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Genetic bases and pathogenic mechanisms of nephronophthisis
DDMEC-396; No of Pages 9
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. xxx, No. xx 2013
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – University of Rochester Medical Center, Rochester, NY.
DISEASE Mechanism underlying inherited renal cystic diseases MECHANISMS
Genetic bases and pathogenic mechanisms of nephronophthisis Marion Delous1,2, Helori M. Gaude´2, Sophie Saunier2,* 1 2
Max-Planck-Institute for Heart and Lung Development, Bad Nauheim, Germany1 INSERM, U983, Paris Descartes, Sorbonne Paris Cite´ University, Imagine Institute, 75015 Paris, France
Nephronophthisis is a recessive cystic kidney disorder that belongs to the group of ciliopathies. Most of the causal gene products localize at the primary cilium, as components of either the transition zone or the retrograde intraflagellar transport IFT-A complex, where they control ciliary protein trafficking and modulate responses to various signaling pathways. In this review, we summarize the current literature on nephronophthisis-related disease genetics and outline the
Section editors Thomas: Benzing and Bernhard Schermer – Kidney Research Center Cologne, Germany. located at the apical surface, which are essential for extracellular chemical and mechanical signal transduction, ensuring tissue morphogenesis and homeostasis [2]. In this review, we cover the latest insights into the molecular and cellular mechanisms underlying NPH.
essential pathophysiological mechanisms underlying Basic genetics of NPH and related ciliopathies (NPH-RC) NPHP genes involved in NPH-RC
these disorders.
Introduction Nephronophthisis (NPH) is the major genetic cause of chronic kidney diseases in children [1], characterized by tubular atrophy with thickened tubular basement membrane, severe interstitial fibrosis and formation of medullary cysts. The juvenile form leads to end-stage renal disease within the first decades of life. Extra-renal symptoms, such as retinal dystrophy, cerebellar vermis hypoplasia/aplasia, hepatic fibrosis, skeletal abnormalities and situs inversus are present in a significant proportion of NPH patients and their association with NPH represents distinct clinical entities. More than 15 NPH-related genes encoding NPHP proteins have been identified. NPHP proteins localize at cell junctions and the primary cilium, thus defining NPH and associated disorders as ciliopathies. Primary cilia are cellular organelles *Corresponding author.: S. Saunier ([email protected]) 1 Current address. 1740-6765/$ ß 2013 Elsevier Ltd. All rights reserved.
Nephronophthisis is a clinically and genetically heterogeneous recessive disorder. Genetic studies combining positional cloning and candidate gene approaches allowed the identification of eleven genes mutated in patients with NPHRC, NPHP1-11 [3] (Table 1). Each NPHP protein is localized at the primary cilium where they work in complex with other ciliopathy-related proteins to maintain cilia structure and function [4]. The identification of additional genes involved in NPH-RC has been facilitated by a combination of ciliary interactome and whole/ciliopathy candidate exome sequencing strategies. Notably, mutations in genes encoding components of the retrograde intraflagellar transport complex A (IFT-A) have been identified in patients with skeletal ciliopathies associated with NPH (Table 1). Mutations of IFT140 cause Mainzer–Saldino Syndrome [5], characterized by cone-shaped epiphysis of the phalanges, NPH and retinal dystrophy. Conditional knockout of Ift140 in mouse collecting ducts results
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Protein
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Isolated NPH
Infantile NPH
NPH-related ciliopathies With liver fibrosis
With situs inversus
With cardiac defects
Other ciliopathies without NPH SLS
JBTS-B
U
U
NPHP1
Nephrocystin-1
UU
INVS/NPHP2
Inversin, nephrocystin-2
U
UU
U
UU
U
NPHP3
Nephrocystin-3
U
U(tr)
U(hyp)
U
U
NPHP4
Nephrocystin-4
U
IQCB1/NPHP5
IQ-motif containing protein, nephrocystin-5
UU
CEP290/NPHP6
Centrosomal protein 290
U
GLIS7/NPHP7
GLI similar 2
RPGRIP1L/NPHP8/ MKS5
RPGRIP1-like
NEK8/NPHP9
NIMA-related kinase 8
SDCCAG8/NPHP10/ SLSN7
Seralogically defined colon cancer antigen 8
TMEM67/NPHP11/ MKS3
Transmembrane protein 67
TTC21B/NPHP12 [7]
IFT139
U
WDR19/NPHP13 [8]
IFT144
U
CEP164/NPHP15 [10]
Centrosomal protein 164
TMEM216/MKS2/ JBTS2 [56]
Transmembrane protein 216
MSS
JATD
SS
BBS-like
LCA
JATD
JBTS
COACH
U
U (tr)
U U
MKS
U
UU
U
UU
U
U U (hyp)
U
U (hyp)
U (tr)
U
U
U U
AHI1 [14]
Jouberin
U
CC2D2A/MKS6 [56]
Coiled coil and C2 domain containing 2A
U
ZNF423/NPHP14 [10]
ZNF423
U
U
U
U
U U U
U
U
U
U U U
U
U
U
U
U: Involvement; UU: main involvement; hyp, hypomorphe mutation; tr, truncating mutation; NPH, nephronophthisis; SLS, Senior–Løken Syndrome; JBTS-B, Joubert Syndrome type B; MKS, Meckel–Gruber Syndrome; MSS, Mainzer–Saldino Syndrome; JATD, Jeune Asphyxiating Thoracic Dystrophy; SS, Sensenbrenner Syndrome; BBS, Bardet–Biedl Syndrome; LCA, Leber’s congenital amaurosis; COACH, Cerebellar vermis hypoplasia/aplasia, Oligophrenia, Ataxia, Coloboma and Hepatic fibrosis.
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Ataxin 10
U (tr)
U
U
IFT140
U
U
U
ATXN10 [4]
U (tr)
U U
IFT140 [5]
U
DDMEC-396; No of Pages 9
GENE [3]
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Table 1. Genes mutated in isolated NPH and NPH-RC
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Drug Discovery Today: Disease Mechanisms | Mechanism underlying inherited renal cystic diseases
in cystic kidneys, which in contrast with other models of polycystic kidney disease, are not due to mitotic axis orientation defects, but rather to increased cell proliferation coupled to an alteration of several signaling pathways (canonical Wnt, Sonic Hedgehog (Shh)) [6]. TTC21B/NPHP12, encoding IFT139, is involved in isolated NPH or NPH associated with Jeune Asphyxiating Thoracic Dystrophy (JATD) [7]. Pathogenic mutations of WDR19/NPHP13, encoding IFT144, are associated with a large variety of phenotypes: isolated NPH, JATD and Sensenbrenner Syndrome (SS), characterized by cranioectodermal dysplasia [8]. Mutations in the other IFTA genes IFT122, IFT43 and WDR35 (IFT121 homolog), have also been reported in patients with skeletal ciliopathies associated with chronic renal failure [9] (our unpublished data). Finally, mutations in two additional genes, CEP164/ NPHP15 and ZNF423/NPHP14, have been recently reported in patients with Senior–Løken Syndrome (SLS), Joubert Syndrome (JBTS), Bardet–Biedl Syndrome (BBS)-like and infantile NPH [10] (Table 1). These genes, involved in DNA damage response (DDR) signaling, point to a novel pathophysiological mechanism in these disorders (see below).
