Flora Silbermann

The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.

The gene mutated in juvenile nephronophthisis type 4 encodes a novel protein that interacts with nephrocystin

Nephronophthisis, the most common genetic cause of chronic renal failure in children, is a progressive tubulo-interstitial kidney disorder that is inherited as an autosomal recessive trait. The disease is characterized by polyuria, growth retardation and deterioration of renal function during childhood or adolescence. The most prominent histological features are modifications of the tubules with thickening of the basement membrane, interstitial fibrosis and, in the advanced stages, medullary cysts. Nephronophthisis can also be associated with conditions affecting extrarenal organs, such as retinitis pigmentosa (Senior–Løken syndrome) and ocular motor apraxia (Cogan syndrome). Three loci are associated with the juvenile, infantile and adolescent forms, on chromosomes 2q13 (NPHP1; refs 5,6), 9q22 (NPHP2; ref. 7) and 3q21 (NPHP3; ref. 8), respectively. NPHP1, the only gene identified so far, encodes nephrocystin, which contains a Src homology 3 (SH3) domain and interacts with intracytoplasmic proteins involved in cell adhesion. Recently, a second locus associated with the juvenile form of the disease, NPHP4, was mapped to chromosome 1p36 (ref. 14). We carried out haplotype analysis of families affected with nephronophthisis that were not linked to the NPHP1, NPHP2 or NPHP3 loci, using markers covering this region. This allowed us to reduce the NPHP4 interval to a one centimorgan interval between D1S2795 and D1S2870, which contains six genes. We identified five different mutations in one of these genes, designated NPHP4, in unrelated individuals with nephronophthisis. The NPHP4 gene encodes a 1,250–amino acid protein of unknown function that we named nephrocystin-4. We demonstrated the interaction of nephrocystin-4 with nephrocystin suggesting that these two proteins participate in a common signaling pathway.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end-stage renal failure. The disease is caused by mutations in NPHP1-9 genes, which encode the nephrocystins, proteins localized to cell–cell junctions and centrosome/primary cilia. Here, we show that nephrocystin mRNA expression is dramatically increased during cell polarization, and shRNA-mediated knockdown of either NPHP1 or NPHP4 in MDCK cells resulted in delayed tight junction (TJ) formation, abnormal cilia formation and disorganized multi-lumen structures when grown in a three-dimensional collagen matrix. Some of these phenotypes are similar to those reported for cells depleted of the TJ proteins PALS1 or Par3, and interestingly, we demonstrate a physical interaction between these nephrocystins and PALS1 as well as their partners PATJ and Par6 and show their partial co-localization in human renal tubules. Taken together, these results demonstrate that the nephrocystins play an essential role in epithelial cell organization, suggesting a plausible mechanism by which the in vivo histopathologic features of NPH might develop.

Integrin Alpha 8 Recessive Mutations Are Responsible for Bilateral Renal Agenesis in Humans

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.

A Homozygous Missense Mutation in the Ciliary Gene TTC21B Causes Familial FSGS

Several genes, mainly involved in podocyte cytoskeleton regulation, have been implicated in familial forms of primary FSGS. We identified a homozygous missense mutation (p.P209L) in the TTC21B gene in seven families with FSGS. Mutations in this ciliary gene were previously reported to cause nephronophthisis, a chronic tubulointerstitial nephropathy. Notably, tubular basement membrane thickening reminiscent of that observed in nephronophthisis was present in patients with FSGS and the p.P209L mutation. We demonstrated that the TTC21B gene product IFT139, an intraflagellar transport-A component, mainly localizes at the base of the primary cilium in developing podocytes from human fetal tissue and in undifferentiated cultured podocytes. In contrast, in nonciliated adult podocytes and differentiated cultured cells, IFT139 relocalized along the extended microtubule network. We further showed that knockdown of IFT139 in podocytes leads to primary cilia defects, abnormal cell migration, and cytoskeleton alterations, which can be partially rescued by p.P209L overexpression, indicating its hypomorphic effect. Our results demonstrate the involvement of a ciliary gene in a glomerular disorder and point to a critical function of IFT139 in podocytes. Altogether, these data suggest that this homozygous TTC21B p.P209L mutation leads to a novel hereditary kidney disorder with both glomerular and tubulointerstitial damages.

CONTROL OF THE WNT PATHWAYS BY NEPHROCYSTIN-4 IS REQUIRED FOR MORPHOGENESIS OF THE ZEBRAFISH PRONEPHROS

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-β-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-β-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.

Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity

Rpgrip1l is required for planar localization of the basal body and acts within a ciliopathy protein complex by stabilizing dishevelled.

Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.

Relationship between inhibition of cell growth and of transferrin receptor expression by interferon (IFN) alpha: studies in IFN-sensitive and IFN-resistant Daudi cells.

We previously showed that treatment of different cell lines with interferon-alpha (IFN-alpha) concurrently inhibited both cell growth and the rise observed in 125I-labelled transferrin binding when cells are exposed to culture conditions that stimulate proliferation. To gain insight into the relationship between these two IFN-induced inhibitory processes, we investigated the effect of IFN-alpha on the binding of 125I-labelled transferrin to Daudi cells sensitive or resistant to its antiproliferative action. We found a close correlation between the ability of IFN-alpha to inhibit cell growth and to inhibit transferrin receptor expression. Since growth inhibition induced by other agents is not always accompanied by an inhibition of transferrin receptor expression, the previous and present observations suggest that the inhibitory effect of IFN on this expression is at least one of the mechanisms by which IFN inhibits cell proliferation. We also observed that IFN-alpha did not modify transferrin receptor biosynthesis in IFN-sensitive Daudi cells, suggesting that IFN-alpha may change the processing of the transferrin receptor molecules, making them unable to bind transferrin.