MKS1 regulates ciliary INPP5E levels in Joubert syndrome

Background Joubert syndrome (JS) is a recessive ciliopathy characterised by a distinctive brain malformation \u27the molar tooth sign\u27. Mutations in \u3e 27 genes cause JS, and mutations in 12 of these genes also cause Meckel-Gruber syndrome (MKS). The goals of this work are to describe the clinical features of MKS1- related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS. Methods We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a three-dimensional (3D) spheroid rescue assay to test the effects of diseaserelated MKS1 mutations. Results We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1- related JS carry \u3e1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids. Conclusions MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalisation, is a key mechanism underlying JS, downstream of MKS1 and ARL13B

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis

©2015. To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p. R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function ofKCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel–Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome1 (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral–facial–digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture

Are renal ciliopathies (replication) stressed out?

Juvenile renal failure is commonly caused by the ciliopathy nephronophthisis (NPHP). Since all NPHP genes regulate cilia function, it has been assumed that NPHP onset is due to cilia loss. However, recent data suggest that DNA damage caused by replication stress, possibly concomitant with or upstream of cilia dysfunction, causes NPHP

Are renal ciliopathies (replication) stressed out?

Juvenile renal failure is commonly caused by the ciliopathy nephronophthisis (NPHP). Since all NPHP genes regulate cilia function, it has been assumed that NPHP onset is due to cilia loss. However, recent data suggest that DNA damage caused by replication stress, possibly concomitant with or upstream of cilia dysfunction, causes NPHP. Renal ciliopathies: not just loss of cilia The leading genetic causes of pediatric as well as adult kidney failure can be traced back to an organelle called the cilium. Cilia loss of function is thought to be the cellular defect responsible for two classes of renal ciliopathies: the common autosomal dominant polycystic kidney disease (ADPKD), which affects adults; and the rare, recessive pediatric/juvenile ciliopathies collectively referred to as NPHP-related ciliopathies (NPHP-RCs) (Box 1). For example, all 19 NPHP-associated genes reported to date encode gene products known to localize to primary cilia and regulate ciliary structure or function. In addition, many NPHPassociated proteins possess extraciliary functions that potentially contribute to the development of disease, and these functions have only recently been explored. Recent molecular evidence argues that the nuclear/DNA damage response (DDR) functions of some NPHP proteins may be critical in disease onset or progression. DDR signaling includes mechanisms to detect DNA damage (lesions), signal the presence of damage, arrest the cell cycle, and promote repair. The question is raised of whether nuclear effects of NPHP gene mutations are upstream, downstream, or independent of cilia dysfunction Replication stress at the root of the stalk Recent studies with cells depleted for CEP164, SDCCAG8, and NEK8 are beginning to address the relation between the DDR and NPHP onset What are the molecular mechanisms linking NPHP proteins to DNA damage? Depletion of NEK8 results in DDR signaling through enhanced cyclin A-associated cyclin-dependent kinase (CDK) activity, leading to replication stress, and activated ATR-CHK1 signaling upon replication stress induction in S phase. Similarly, live-cell imaging of CEP164-depleted cells showed delayed S phase progression that could be rescued with wild type but not CEP164 patient allele

Nephronophthisis : should we target cysts or fibrosis?

Ciliopathy nephronophthisis (NPHP), a common cause of end-stage renal disease (ESRD) in children and young adults, is characterized by disintegration of the tubular basement membrane accompanied by irregular thickening and attenuation, interstitial fibrosis and tubular atrophy, and occasionally cortico-medullary cyst formation. Pharmacological approaches that delay the development of ESRD could potentially extend the window of therapeutic opportunity for this group of patients, generating time to find an appropriate donor or even for new treatments to mature. In this review we provide an overview of compounds that have been tested to ameliorate kidney cysts and/or fibrosis. We also revisit paclitaxel as a potential strategy to target fibrosis in NPHP. At low dosage this chemotherapy drug shows promising results in rodent models of renal fibrosis. Possible adverse events and safety of paclitaxel treatment in pediatric patients would need to be investigated, as would the efficacy, optimum dose, and administration schedule for the treatment of renal fibrosis in NPHP patients. Paclitaxel is an approved drug for human use with known pharmacokinetics, which could potentially be used in other ciliopathies through targeting the microtubule skeleton

Nephronophthisis : should we target cysts or fibrosis?