Genotype–phenotype correlations Altogether, mutations in the identified causative genes account for approximately 30% of the published NPH-RC patient cohort [11] and 44% of the patients in our cohort at Necker Hospital (860 patients from 792 families). Genotype– phenotype correlation analyses have demonstrated a genetic overlap, and highly variable phenotypes are associated with mutations in these genes (Table 1 and Fig. 1). The common cause of juvenile NPH, which underlies 56% (published cohort) [12] to 62% (Necker Hospital cohort, unpublished data) of patients with identified mutations, is the homozygous deletion of NPHP1, due to unequal recombination between duplicated regions flanking the gene [13]. Mutations in the other NPHP genes account for lower proportions of cases in both cohorts: <10% for NPHP4, IQCB1/NPHP5, CEP290/NPHP6, TMEM67/NPHP11 and TTC21B/NPHP12, <5% for the other genes, with sometimes only one family associated with recessive mutations of a gene (i.e. GLIS2/ NPHP7 and ATXN10) [14] (Table 1). Some NPHP genes are preferentially associated with specific clinical features, thus facilitating diagnosis. For instance, NPHP1 and NPHP4 are
NPH-RC disease spectrum MSS
NPH
JATD
TTC21B
SS
SRP
TTC21B IFT140
Cilia
IFT43 WDR19 IFT122
IFT B
WDR35
DYNC2H1
Inversin compartment
KIF3A/B/C
IFT A
Transition zone
Inversin
RPGRIP1L NPHP4
NPHP1
ATXN10 IQCB1
CEP290 Exocyst
JBTS
MKS
NPHP3
NPHP3
NEK8 SDCCAG8 NPHP1 NPHP4 RPGRIP1L TMEM67
MKS complex
Transition Fibers
Basal Body
SLS
INVS
Transition zone
NPHP3 NEK8
Basal Body
Inversin compartment
NPH
CEP290 ATXN10 IQCB1 CEP164 GLIS2
Degenerative Disease
Dysplastic Disease Drug Discovery Today: Mechanisms
Figure 1. NPHP proteins at the primary cilium and nephronophthisis-related ciliopathy (NPH-RC) disease spectrum. Proteins encoded by genes involved in NPH-RC share a common localization at the base of the cilium (left panel) where they assemble within different subdomains: ATXN10/CEP290/IQCB1 at the basal body (light purple), NPHP1/NPHP4/RPGRIP1L at the transition zone (light brown) and inversin/NPHP3/NEK8 at the inversin compartment (light orange). Together with the MKS complex (grey), these four complexes establish a gate that selects ciliary cargo and helps them dock on IFTs to ensure cilia function and maintenance. A second class of proteins associated with NPH-RC belongs to the retrograde IFT-A complex (light pink). The ciliary membrane composition controlled in part by the NPHP and MKS proteins is tissue-dependent, thus explaining the variability of the organ involvement in NPH-RC. NPH-associated disorders represent a spectrum (right panel) ranging from mild degenerative diseases (isolated NPH) to severe dysplastic diseases with fetal presentation. NPH: Nephronophthisis, MSS: Mainzer–Saldino Syndrome, JATD: Jeune Asphyxiating Thoracic Dystrophy, SS: Sensenbrenner Syndrome, SRP: Short-Rib-Polydactyly Syndrome, SLS: Senior–Løken Syndrome, JBTS: Joubert Syndrome, MKS: Meckel–Gruber Syndrome.
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DDMEC-396; No of Pages 9 Drug Discovery Today: Disease Mechanisms | Mechanism underlying inherited renal cystic diseases
generally linked to isolated NPH, whereas INVS/NPHP2 (encoding inversin) is associated with infantile NPH (endstage renal disease before 3-years-old). Mutations of IQCB1/ NPHP5 are tightly linked to NPH patients with severe ocular alteration (SLS) and the age at which renal symptoms present is highly variable [15]; mutations of IFT-A genes are generally observed in NPH patients with skeletal anomalies (MSS, JATD, SS) [16] (Table 1). The broad range of phenotypes associated with mutations of any given NPHP gene can be explained in part by the variability of organ-specific functions of the cilia (i.e. visual function of the photoreceptor cells, solute exchange function of the tubular epithelial cells). However, the nature of the mutations can also influence phenotypes. Hypomorphic TMEM67/NPHP11 or NPHP3 mutations cause juvenile NPH with limited extrarenal involvement, while truncating mutations lead to the severe developmental features of Meckel–Gruber Syndrome (MKS; central nervous system malformations, renal cystic dysplasia, polydactyly) (Table 1 and Fig. 1). Similarly, expressivity of bone and retinal phenotypes seems to depend on the nature of the IFT-A mutations: a mild phenotype such as isolated NPH is associated with hypomorphic mutations in TTC21B/ NPHP12 (IFT139) or WDR19/NPHP13 (IFT144), while truncating mutations of WDR19/NPHP13 result in SS. In agreement with this correlation, functional analyses of cilia in patient fibroblasts and mutant mouse tissues revealed subtle defects associated with hypomorphic IFT-A alleles, whereas total allele loss dramatically affected cilia structure and disrupted downstream signaling pathways [5,6,8,17]. Finally, for several NPHP genes, a same mutation can lead to a large spectrum of defects. Some CEP290/NPHP6 mutations cause either isolated Leber congenital amaurosis (LCA) or MKS [18], suggesting that modifier genes can modulate the phenotypic expressivity of NPH-RC mutations. Indeed, all the mutations or variants of ciliopathyrelated genes carried by one patient are expected to contribute to the manifestation of the phenotype. This concept is referred as mutational load [19]. Hence, heterozygous pathogenic mutations of TTC21B/NPHP12 (IFT139) are enriched in ciliopathy cases (5%) [7], suggesting that they have a modifier effect across the ciliopathy spectrum in addition to their causal role in NPH/JATD patients. Extending next generation exome/genome sequencing to large cohorts of ciliopathy patients will allow identification of further genes involved and decipher the role of modifiers in NPHP-RC.
Functions of NPHP proteins Several recent studies have significantly advanced our understanding of the signaling pathways and pathophysiological mechanisms involving NPHP proteins. e4
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NPHP proteins as cilia gatekeepers Despite their localization at the primary cilia/centrosome, most nephrocystins are not essential for cilium formation per se. Indeed, loss of function of the majority of the NPHP proteins in mammalian cells, animal models or patient tissues, does not affect ciliogenesis [4,20,21] (our unpublished data). However, NPHP proteins play a crucial role, together with the MKS-related proteins, in establishing a ciliary gate that is essential for cilium function and maintenance (Table 2). The combination of proteomic analyses and in vivo studies in Caenorhabditis elegans showed that NPHP proteins are organized in complexes at different subdomains of the cilium base [4,22] (Fig. 1). The first module, consisting of NPHP1, NPHP4 and RPGRIP1L/NPHP8, is located at the transition zone (TZ), a conserved structure characterized by Y-shaped linkers that link the microtubule doublets of the axoneme to the membrane (ciliary necklace). These proteins participate in both the integrity and stability of the TZ-protein complexes. Adjacent to the TZ is the ‘Inversin compartment’ that consists of the inversinNPHP3-NEK8/NPHP9 complex [21] and the centrosome, where IQCB1/NPHP5 and CEP290/NPHP6 form another module with ATXN10 [4]. In Chlamydomonas reinhardtii, Cep290 links the flagellar membrane to the TZ [22], a function also shared by RPGRIP1L/NPHP8 and inversin, which connect NPHP modules to the MKS-related proteins at the ciliary membrane [4,23–25]. Altogether, NPHP proteins participate in the organization of the TZ, whereas IQCB1/ NPHP5, by interacting with the exocyst, may help to its recruitment at the cilia base, thereby promoting membrane trafficking towards the cilium [4]. The correct targeting of TZrelated proteins is thus essential for cilia function and regulators of this process have been recently identified, such as UNC119 that binds the myristoylated NPHP3 protein and allows its ciliary targeting [26]. The second class of NPH-RC associated proteins belongs to the retrograde IFT-A complex that transports proteins from the tip to the base of the cilia [27]. IFT proteins, present at the transition fibers, functionally interact with TZ components. Indeed, the MKS complex along with inversin interacts with IFT particles in zebrafish [28] and loss of Nphp1 in mouse photoreceptor cells leads to a mislocalization of IFTs [29], suggesting that the TZ complex acts as a molecular filter that selects and retains cilia components, including IFT proteins and their cargos (Fig. 2). The variability of the organs involved in the ciliopathy spectrum suggests that alterations of some of the functions of NPHP-MKS and IFT-A complexes are tissue specific [24]. Thus, the complexity of phenotypes of the patients with NPHP and IFT-A mutations is likely due to the loss of specific membrane proteins in the mutant cilia. By modifying the ciliary protein composition, mutations of NPHP/IFTA components may alter the pathways that are controlled by ciliary receptors,
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Subcellular localization [3,27,57]
Functions [3,27,38,57–59]
Models Zebrafish
Mouse [60]
Nephrocystin-1
BB, TZ, cilia, cell junctions
Formation and function of TZ, cell junction regulator
Pronephric cysts, cloaca obstruction [44]
Retinal degeneration, male sterility (Nphp1tm1.1hung); retinal degeneration (Nphp1tm1jgg)
