Mutations in CSPP1 Lead to Classical Joubert Syndrome

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.     

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies.     

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.     

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.     

Role of cilia in structural birth defects: Insights from ciliopathy mutant mouse models

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia‐related genes from many large‐scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs. Birth Defects Research (Part C) 102:115–125, 2014. © 2014 Wiley Periodicals, Inc.     

Mutations of CEP83 Cause Infantile Nephronophthisis and Intellectual Disability

Ciliopathies are a group of hereditary disorders associated with defects in cilia structure and function. The distal appendages (DAPs) of centrioles are involved in the docking and anchoring of the mother centriole to the cellular membrane during ciliogenesis. The molecular composition of DAPs was recently elucidated and mutations in two genes encoding DAPs components (CEP164/NPHP15, SCLT1) have been associated with human ciliopathies, namely nephronophthisis and orofaciodigital syndrome. To identify additional DAP components defective in ciliopathies, we independently performed targeted exon sequencing of 1,221 genes associated with cilia and 5 known DAP protein-encoding genes in 1,255 individuals with a nephronophthisis-related ciliopathy. We thereby detected biallelic mutations in a key component of DAP-encoding gene, CEP83, in seven families. All affected individuals had early-onset nephronophthisis and four out of eight displayed learning disability and/or hydrocephalus. Fibroblasts and tubular renal cells from affected individuals showed an altered DAP composition and ciliary defects. In summary, we have identified mutations in CEP83, another DAP-component-encoding gene, as a cause of infantile nephronophthisis associated with central nervous system abnormalities in half of the individuals.     

CSPP Is a Ciliary Protein Interacting with Nephrocystin 8 and Required for Cilia Formation

We described previously the cell cycle- and microtubule-related functions of two splice isoforms of the centrosome spindle pole-associated protein (CSPP and CSPP-L). Here, we show that endogenous CSPP isoforms not only localize to centrosomes and the midbody in cycling cells but also extend to the cilia axoneme in postmitotic resting cells. They are required for ciliogenesis in hTERT-RPE1 cells in vitro and are expressed in ciliated renal, retinal, and respiratory cells in vivo. We report that CSPP isoforms require their common C-terminal domain to interact with Nephrocystin 8 (NPHP8/RPGRIP1L) and to form a ternary complex with NPHP8 and NPHP4. We find CSPP-L to be required for the efficient localization of NPHP8 but not NPHP4 to the basal body. The ciliogenesis defect in hTERT-RPE1 cells is, however, not mediated through loss of NPHP8. Similar to the effects of ectopical expression of CSPP-L, cilia length increased in NPHP8-depleted cells. Our results thus suggest that CSPP proteins may be involved in further cytoskeletal organization of the basal body and its primary cilium. To conclude, we have identified a novel, nonmitotic function of CSPP proteins placing them into a ciliary protein network crucial for normal renal and retinal tissue architecture and physiology.     

Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1^Gt/Gt). Surprisingly, Azi1^Gt/Gt MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.     

Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1^−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1^−/− cells.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation

Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.     

The NIMA-related kinase NEK1 cycles through the nucleus

Mutations in NEK1 in mice are causal for cystic kidneys, and model the ciliopathy polycystic kidney disease caused by abnormal ciliary structure or signaling. NEK1 has previously been shown to localize near centrosomes and to play a role in centrosomal stability and ciliogenesis. Recent data suggest that the etiology of kidney cysts involves aberrant signaling from the primary cilium to the nucleus. Here we demonstrate that NEK1 contains functional nuclear localization signals, is exported from the nucleus via a nuclear export signal-dependent pathway and that the protein cycles through the nucleus. Our data suggest that NEK1 is a candidate to transduce messages from the ciliary-basal body region to the regulation of nuclear gene expression.     

