef and rasgef1b, have been identified and high throughput analysis revealed tissue specific embryonic expression for rasgef1b. Here, we show that three rasgef1b-transcripts are generated from two transcriptional start sites and by alternative splicing. Detailed expression analyses show that rasgef1b is expressed in a subset of adaxial cells, in the anterior part of somites, in the rostral part of the mid-hindbrain boundary and in the rhombomere boundaries. In the larva, rasgef1b is further expressed in the pallium and the inner nuclear layer of the retina. We also find that rasgef1b is expressed maternally and that the ubiquitous distribution of maternal transcripts disappears shortly after mid-blastula transition. At early epiboly stages, rasgef1b expression is restricted to the margin with low levels of expression on the ventral and high levels of expression on the dorsal side. Finally, we show that early zygotic expression is regulated by Nodal and FGF signals and that these signals have different activities in regulating the level and distribution of early zygotic rasgef1b mRNA expression.

The neuronal transcription factor NPAS4 is a strong inducer of sprouting angiogenesis and tip cell formation.

RATIONALE: Regarding branching morphogenesis, neurogenesis and angiogenesis share common principle mechanisms and make use of the same molecules. Therefore, the investigation of neuronal molecules involved in vascular morphogenesis provides new possibilities for pro-angiogenic approaches in cardiovascular diseases. OBJECTIVE: In this study, we investigated the role of the neuronal transcription factor NPAS4 in angiogenesis. METHODS AND RESULTS: Here, we demonstrate that the neuronal transcription factor NPAS4 is expressed in endothelial cells of different origin using reverse transcription PCR and western blot analysis. To investigate how NPAS4 affects endothelial cell function, NPAS4 was overexpressed by plasmid transfection or depleted from human umbilical vein endothelial cells (HUVECs) by specific siRNAs. In vitro HUVEC sprouting assays showed that sprouting and branching of endothelial cells was enhanced by NPAS4 overexpression. Consistently, silencing of NPAS4 resulted in reduced HUVEC sprouting and branching. Mechanistically, we identified as target gene vascular endothelial adhesion molecule VE-cadherin to be involved in the pro-angiogenic function of NPAS4. In endothelial cell mosaic spheroid sprouting assays, NPAS4 was involved in tip cell formation. In vivo experiments in mouse and zebrafish confirmed our in vitro findings. NPAS4-deficient mice displayed reduced ingrowth of endothelial cells in the Matrigel plug assay. Consistent with a regulatory role of NPAS4 in endothelial cell function silencing of NPAS4 in zebrafish by specific morpholinos resulted in perturbed intersegmental vessels growth. CONCLUSIONS: NPAS4 is expressed in endothelial cells, regulates VE-cadherin expression and regulates sprouting angiogenesis.

Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

A complex of BBS1 and NPHP7 is required for cilia motility in zebrafish.

Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, situs inversus and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish bbs1 and nphp7.2 caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of zbbs1 and znphp7.2 enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between zbbs1 and znphp7.2 in vivo. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3.

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis.

Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.