INVS/NPHP2
Inversin, nephrocystin-2
BB, Inv compartment, apical membrane, nucleus
Anchoring of NPHP3 and NEK8 at TZ, canonical Wnt inhibitor and PCP activator, cell cycle regulator
Pronephric cysts, laterality and CE defects [40]
Situs-inversus, jaundice, enlarged cystic kidneys (invsInv)
NPHP3
Nephrocystin-3
Inv compartment, nucleus, cell junctions
Modulator of canonical Wnt/ PCP, cilia function
Hydrocephaly, pronephric cysts, laterality and CE defects [61]
Cystic kidneys (Nphp3pcy); laterality defects, congenital heart defects, embryonic lethality (Nphp3tm1Cbe)
NPHP4
Nephrocystin-4
BB, TZ, cilia, cell junctions
Formation and function of TZ, canonical Wnt inhibitor and PCP activator, cell junction regulator
Pronephric cysts, cloaca obstruction, laterality and CE defects, retinal anomalies [39,44]
Male sterility, retinal degeneration (Nphp4nmf192)
IQCB1/NPHP5
Nephrocystin-5
BB, cilia, cell junctions
RPGR/calmodulin complex, ciliogenesis
Hydrocephaly, pronephric cysts, laterality and CE defects [62]
None
CEP290/NPHP6
Centrosomal protein 290
TZ, BB
Formation and function of TZ, RPGR complex trafficking, cilia function, positive modulator of Hh
CE defects, pronephric cysts, retinal and cerebellar defects [63]
Anosmia, retinal degeneration (Cep290rd16); cerebellar hypoplasia, retinal degeneration (Cep290tm1jgg)
GLIS2/NPHP7
GLI similar 2
Cilia, nucleus
Transcription factor, negative modulator of Hh and canonical Wnt
None
Renal atrophy and fibrosis (Glis2tm1Amj); (Glis2tm1Tre)
RPGRIP1L/NPHP8/MKS5
RPGRIP1-like
TZ, cilia, cell junctions
Formation and function of TZ, modulator of Hh and canonical Wnt/PCP
Hydrocephaly, laterality and CE defects [42]
Exencephaly, polydactyly, laterality defects, cystic kidneys, birth lethality (Rpgrip1LtmUrt)
NEK8/NPHP9
NIMA-related kinase 8
Inv compartment, BB
Modulator of PC1 and PC2, cell cycle regulator
Pronephric cysts, laterality and CE defects [64]
Cystic kidneys (Nek8jck)
SDCCAG8/NPHP10/SLSN7
SDCCAG8
BB, TZ, cell junctions, nuclear foci
Cilia function
Pronephric cysts, hydrocephaly, CE defects [65]
None
TMEM67/NPHP11/MKS3
Transmembrane protein 67
ciliary membrane, TZ, cell junctions
Formation and function of TZ, intraciliary transport
Pronephric cysts, eye formation and CE defects [66]
Cystic kidneys, postnatal lethality (Tmem67tm1Dgen)
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NPHP1
DDMEC-396; No of Pages 9
Protein
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Gene
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Table 2. NPH-RC-related genes and their protein product functions and animal model-associated phenotypes
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Exencephaly, skeletal dysplasia, anopthalmia, hearing defects (Ift144twt)
Cystic kidneys (Ift140 HoxB7-Cre) [6]
None
None
Pronephric cysts, hydrocephaly, retinal dyslasia, laterality and CE defects, cell death [10]
Retrograde transport and cilia function, negative modulator of Hh
Retrograde transport and cilia function, modulator of Hh/ Wnt/Hippo [6]
DNA damage response [10]
BB, cilia
BB, cilia
BB, nuclear foci [10]
IFT144
IFT140
Centrosomal protein 164
WDR19/NPHP13
IFT140
CEP164/NPHP15
CE: convergence-extension defects (curved and shortened body axis, broad and kinked notochords, misshapen somites); BB: basal body; TZ: transition zone; Hh: Hedgehog signaling pathway; PCP: planar cell polarity; PC1: polycystin-1; PC2: polycystin-2, RPGR: retinitis pigmentosa GTPase regulator.
Polydactyly, split and fused ribs, neural tube, eye and forebrain defects, embryonic lethality (Ttc21baln)
None
Mouse [60] Zebrafish
CE defects [7] Retrograde transport and cilia function, negative modulator of Hh BB, cilia IFT139 TTC21B/NPHP12
Gene
Table 2 (Continued )
Protein
Models Functions [3,27,38,57–59] Subcellular localization [3,27,57]
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such as G coupled receptors [Smoothened receptor (Hh pathway), vasopressin type 2 receptor (V2R) or somatostatin receptor 3 (SSTR3)], Arl13B, type III adenylyl cyclase (ACIII) or ADPKD-related protein polycystin-2 (TRPP2) [30–32]. For instance, hypomorphic mutations of Ift144 in mice leads to defects in Sonic Hedgehog (Shh)-dependent patterning of the neural tube through a reduction of ciliary ACIII, a negative regulator of the pathway [17]. Alterations of the Hh pathway are also associated with mutations in genes involved in Meckel and Joubert syndromes characterized by limb (polydactyly) and neural tube defects [33,34]. In renal cells depleted for basal body-related proteins involved in Bardet–Biedl Syndrome, the absence of V2R at the primary cilium impairs the cellular responses to luminal vasopressin and leads to water reabsorption defects [35]. Then, defects in ciliary protein composition represent a major pathogenic mechanism in ciliopathies that affects ciliary signaling pathways essential for tissue homeostasis and development.
NPHP1 and cilia disassembly Polo-like kinase 1 (Plk1), a regulator of cell cycle progression, represents a key link between the non-canonical Wnt5a-Dvl2 signaling pathway and the HEF1-dependent Aurora A activation that controls cilia disassembly [36]. Interestingly, Plk1 localizes at the TZ where it interacts with and phosphorylates NPHP1, suggesting that NPHP1 also plays a role in cilia disassembly [37]. However, the exact function of phosphorylated NPHP1 in the HEF1-Aurora A pathway remains to be investigated. It is tempting to speculate that, as components of the cilia gate, NPHP proteins may act as sensors of cilia disassembly signals and their tight regulation by Plk1 would thus trigger and facilitate the reentry in the cell cycle necessary for tissue maintenance and homeostasis.
Canonical Wnt/b-catenin and non-canonical Wnt/PCP signaling pathways Along with other ciliary proteins, several NPHP proteins, notably inversin, NPHP3, NPHP4, CEP290/NPHP6 and RPGRIP1L/NPHP8, are involved in both canonical and non-canonical Wnt signaling pathways implicated in development, epithelial cell renewal and planar cell polarity (PCP) (Table 2). Indeed, both overactivation and downregulation of the canonical Wnt/b-catenin signaling in animal models, as well as loss of the Wnt/PCP signaling, lead to cyst formation [38]. A key regulator of both Wnt pathways is Dishevelled (Dvl). Interestingly, we and others have shown that the inversin-NPHP4-RPGRIP1L protein complex, present at both the cilium base and cell junctions, finely tunes Dvl protein level and cellular localization. Inversin and NPHP4 act as negative regulators of the canonical Wnt pathway by addressing cytosolic Dvl to the proteasome [39,40] and by stabilizing the ubiquitin ligase Jade-1, a negative regulator of b-catenin [41]. In contrast, we have shown that RPGRIP1L
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Cargo
Retrograde IFT
IFT-A
Cilia-dependent pathways
Fz Dvl NPHP
Y YY
YY Y
IFT-A
Wnt
Plk1
Dvl
Dvl stability Regulation
Crb
Cilia Gatekeepers
NPHP MKS
Cilia disassembly Regulation
Tight Junctions
NPHP Cell-cell junction Maintenance
Wnt/PCP pathway
Lats1
Junction Integrity
Hippo signaling
TGF-β Wnt/βcatenin Cell-matrix signal Transduction Pyk2 NPHP Cas
Adherent Junctions
NPHP4
DDR
DEDIFFERENTIATION PROLIFERATION CEP164 APOPTOSIS ECM
Focal Adhesions Drug Discovery Today: Mechanisms
Figure 2. Signaling pathways and physiopathological mechanisms involving NPHP proteins. At the primary cilia, NPHP proteins are present at the transition zone where they associate with MKS complex and IFT-A components to form the ciliary gate. Along the axoneme, IFT-A components are involved in the retrograde transport of ciliary cargos. By determining membrane composition and governing retrograde transport, the cilia gatekeeper ensures correct transduction of signals sensed by the primary cilium. At the transition zone (TZ), NPHP proteins interact with Plk1 suggesting a role in regulation of cilia disassembly. By interacting with the Wnt signaling downstream protein Dvl at the membrane and TZ, NPHP proteins regulate its stability and protein level to promote Wnt/PCP signaling. At cell–cell junctions, NPHP proteins interact with components of tight and adherens junctions participating in their stability and integrity. A similar role at focal adhesions is suggested by the association of NPHP proteins with signal transduction molecules involved in signal transduction (i.e. p130Cas, Pyk2). Both PCP and cell junction formation regulate Hippo signaling in NPHP protein-dependent and -independent manners, to maintain the downstream target TAZ in the cytoplasm, preventing the transcriptional activation of TGF-b and Wnt/b-catenin target genes. Conversely, NPHP4 can also associate with the Hippo signaling-related kinase Lats1 to inhibit its association with TAZ and allow the nuclear accumulation of TAZ to promote cell proliferation. Finally, some NPHP proteins were recently shown to play a role in DNA damage response. Collectively, the signaling pathways involving NPHP proteins lead to the maintenance of the renal epithelium by regulating differentiation, cell proliferation and apoptosis. AJs, adherens junctions; Cas, p130Cas; Crb, Crumbs complex; Dvl, dishevelled; DDR; DNA damage response; ECM, extracellular matrix; Fz, frizzled; IFT, intraflagellar transport; PCP, planar cell polarity; TJs, tight junctions.