Identification of a Ninein (NIN) mutation in a family with spondyloepimetaphyseal dysplasia with joint laxity (leptodactylic type)-like phenotype

Spondyloepimetaphyseal dysplasia with joint laxity-leptodactylic type (SEMDJL2) is an autosomal dominant skeletal dysplasia which is characterized by midface hypoplasia, short stature, joint laxity with dislocations, genua valga, progressive scoliosis, and slender fingers. Recently, heterozygous missense mutations in KIF22, a gene which encodes a member of the kinesin-like protein family, have been identified in sporadic as well as familial cases of SEMDJL2. In the present study homozygosity mapping and whole-exome sequencing were combined to analyze a consanguineous family with a phenotype resembling SEMDJL2. We identified homozygous missense mutations in the two nearby genes NIN (Ninein) and POLE2 (DNA polymerase epsilon subunit B) which segregate with the disease in the family and were not present in 500 healthy control individuals and in the 1094 control individuals contained within the 1000-genomes database. We present several lines of evidence that mutant Ninein is most likely causative for the SEMDJL2-like phenotype. The centrosomal protein NIN shows a functional relationship with KIF22 and other proteins associated with chromosome congression/movement, centrosomal function, and ciliogenesis, which have been associated with skeletal dysplasias. Moreover, compound heterozygous missense mutations at more N-terminal positions of Ninein have very recently been identified in a family with microcephalic primordial dwarfism. Together with the present report this strongly supports a fundamental role of Ninein in skeletal development.     

CYLD mediates ciliogenesis in multiple organs by deubiquitinating Cep70 and inactivating HDAC6

Cilia are hair-like organelles extending from the cell surface with important sensory and motility functions. Ciliary defects can result in a wide range of human diseases known as ciliopathies. However, the molecular mechanisms controlling ciliogenesis remain poorly defined. Here we show that cylindromatosis (CYLD), a tumor suppressor protein harboring deubiquitinase activity, plays a critical role in the assembly of both primary and motile cilia in multiple organs. CYLD knockout mice exhibit polydactyly and various ciliary defects, such as failure in basal body anchorage and disorganization of basal bodies and axenomes. The ciliary function of CYLD is partially attributed to its deconjugation of the polyubiquitin chain from centrosomal protein of 70 kDa (Cep70), a requirement for Cep70 to interact with γ-tubulin and localize at the centrosome. In addition, CYLD-mediated inhibition of histone deacetylase 6 (HDAC6), which promotes tubulin acetylation, constitutes another mechanism for the ciliary function of CYLD. Small-molecule inhibitors of HDAC6 could partially rescue the ciliary defects in CYLD knockout mice. These findings highlight the importance of protein ubiquitination in the modulation of ciliogenesis, identify CYLD as a crucial regulator of this process, and suggest the involvement of CYLD deficiency in ciliopathies.     

Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral‐facial‐digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein Cep164. Myosin VI depletion in non‐tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be, at least partially, ascribed to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.     

NEK1 mutations cause short-rib polydactyly syndrome type majewski

Defects of ciliogenesis have been implicated in a wide range of human phenotypes and play a crucial role in signal transduction and cell-cycle coordination. We used homozygosity mapping in two families with autosomal-recessive short-rib polydactyly syndrome Majewski type to identify mutations in NEK1 as an underlying cause of this lethal osteochondrodysplasia. NEK1 encodes a serine/threonine kinase with proposed function in DNA double-strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis. We found that absence of functional full-length NEK1 severely reduces cilia number and alters ciliar morphology in vivo. We further substantiate a proposed digenic diallelic inheritance of ciliopathies by the identification of heterozygous mutations in NEK1 and DYNC2H1 in an additional family. Notably, these findings not only increase the broad spectrum of ciliar disorders, but suggest a correlation between the degree of defective microtubule or centriole elongation and organization and the severity of the resulting phenotype.     

A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alstr?m syndrome. Alstr?m syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alstr?m syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alstr?m syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alstr?m syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.     

DCDC2 Mutations Cause a Renal-Hepatic Ciliopathy by Disrupting Wnt Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.