Ciliopathy nephronophthisis (NPHP), a common cause of end-stage renal disease (ESRD) in children and young adults, is characterized by disintegration of the tubular basement membrane accompanied by irregular thickening and attenuation, interstitial fibrosis and tubular atrophy, and occasionally cortico-medullary cyst formation. Pharmacological approaches that delay the development of ESRD could potentially extend the window of therapeutic opportunity for this group of patients, generating time to find an appropriate donor or even for new treatments to mature. In this review we provide an overview of compounds that have been tested to ameliorate kidney cysts and/or fibrosis. We also revisit paclitaxel as a potential strategy to target fibrosis in NPHP. At low dosage this chemotherapy drug shows promising results in rodent models of renal fibrosis. Possible adverse events and safety of paclitaxel treatment in pediatric patients would need to be investigated, as would the efficacy, optimum dose, and administration schedule for the treatment of renal fibrosis in NPHP patients. Paclitaxel is an approved drug for human use with known pharmacokinetics, which could potentially be used in other ciliopathies through targeting the microtubule skeleton

Primary cilia suppress Ripk3-mediated necroptosis

Abstract Cilia are sensory organelles that project from the surface of almost all cells. Nephronophthisis (NPH) and NPH-related ciliopathies are degenerative genetic diseases caused by mutation of cilia-associated genes. These kidney disorders are characterized by progressive loss of functional tubular epithelial cells which is associated with inflammation, progressive fibrosis, and cyst formation, ultimately leading to end-stage renal disease. However, disease mechanisms remain poorly understood. Here, we show that targeted deletion of cilia in renal epithelial cells enhanced susceptibility to necroptotic cell death under inflammatory conditions. Treatment of non-ciliated cells with tumor necrosis factor (TNF) α and the SMAC mimetic birinapant resulted in Ripk1-dependent cell death, while viability of ciliated cells was almost not affected. Cell death could be enhanced and shifted toward necroptosis by the caspase inhibitor emricasan, which could be blocked by inhibitors of Ripk1 and Ripk3. Moreover, combined treatment of ciliated and non-ciliated cells with TNFα and cycloheximide induced a cell death response that could be partially rescued with emricasan in ciliated cells. In contrast, non-ciliated cells responded with pronounced cell death that was blocked by necroptosis inhibitors. Consistently, combined treatment with interferon-γ and emricasan induced cell death only in non-ciliated cells. Mechanistically, enhanced necroptosis induced by loss of cilia could be explained by induction of Ripk3 and increased abundance of autophagy components, including p62 and LC3 associated with the Ripk1/Ripk3 necrosome. Genetic ablation of cilia in renal tubular epithelial cells in mice resulted in TUNEL positivity and increased expression of Ripk3 in kidney tissue. Moreover, loss of Nphp1, the most frequent cause of NPH, further increased susceptibility to necroptosis in non-ciliated epithelial cells, suggesting that necroptosis might contribute to the pathogenesis of the disease. Together, these data provide a link between cilia-related signaling and cell death responses and shed new light on the disease pathogenesis of NPH-related ciliopathies

Reduced cilia frequencies in human renal cell carcinomas versus neighboring parenchymal tissue

<p>Abstract</p> <p>Background</p> <p>Cilia are essential organelles in multiple organ systems, including the kidney where they serve as important regulators of renal homeostasis. Renal nephron cilia emanate from the apical membrane of epithelia, extending into the lumen where they function in flow-sensing and ligand-dependent signaling cascades. Ciliary dysfunction underlies renal cyst formation that is in part caused by deregulation of planar cell polarity and canonical Wnt signaling. Renal cancer pathologies occur sporadically or in heritable syndromes caused by germline mutations in tumor suppressor genes including <it>VHL</it>. Importantly, Von Hippel-Lindau (VHL) patients frequently develop complex renal cysts that can be considered a premalignant stage. One of the well-characterized molecular functions of VHL is its requirement for the maintenance of cilia. In this study, tissue from 110 renal cancer patients who underwent nephrectomy was analyzed to determine if lower ciliary frequency is a common hallmark of renal tumorigenesis by comparing cilia frequencies in both tumor and adjacent parenchymal tissue biopsies from the same kidney.</p> <p>Methods</p> <p>We stained sections of human renal material using markers for cilia. Preliminary staining was performed using an immunofluorescent approach and a combination of acetylated-α-tubulin and pericentrin antibodies and DAPI. After validation of an alternative, higher throughput approach using acetylated-α-tubulin immunohistochemistry, we continued to manually quantify cilia in all tissues. Nuclei were separately counted in an automated fashion in order to determine ciliary frequencies. Similar staining and scoring for Ki67 positive cells was performed to exclude that proliferation obscures cilia formation potential.</p> <p>Results</p> <p>Samples from renal cell carcinoma patients deposited in our hospital tissue bank were previously used to compose a tissue microarray containing three cores of both tumor and parenchymal tissue per patient. Cilia frequencies in a total of eighty-nine clear cell, eight papillary, five chromophobe renal cell carcinomas, two sarcomatoid renal tumors and six oncocytomas were determined. A marked decrease of primary cilia across renal cell carcinoma subtypes was observed compared to adjacent nontumorigenic tissue.</p> <p>Conclusions</p> <p>Our study shows that cilia are predominantly lost in renal cell carcinomas compared to tissue of the tumor parenchyma. These results suggest that ciliary loss is common in renal tumorigenesis, possibly participating in the sequence of cellular events leading to malignant tumor development. Future therapies aimed at restoring or circumventing cilia signaling might therefore aid in current treatment efficacy.</p