counteracts the inversin-NPHP4-dependant proteasomal degradation of Dvl and promotes Dvl stabilization at the cilium base to favor PCP, in mice and zebrafish [42].
Role of NPHP proteins at the cell junctions Besides its localization at the TZ, the NPHP1-NPHP4 complex is present at cell–cell junctions where it associates with the polarity-related proteins, PALS1/PATJ and Par6 [20,43]. Depletion of these proteins leads to an alteration in cell junction formation and epithelial morphogenesis. These defects are illustrated in vivo by cloaca malformation in nphp4-depleted zebrafish embryos, that worsen in the nphp4;
par6 double morphants [39,44]. In addition, NPHP1-NPHP4 complex interacts with and regulates focal adhesion regulators, including p130Cas and Pyk2 [45,46]. Indeed, loss of NPHP4 or overexpression of NPHP1 in renal epithelial cells triggers overactivation of Pyk2 and p130Cas, and actin cytoskeleton reorganization [45,47] (our unpublished data). Interestingly, renal fibrosis induced by mechanical stretch in renal tubular cells is significantly reduced in pyk2 / mice [48]. Hence, development of cyst and interstitial fibrosis in NPH patients could occur, in part, through the dysregulation of cell polarity and focal complexes due to the absence of NPHP proteins. www.drugdiscoverytoday.com
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Hippo signaling
Conclusion
Recently, attention has become focused on the Hippo pathway, an integrative pathway modulated by cell junction formation and polarity, and whose transcriptional output governs organ size [49]. In particular, the Hippo transcription coactivators TAZ/YAP promote cell proliferation and survival as well as cell migration and differentiation, through interplays with other pathways such as TGF-b and Wnt/b-catenin. Overexpression of NPHP4 and NPHP9/ NEK8 in mammalian cells has been shown to negatively regulate the Hippo pathway by preventing Lats1 kinase from phosphorylating YAP/TAZ, leading to the nuclear accumulation of the latter and activation of cell proliferation [50,51]. Hence, loss of NPHP4 or NPHP9 could decrease cell proliferation during kidney development, explaining the small size of kidneys observed in most of the NPH patients. However, defects in cell–cell junctions observed in NPHP1and NPHP4-depleted tubular renal cells, along with the association of these NPHP proteins with the polarity Crumbs complex components PALS1 and PATJ, suggest that NPHP1 and NPHP4 may also act as positive modulators of Hippo signaling [20]. Therefore, in NPH patients, NPHPdependant loss of cell junction integrity could lead to a downregulation of the Hippo signaling and subsequent dysregulation of the Wnt and profibrotic TGF-b pathways during the course of the disease, contributing to the appearance of renal fibrosis. Further studies in animal models or with NPH patient tissues are necessary to confirm these hypotheses.
In conclusion, high-throughput exome sequencing in parallel with functional analyses in cellular, animal models and patient tissues have been used to gain deeper insight into the pathogenesis of NPH-RC. The role of NPHP protein complexes as gatekeepers at the cilium base, the involvement of another class of ciliary actors, the intraflagellar transport IFT-A components, and the cooperative role of these two groups of proteins for regulation of ciliary protein trafficking and signaling in kidney are key concepts that have emerged in the field over the last two years. It is now recognized that the NPH-related proteins represent the major intermediates that integrate extracellular cues (cell stretching or fluid flow) and modulate cell signaling pathways (Wnt, Shh, and Hippo) at the cilium and/or at cell contacts to control essential processes within renal epithelia required for proper tissue organization and maintenance. These findings have provided new avenues for the discovery of therapeutic targets that may regulate ciliary or membrane protein composition. Finally, as the genetic causes have not been elucidated in half of the cases, it is expected that identification of the remaining genes will complete the picture for the pathways already implicated, but will also likely unveil other disease mechanisms not yet suspected, as it happened recently with the involvement of DDR in the pathophysiology of NPH.
DNA damage response (DDR) Very recently, another signaling pathway involving NPHP proteins, independent of their role at the primary cilium, has been identified: the DDR pathway. Mutations in two genes involved in DDR, namely ZNF423/NPHP14 and CEP164/ NPHP15, cause NPHP-RC (Table 1). CEP164/NPHP15 plays a crucial role in the early steps of ciliogenesis [52] but it is also involved in the maintenance of genomic stability [53]. CEP164/NPHP15 interacts with and is phosphorylated by ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases and, upon DNA damage, translocates to nuclear foci where many DNA repair and checkpoint proteins form complexes at the sites of damage [53]. Interestingly, other NPHP proteins, such as SDCCAG8/NPHP10, are also involved in DDR signaling [10]. SDCCAG8/NPHP10 localizes at the nuclear foci along with CEP164/NPHP15 and IQCB1/NPHP5, the latter interacting with the AAA+ ATPase RuvBl1 whose zebrafish mutants present with a NPH-like phenotype [54]. These findings link NPH-RC disorders to mechanisms of DDR. In this context, the apoptosis observed in several mouse model tissues (e.g. retina of Nphp1 null mice) or in kidneys of patients carrying NPHP1 homozygous deletion could involve DDR [55]. e8
Conflict of interest The author(s) have no conflict of interest to declare.
Acknowledgements Thanks to Ce´cile Jeanpierre and Scott J. Harvey for helpful discussion and critical reading of the manuscript. M.D. thanks Didier Y. Stainier for his support during the writing of this review. The work of the authors is supported by the Inserm, ANR (S.S. grants R09087KS and RPV11012KK), and Socie´te´ de Ne´phrologie (H.M.G.).
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39. Burckle C, et al. Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros. Hum Mol Genet 2011;20:2611–27. 40. Simons M, et al. Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat Genet 2005;37:537–43. 41. Borgal L, et al. The ciliary protein nephrocystin-4 translocates the canonical Wnt-regulator jade-1 to the nucleus to negatively regulate betacatenin signaling. J Biol Chem 2012;287:25370–80. 42. Mahuzier A, et al. Dishevelled stabilization by the ciliopathy Rpgrip1L is essential for planar cell polarity. J Cell Biol 2012;198:927–40. 43. Donaldson JC, et al. Nephrocystin-conserved domains involved in targeting to epithelial cell–cell junctions, interaction with filamins, and establishing cell polarity. J Biol Chem 2002;277:29028–35. 44. Slanchev K, et al. Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish. Hum Mol Genet 2011;20:3119–28. 45. Benzing T, et al. Nephrocystin interacts with Pyk2, p130(Cas), and tensin and triggers phosphorylation of Pyk2. Proc Natl Acad Sci USA 2001;98:9784–9. 46. Mollet G, et al. Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes. Hum Mol Genet 2005;14:645–56. 47. Liebau MC, et al. Nephrocystin-4 regulates Pyk2-induced tyrosine phosphorylation of nephrocystin-1 to control targeting to monocilia. J Biol Chem 2011;286:14237–45. 48. Sonomura K, et al. The kinase Pyk2 is involved in renal fibrosis by means of mechanical stretch-induced growth factor expression in renal tubules. Kidney Int 2011;81:449–57. 49. Zhao B, et al. Hippo signaling at a glance. J Cell Sci 2010;123(Pt 23):4001–6. 50. Habbig S, et al. NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway. J Cell Biol 2011;193:633–42. 51. Habbig S, et al. The ciliopathy disease protein NPHP9 promotes nuclear delivery and activation of the oncogenic transcriptional regulator TAZ. Hum Mol Genet 2012;21:5528–38. 52. Schmidt KN, et al. Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. J Cell Biol 2012;199:1083–101. 53. Sivasubramaniam S, et al. Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1. Genes Dev 2008;22:587–600. 54. Sun Z, et al. A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development 2004;131:4085–93. 55. Wodarczyk C, et al. Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals. PLoS One 2010;5:e12719. 56. Logan CV, et al. Molecular genetics and pathogenic mechanisms for the severe ciliopathies: insights into neurodevelopment and pathogenesis of neural tube defects. Mol Neurobiol 2011;43:12–26. 57. Czarnecki PG, Shah JV. The ciliary transition zone: from morphology and molecules to medicine. Trends Cell Biol 2012;22:201–10. 58. Reiter JF, et al. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep 2012;13:608–18. 59. Goetz SC, Anderson KV. The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 2010;11:331–44. 60. Norris DP, Grimes DT. Mouse models of ciliopathies: the state of the art. Dis Model Mech 2012;5:299–312. 61. Zhou W, et al. Nephrocystin-3 is required for ciliary function in zebrafish embryos. Am J Physiol Renal Physiol 2010;299:F55–62. 62. Schafer T, et al. Genetic and physical interaction between the NPHP5 and NPHP6 gene products. Hum Mol Genet 2008;17:3655–62. 63. Sayer JA, et al. The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat Genet 2006;38:674–81. 64. Liu S, et al. A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish. Development 2002;129:5839–46. 65. Otto EA, et al. Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat Genet 2010;42:840–50. 66. Leitch CC, et al. Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat Genet 2008;40:443–8.
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Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. xxx, No. xx 2013
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – University of Rochester Medical Center, Rochester, NY.
DISEASE Mechanism underlying inherited renal cystic diseases MECHANISMS
Genetic bases and pathogenic mechanisms of nephronophthisis Marion Delous1,2, Helori M. Gaude´2, Sophie Saunier2,* 1 2
Max-Planck-Institute for Heart and Lung Development, Bad Nauheim, Germany1 INSERM, U983, Paris Descartes, Sorbonne Paris Cite´ University, Imagine Institute, 75015 Paris, France
Nephronophthisis is a recessive cystic kidney disorder that belongs to the group of ciliopathies. Most of the causal gene products localize at the primary cilium, as components of either the transition zone or the retrograde intraflagellar transport IFT-A complex, where they control ciliary protein trafficking and modulate responses to various signaling pathways. In this review, we summarize the current literature on nephronophthisis-related disease genetics and outline the
Section editors Thomas: Benzing and Bernhard Schermer – Kidney Research Center Cologne, Germany. located at the apical surface, which are essential for extracellular chemical and mechanical signal transduction, ensuring tissue morphogenesis and homeostasis [2]. In this review, we cover the latest insights into the molecular and cellular mechanisms underlying NPH.
essential pathophysiological mechanisms underlying Basic genetics of NPH and related ciliopathies (NPH-RC) NPHP genes involved in NPH-RC
these disorders.
Introduction Nephronophthisis (NPH) is the major genetic cause of chronic kidney diseases in children [1], characterized by tubular atrophy with thickened tubular basement membrane, severe interstitial fibrosis and formation of medullary cysts. The juvenile form leads to end-stage renal disease within the first decades of life. Extra-renal symptoms, such as retinal dystrophy, cerebellar vermis hypoplasia/aplasia, hepatic fibrosis, skeletal abnormalities and situs inversus are present in a significant proportion of NPH patients and their association with NPH represents distinct clinical entities. More than 15 NPH-related genes encoding NPHP proteins have been identified. NPHP proteins localize at cell junctions and the primary cilium, thus defining NPH and associated disorders as ciliopathies. Primary cilia are cellular organelles *Corresponding author.: S. Saunier ([email protected]) 1 Current address. 1740-6765/$ ß 2013 Elsevier Ltd. All rights reserved.
Nephronophthisis is a clinically and genetically heterogeneous recessive disorder. Genetic studies combining positional cloning and candidate gene approaches allowed the identification of eleven genes mutated in patients with NPHRC, NPHP1-11 [3] (Table 1). Each NPHP protein is localized at the primary cilium where they work in complex with other ciliopathy-related proteins to maintain cilia structure and function [4]. The identification of additional genes involved in NPH-RC has been facilitated by a combination of ciliary interactome and whole/ciliopathy candidate exome sequencing strategies. Notably, mutations in genes encoding components of the retrograde intraflagellar transport complex A (IFT-A) have been identified in patients with skeletal ciliopathies associated with NPH (Table 1). Mutations of IFT140 cause Mainzer–Saldino Syndrome [5], characterized by cone-shaped epiphysis of the phalanges, NPH and retinal dystrophy. Conditional knockout of Ift140 in mouse collecting ducts results
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Protein
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Isolated NPH
Infantile NPH
NPH-related ciliopathies With liver fibrosis
With situs inversus
With cardiac defects
Other ciliopathies without NPH SLS
JBTS-B
U
U
NPHP1
Nephrocystin-1
UU
INVS/NPHP2
Inversin, nephrocystin-2
U
UU
U
UU
U
NPHP3
Nephrocystin-3
U
U(tr)
U(hyp)
U
U
NPHP4
Nephrocystin-4
U
IQCB1/NPHP5
IQ-motif containing protein, nephrocystin-5
UU
CEP290/NPHP6
Centrosomal protein 290
U
GLIS7/NPHP7
GLI similar 2
RPGRIP1L/NPHP8/ MKS5
RPGRIP1-like
NEK8/NPHP9
NIMA-related kinase 8
SDCCAG8/NPHP10/ SLSN7
Seralogically defined colon cancer antigen 8
TMEM67/NPHP11/ MKS3
Transmembrane protein 67
TTC21B/NPHP12 [7]
IFT139
U
WDR19/NPHP13 [8]
IFT144
U
CEP164/NPHP15 [10]
Centrosomal protein 164
TMEM216/MKS2/ JBTS2 [56]
Transmembrane protein 216
MSS
JATD
SS
BBS-like
LCA
JATD
JBTS
COACH
U
U (tr)
U U
MKS
U
UU
U
UU
U
U U (hyp)
U
U (hyp)
U (tr)
U
U
U U
AHI1 [14]
Jouberin
U
CC2D2A/MKS6 [56]
Coiled coil and C2 domain containing 2A
U
ZNF423/NPHP14 [10]
ZNF423
U
U
U
U
U U U
U
U
U
U U U
U
U
U
U
U: Involvement; UU: main involvement; hyp, hypomorphe mutation; tr, truncating mutation; NPH, nephronophthisis; SLS, Senior–Løken Syndrome; JBTS-B, Joubert Syndrome type B; MKS, Meckel–Gruber Syndrome; MSS, Mainzer–Saldino Syndrome; JATD, Jeune Asphyxiating Thoracic Dystrophy; SS, Sensenbrenner Syndrome; BBS, Bardet–Biedl Syndrome; LCA, Leber’s congenital amaurosis; COACH, Cerebellar vermis hypoplasia/aplasia, Oligophrenia, Ataxia, Coloboma and Hepatic fibrosis.
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Ataxin 10
U (tr)
U
U
IFT140
U
U
U
ATXN10 [4]
U (tr)
U U
IFT140 [5]
U
DDMEC-396; No of Pages 9
GENE [3]
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Table 1. Genes mutated in isolated NPH and NPH-RC
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in cystic kidneys, which in contrast with other models of polycystic kidney disease, are not due to mitotic axis orientation defects, but rather to increased cell proliferation coupled to an alteration of several signaling pathways (canonical Wnt, Sonic Hedgehog (Shh)) [6]. TTC21B/NPHP12, encoding IFT139, is involved in isolated NPH or NPH associated with Jeune Asphyxiating Thoracic Dystrophy (JATD) [7]. Pathogenic mutations of WDR19/NPHP13, encoding IFT144, are associated with a large variety of phenotypes: isolated NPH, JATD and Sensenbrenner Syndrome (SS), characterized by cranioectodermal dysplasia [8]. Mutations in the other IFTA genes IFT122, IFT43 and WDR35 (IFT121 homolog), have also been reported in patients with skeletal ciliopathies associated with chronic renal failure [9] (our unpublished data). Finally, mutations in two additional genes, CEP164/ NPHP15 and ZNF423/NPHP14, have been recently reported in patients with Senior–Løken Syndrome (SLS), Joubert Syndrome (JBTS), Bardet–Biedl Syndrome (BBS)-like and infantile NPH [10] (Table 1). These genes, involved in DNA damage response (DDR) signaling, point to a novel pathophysiological mechanism in these disorders (see below).
Genotype–phenotype correlations Altogether, mutations in the identified causative genes account for approximately 30% of the published NPH-RC patient cohort [11] and 44% of the patients in our cohort at Necker Hospital (860 patients from 792 families). Genotype– phenotype correlation analyses have demonstrated a genetic overlap, and highly variable phenotypes are associated with mutations in these genes (Table 1 and Fig. 1). The common cause of juvenile NPH, which underlies 56% (published cohort) [12] to 62% (Necker Hospital cohort, unpublished data) of patients with identified mutations, is the homozygous deletion of NPHP1, due to unequal recombination between duplicated regions flanking the gene [13]. Mutations in the other NPHP genes account for lower proportions of cases in both cohorts: <10% for NPHP4, IQCB1/NPHP5, CEP290/NPHP6, TMEM67/NPHP11 and TTC21B/NPHP12, <5% for the other genes, with sometimes only one family associated with recessive mutations of a gene (i.e. GLIS2/ NPHP7 and ATXN10) [14] (Table 1). Some NPHP genes are preferentially associated with specific clinical features, thus facilitating diagnosis. For instance, NPHP1 and NPHP4 are
NPH-RC disease spectrum MSS
NPH
JATD
TTC21B
SS
SRP
TTC21B IFT140
Cilia
IFT43 WDR19 IFT122
IFT B
WDR35
DYNC2H1
Inversin compartment
KIF3A/B/C
IFT A
Transition zone
Inversin
RPGRIP1L NPHP4
NPHP1
ATXN10 IQCB1
CEP290 Exocyst
JBTS
MKS
NPHP3
NPHP3
NEK8 SDCCAG8 NPHP1 NPHP4 RPGRIP1L TMEM67
MKS complex
Transition Fibers
Basal Body
SLS
INVS
Transition zone
NPHP3 NEK8
Basal Body
Inversin compartment
NPH
CEP290 ATXN10 IQCB1 CEP164 GLIS2
Degenerative Disease
Dysplastic Disease Drug Discovery Today: Mechanisms
Figure 1. NPHP proteins at the primary cilium and nephronophthisis-related ciliopathy (NPH-RC) disease spectrum. Proteins encoded by genes involved in NPH-RC share a common localization at the base of the cilium (left panel) where they assemble within different subdomains: ATXN10/CEP290/IQCB1 at the basal body (light purple), NPHP1/NPHP4/RPGRIP1L at the transition zone (light brown) and inversin/NPHP3/NEK8 at the inversin compartment (light orange). Together with the MKS complex (grey), these four complexes establish a gate that selects ciliary cargo and helps them dock on IFTs to ensure cilia function and maintenance. A second class of proteins associated with NPH-RC belongs to the retrograde IFT-A complex (light pink). The ciliary membrane composition controlled in part by the NPHP and MKS proteins is tissue-dependent, thus explaining the variability of the organ involvement in NPH-RC. NPH-associated disorders represent a spectrum (right panel) ranging from mild degenerative diseases (isolated NPH) to severe dysplastic diseases with fetal presentation. NPH: Nephronophthisis, MSS: Mainzer–Saldino Syndrome, JATD: Jeune Asphyxiating Thoracic Dystrophy, SS: Sensenbrenner Syndrome, SRP: Short-Rib-Polydactyly Syndrome, SLS: Senior–Løken Syndrome, JBTS: Joubert Syndrome, MKS: Meckel–Gruber Syndrome.
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generally linked to isolated NPH, whereas INVS/NPHP2 (encoding inversin) is associated with infantile NPH (endstage renal disease before 3-years-old). Mutations of IQCB1/ NPHP5 are tightly linked to NPH patients with severe ocular alteration (SLS) and the age at which renal symptoms present is highly variable [15]; mutations of IFT-A genes are generally observed in NPH patients with skeletal anomalies (MSS, JATD, SS) [16] (Table 1). The broad range of phenotypes associated with mutations of any given NPHP gene can be explained in part by the variability of organ-specific functions of the cilia (i.e. visual function of the photoreceptor cells, solute exchange function of the tubular epithelial cells). However, the nature of the mutations can also influence phenotypes. Hypomorphic TMEM67/NPHP11 or NPHP3 mutations cause juvenile NPH with limited extrarenal involvement, while truncating mutations lead to the severe developmental features of Meckel–Gruber Syndrome (MKS; central nervous system malformations, renal cystic dysplasia, polydactyly) (Table 1 and Fig. 1). Similarly, expressivity of bone and retinal phenotypes seems to depend on the nature of the IFT-A mutations: a mild phenotype such as isolated NPH is associated with hypomorphic mutations in TTC21B/ NPHP12 (IFT139) or WDR19/NPHP13 (IFT144), while truncating mutations of WDR19/NPHP13 result in SS. In agreement with this correlation, functional analyses of cilia in patient fibroblasts and mutant mouse tissues revealed subtle defects associated with hypomorphic IFT-A alleles, whereas total allele loss dramatically affected cilia structure and disrupted downstream signaling pathways [5,6,8,17]. Finally, for several NPHP genes, a same mutation can lead to a large spectrum of defects. Some CEP290/NPHP6 mutations cause either isolated Leber congenital amaurosis (LCA) or MKS [18], suggesting that modifier genes can modulate the phenotypic expressivity of NPH-RC mutations. Indeed, all the mutations or variants of ciliopathyrelated genes carried by one patient are expected to contribute to the manifestation of the phenotype. This concept is referred as mutational load [19]. Hence, heterozygous pathogenic mutations of TTC21B/NPHP12 (IFT139) are enriched in ciliopathy cases (5%) [7], suggesting that they have a modifier effect across the ciliopathy spectrum in addition to their causal role in NPH/JATD patients. Extending next generation exome/genome sequencing to large cohorts of ciliopathy patients will allow identification of further genes involved and decipher the role of modifiers in NPHP-RC.
Functions of NPHP proteins Several recent studies have significantly advanced our understanding of the signaling pathways and pathophysiological mechanisms involving NPHP proteins. e4
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NPHP proteins as cilia gatekeepers Despite their localization at the primary cilia/centrosome, most nephrocystins are not essential for cilium formation per se. Indeed, loss of function of the majority of the NPHP proteins in mammalian cells, animal models or patient tissues, does not affect ciliogenesis [4,20,21] (our unpublished data). However, NPHP proteins play a crucial role, together with the MKS-related proteins, in establishing a ciliary gate that is essential for cilium function and maintenance (Table 2). The combination of proteomic analyses and in vivo studies in Caenorhabditis elegans showed that NPHP proteins are organized in complexes at different subdomains of the cilium base [4,22] (Fig. 1). The first module, consisting of NPHP1, NPHP4 and RPGRIP1L/NPHP8, is located at the transition zone (TZ), a conserved structure characterized by Y-shaped linkers that link the microtubule doublets of the axoneme to the membrane (ciliary necklace). These proteins participate in both the integrity and stability of the TZ-protein complexes. Adjacent to the TZ is the ‘Inversin compartment’ that consists of the inversinNPHP3-NEK8/NPHP9 complex [21] and the centrosome, where IQCB1/NPHP5 and CEP290/NPHP6 form another module with ATXN10 [4]. In Chlamydomonas reinhardtii, Cep290 links the flagellar membrane to the TZ [22], a function also shared by RPGRIP1L/NPHP8 and inversin, which connect NPHP modules to the MKS-related proteins at the ciliary membrane [4,23–25]. Altogether, NPHP proteins participate in the organization of the TZ, whereas IQCB1/ NPHP5, by interacting with the exocyst, may help to its recruitment at the cilia base, thereby promoting membrane trafficking towards the cilium [4]. The correct targeting of TZrelated proteins is thus essential for cilia function and regulators of this process have been recently identified, such as UNC119 that binds the myristoylated NPHP3 protein and allows its ciliary targeting [26]. The second class of NPH-RC associated proteins belongs to the retrograde IFT-A complex that transports proteins from the tip to the base of the cilia [27]. IFT proteins, present at the transition fibers, functionally interact with TZ components. Indeed, the MKS complex along with inversin interacts with IFT particles in zebrafish [28] and loss of Nphp1 in mouse photoreceptor cells leads to a mislocalization of IFTs [29], suggesting that the TZ complex acts as a molecular filter that selects and retains cilia components, including IFT proteins and their cargos (Fig. 2). The variability of the organs involved in the ciliopathy spectrum suggests that alterations of some of the functions of NPHP-MKS and IFT-A complexes are tissue specific [24]. Thus, the complexity of phenotypes of the patients with NPHP and IFT-A mutations is likely due to the loss of specific membrane proteins in the mutant cilia. By modifying the ciliary protein composition, mutations of NPHP/IFTA components may alter the pathways that are controlled by ciliary receptors,
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Subcellular localization [3,27,57]
Functions [3,27,38,57–59]
Models Zebrafish
Mouse [60]
Nephrocystin-1
BB, TZ, cilia, cell junctions
Formation and function of TZ, cell junction regulator
Pronephric cysts, cloaca obstruction [44]
Retinal degeneration, male sterility (Nphp1tm1.1hung); retinal degeneration (Nphp1tm1jgg)
INVS/NPHP2
Inversin, nephrocystin-2
BB, Inv compartment, apical membrane, nucleus
Anchoring of NPHP3 and NEK8 at TZ, canonical Wnt inhibitor and PCP activator, cell cycle regulator
Pronephric cysts, laterality and CE defects [40]
Situs-inversus, jaundice, enlarged cystic kidneys (invsInv)
NPHP3
Nephrocystin-3
Inv compartment, nucleus, cell junctions
Modulator of canonical Wnt/ PCP, cilia function
Hydrocephaly, pronephric cysts, laterality and CE defects [61]
Cystic kidneys (Nphp3pcy); laterality defects, congenital heart defects, embryonic lethality (Nphp3tm1Cbe)
NPHP4
Nephrocystin-4
BB, TZ, cilia, cell junctions
Formation and function of TZ, canonical Wnt inhibitor and PCP activator, cell junction regulator
Pronephric cysts, cloaca obstruction, laterality and CE defects, retinal anomalies [39,44]
Male sterility, retinal degeneration (Nphp4nmf192)
IQCB1/NPHP5
Nephrocystin-5
BB, cilia, cell junctions
RPGR/calmodulin complex, ciliogenesis
Hydrocephaly, pronephric cysts, laterality and CE defects [62]
None
CEP290/NPHP6
Centrosomal protein 290
TZ, BB
Formation and function of TZ, RPGR complex trafficking, cilia function, positive modulator of Hh
CE defects, pronephric cysts, retinal and cerebellar defects [63]
Anosmia, retinal degeneration (Cep290rd16); cerebellar hypoplasia, retinal degeneration (Cep290tm1jgg)
GLIS2/NPHP7
GLI similar 2
Cilia, nucleus
Transcription factor, negative modulator of Hh and canonical Wnt
None
Renal atrophy and fibrosis (Glis2tm1Amj); (Glis2tm1Tre)
RPGRIP1L/NPHP8/MKS5
RPGRIP1-like
TZ, cilia, cell junctions
Formation and function of TZ, modulator of Hh and canonical Wnt/PCP
Hydrocephaly, laterality and CE defects [42]
Exencephaly, polydactyly, laterality defects, cystic kidneys, birth lethality (Rpgrip1LtmUrt)
NEK8/NPHP9
NIMA-related kinase 8
Inv compartment, BB
Modulator of PC1 and PC2, cell cycle regulator
Pronephric cysts, laterality and CE defects [64]
Cystic kidneys (Nek8jck)
SDCCAG8/NPHP10/SLSN7
SDCCAG8
BB, TZ, cell junctions, nuclear foci
Cilia function
Pronephric cysts, hydrocephaly, CE defects [65]
None
TMEM67/NPHP11/MKS3
Transmembrane protein 67
ciliary membrane, TZ, cell junctions
Formation and function of TZ, intraciliary transport
Pronephric cysts, eye formation and CE defects [66]
Cystic kidneys, postnatal lethality (Tmem67tm1Dgen)
e5
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NPHP1
DDMEC-396; No of Pages 9
Protein
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Gene
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Table 2. NPH-RC-related genes and their protein product functions and animal model-associated phenotypes
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Exencephaly, skeletal dysplasia, anopthalmia, hearing defects (Ift144twt)
Cystic kidneys (Ift140 HoxB7-Cre) [6]
None
None
Pronephric cysts, hydrocephaly, retinal dyslasia, laterality and CE defects, cell death [10]
Retrograde transport and cilia function, negative modulator of Hh
Retrograde transport and cilia function, modulator of Hh/ Wnt/Hippo [6]
DNA damage response [10]
BB, cilia
BB, cilia
BB, nuclear foci [10]
IFT144
IFT140
Centrosomal protein 164
WDR19/NPHP13
IFT140
CEP164/NPHP15
CE: convergence-extension defects (curved and shortened body axis, broad and kinked notochords, misshapen somites); BB: basal body; TZ: transition zone; Hh: Hedgehog signaling pathway; PCP: planar cell polarity; PC1: polycystin-1; PC2: polycystin-2, RPGR: retinitis pigmentosa GTPase regulator.
Polydactyly, split and fused ribs, neural tube, eye and forebrain defects, embryonic lethality (Ttc21baln)
None
Mouse [60] Zebrafish
CE defects [7] Retrograde transport and cilia function, negative modulator of Hh BB, cilia IFT139 TTC21B/NPHP12
Gene
Table 2 (Continued )
Protein
Models Functions [3,27,38,57–59] Subcellular localization [3,27,57]
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such as G coupled receptors [Smoothened receptor (Hh pathway), vasopressin type 2 receptor (V2R) or somatostatin receptor 3 (SSTR3)], Arl13B, type III adenylyl cyclase (ACIII) or ADPKD-related protein polycystin-2 (TRPP2) [30–32]. For instance, hypomorphic mutations of Ift144 in mice leads to defects in Sonic Hedgehog (Shh)-dependent patterning of the neural tube through a reduction of ciliary ACIII, a negative regulator of the pathway [17]. Alterations of the Hh pathway are also associated with mutations in genes involved in Meckel and Joubert syndromes characterized by limb (polydactyly) and neural tube defects [33,34]. In renal cells depleted for basal body-related proteins involved in Bardet–Biedl Syndrome, the absence of V2R at the primary cilium impairs the cellular responses to luminal vasopressin and leads to water reabsorption defects [35]. Then, defects in ciliary protein composition represent a major pathogenic mechanism in ciliopathies that affects ciliary signaling pathways essential for tissue homeostasis and development.
NPHP1 and cilia disassembly Polo-like kinase 1 (Plk1), a regulator of cell cycle progression, represents a key link between the non-canonical Wnt5a-Dvl2 signaling pathway and the HEF1-dependent Aurora A activation that controls cilia disassembly [36]. Interestingly, Plk1 localizes at the TZ where it interacts with and phosphorylates NPHP1, suggesting that NPHP1 also plays a role in cilia disassembly [37]. However, the exact function of phosphorylated NPHP1 in the HEF1-Aurora A pathway remains to be investigated. It is tempting to speculate that, as components of the cilia gate, NPHP proteins may act as sensors of cilia disassembly signals and their tight regulation by Plk1 would thus trigger and facilitate the reentry in the cell cycle necessary for tissue maintenance and homeostasis.
Canonical Wnt/b-catenin and non-canonical Wnt/PCP signaling pathways Along with other ciliary proteins, several NPHP proteins, notably inversin, NPHP3, NPHP4, CEP290/NPHP6 and RPGRIP1L/NPHP8, are involved in both canonical and non-canonical Wnt signaling pathways implicated in development, epithelial cell renewal and planar cell polarity (PCP) (Table 2). Indeed, both overactivation and downregulation of the canonical Wnt/b-catenin signaling in animal models, as well as loss of the Wnt/PCP signaling, lead to cyst formation [38]. A key regulator of both Wnt pathways is Dishevelled (Dvl). Interestingly, we and others have shown that the inversin-NPHP4-RPGRIP1L protein complex, present at both the cilium base and cell junctions, finely tunes Dvl protein level and cellular localization. Inversin and NPHP4 act as negative regulators of the canonical Wnt pathway by addressing cytosolic Dvl to the proteasome [39,40] and by stabilizing the ubiquitin ligase Jade-1, a negative regulator of b-catenin [41]. In contrast, we have shown that RPGRIP1L
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Cargo
Retrograde IFT
IFT-A
Cilia-dependent pathways
Fz Dvl NPHP
Y YY
YY Y
IFT-A
Wnt
Plk1
Dvl
Dvl stability Regulation
Crb
Cilia Gatekeepers
NPHP MKS
Cilia disassembly Regulation
Tight Junctions
NPHP Cell-cell junction Maintenance
Wnt/PCP pathway
Lats1
Junction Integrity
Hippo signaling
TGF-β Wnt/βcatenin Cell-matrix signal Transduction Pyk2 NPHP Cas
Adherent Junctions
NPHP4
DDR
DEDIFFERENTIATION PROLIFERATION CEP164 APOPTOSIS ECM
Focal Adhesions Drug Discovery Today: Mechanisms
Figure 2. Signaling pathways and physiopathological mechanisms involving NPHP proteins. At the primary cilia, NPHP proteins are present at the transition zone where they associate with MKS complex and IFT-A components to form the ciliary gate. Along the axoneme, IFT-A components are involved in the retrograde transport of ciliary cargos. By determining membrane composition and governing retrograde transport, the cilia gatekeeper ensures correct transduction of signals sensed by the primary cilium. At the transition zone (TZ), NPHP proteins interact with Plk1 suggesting a role in regulation of cilia disassembly. By interacting with the Wnt signaling downstream protein Dvl at the membrane and TZ, NPHP proteins regulate its stability and protein level to promote Wnt/PCP signaling. At cell–cell junctions, NPHP proteins interact with components of tight and adherens junctions participating in their stability and integrity. A similar role at focal adhesions is suggested by the association of NPHP proteins with signal transduction molecules involved in signal transduction (i.e. p130Cas, Pyk2). Both PCP and cell junction formation regulate Hippo signaling in NPHP protein-dependent and -independent manners, to maintain the downstream target TAZ in the cytoplasm, preventing the transcriptional activation of TGF-b and Wnt/b-catenin target genes. Conversely, NPHP4 can also associate with the Hippo signaling-related kinase Lats1 to inhibit its association with TAZ and allow the nuclear accumulation of TAZ to promote cell proliferation. Finally, some NPHP proteins were recently shown to play a role in DNA damage response. Collectively, the signaling pathways involving NPHP proteins lead to the maintenance of the renal epithelium by regulating differentiation, cell proliferation and apoptosis. AJs, adherens junctions; Cas, p130Cas; Crb, Crumbs complex; Dvl, dishevelled; DDR; DNA damage response; ECM, extracellular matrix; Fz, frizzled; IFT, intraflagellar transport; PCP, planar cell polarity; TJs, tight junctions.
counteracts the inversin-NPHP4-dependant proteasomal degradation of Dvl and promotes Dvl stabilization at the cilium base to favor PCP, in mice and zebrafish [42].
Role of NPHP proteins at the cell junctions Besides its localization at the TZ, the NPHP1-NPHP4 complex is present at cell–cell junctions where it associates with the polarity-related proteins, PALS1/PATJ and Par6 [20,43]. Depletion of these proteins leads to an alteration in cell junction formation and epithelial morphogenesis. These defects are illustrated in vivo by cloaca malformation in nphp4-depleted zebrafish embryos, that worsen in the nphp4;
par6 double morphants [39,44]. In addition, NPHP1-NPHP4 complex interacts with and regulates focal adhesion regulators, including p130Cas and Pyk2 [45,46]. Indeed, loss of NPHP4 or overexpression of NPHP1 in renal epithelial cells triggers overactivation of Pyk2 and p130Cas, and actin cytoskeleton reorganization [45,47] (our unpublished data). Interestingly, renal fibrosis induced by mechanical stretch in renal tubular cells is significantly reduced in pyk2 / mice [48]. Hence, development of cyst and interstitial fibrosis in NPH patients could occur, in part, through the dysregulation of cell polarity and focal complexes due to the absence of NPHP proteins. www.drugdiscoverytoday.com
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Hippo signaling
Conclusion
Recently, attention has become focused on the Hippo pathway, an integrative pathway modulated by cell junction formation and polarity, and whose transcriptional output governs organ size [49]. In particular, the Hippo transcription coactivators TAZ/YAP promote cell proliferation and survival as well as cell migration and differentiation, through interplays with other pathways such as TGF-b and Wnt/b-catenin. Overexpression of NPHP4 and NPHP9/ NEK8 in mammalian cells has been shown to negatively regulate the Hippo pathway by preventing Lats1 kinase from phosphorylating YAP/TAZ, leading to the nuclear accumulation of the latter and activation of cell proliferation [50,51]. Hence, loss of NPHP4 or NPHP9 could decrease cell proliferation during kidney development, explaining the small size of kidneys observed in most of the NPH patients. However, defects in cell–cell junctions observed in NPHP1and NPHP4-depleted tubular renal cells, along with the association of these NPHP proteins with the polarity Crumbs complex components PALS1 and PATJ, suggest that NPHP1 and NPHP4 may also act as positive modulators of Hippo signaling [20]. Therefore, in NPH patients, NPHPdependant loss of cell junction integrity could lead to a downregulation of the Hippo signaling and subsequent dysregulation of the Wnt and profibrotic TGF-b pathways during the course of the disease, contributing to the appearance of renal fibrosis. Further studies in animal models or with NPH patient tissues are necessary to confirm these hypotheses.
In conclusion, high-throughput exome sequencing in parallel with functional analyses in cellular, animal models and patient tissues have been used to gain deeper insight into the pathogenesis of NPH-RC. The role of NPHP protein complexes as gatekeepers at the cilium base, the involvement of another class of ciliary actors, the intraflagellar transport IFT-A components, and the cooperative role of these two groups of proteins for regulation of ciliary protein trafficking and signaling in kidney are key concepts that have emerged in the field over the last two years. It is now recognized that the NPH-related proteins represent the major intermediates that integrate extracellular cues (cell stretching or fluid flow) and modulate cell signaling pathways (Wnt, Shh, and Hippo) at the cilium and/or at cell contacts to control essential processes within renal epithelia required for proper tissue organization and maintenance. These findings have provided new avenues for the discovery of therapeutic targets that may regulate ciliary or membrane protein composition. Finally, as the genetic causes have not been elucidated in half of the cases, it is expected that identification of the remaining genes will complete the picture for the pathways already implicated, but will also likely unveil other disease mechanisms not yet suspected, as it happened recently with the involvement of DDR in the pathophysiology of NPH.
DNA damage response (DDR) Very recently, another signaling pathway involving NPHP proteins, independent of their role at the primary cilium, has been identified: the DDR pathway. Mutations in two genes involved in DDR, namely ZNF423/NPHP14 and CEP164/ NPHP15, cause NPHP-RC (Table 1). CEP164/NPHP15 plays a crucial role in the early steps of ciliogenesis [52] but it is also involved in the maintenance of genomic stability [53]. CEP164/NPHP15 interacts with and is phosphorylated by ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases and, upon DNA damage, translocates to nuclear foci where many DNA repair and checkpoint proteins form complexes at the sites of damage [53]. Interestingly, other NPHP proteins, such as SDCCAG8/NPHP10, are also involved in DDR signaling [10]. SDCCAG8/NPHP10 localizes at the nuclear foci along with CEP164/NPHP15 and IQCB1/NPHP5, the latter interacting with the AAA+ ATPase RuvBl1 whose zebrafish mutants present with a NPH-like phenotype [54]. These findings link NPH-RC disorders to mechanisms of DDR. In this context, the apoptosis observed in several mouse model tissues (e.g. retina of Nphp1 null mice) or in kidneys of patients carrying NPHP1 homozygous deletion could involve DDR [55]. e8
Conflict of interest The author(s) have no conflict of interest to declare.
Acknowledgements Thanks to Ce´cile Jeanpierre and Scott J. Harvey for helpful discussion and critical reading of the manuscript. M.D. thanks Didier Y. Stainier for his support during the writing of this review. The work of the authors is supported by the Inserm, ANR (S.S. grants R09087KS and RPV11012KK), and Socie´te´ de Ne´phrologie (H.M.G.).
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Drug Discovery Today: Disease Mechanisms | Mechanism underlying inherited renal cystic diseases
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Please cite this article in press as: Delous M, et al. Genetic bases and pathogenic mechanisms of nephronophthisis, Drug Discov Today: Dis Mech (2013), http://dx.doi.org/ 10.1016/j.ddmec.2013.10.001