CRC 1453 Seminar Series - Distinguished Speaker Professor Katalin Susztak – Perelman School of Medicine, University of Pennsylvania, USA
Topic: Going from kidney function GWAS to kidney disease mechanism
CRC 1453 Seminar Series - Distinguished Speaker Professor Friedhelm Hildebrandt – Harvard Medical School; Boston Children’s Hospital
Topic: Precision Medicine in Early-Onset Chronic Kidney Disease
- P1: Polycystin-2 signalling in Drosophila melanogaster (M. Köttgen)
- P2: Identification and functional characterization of new components of TOR signalling in C. elegans (E. Neumann-Haefelin)
- P3: Cyst formation in zebrafish (A. Kramer-Zucker)
- P4: The role of nephronophthisis proteins in motile cilia and renal tubules of Xenopus laevis. (S. Lienkamp)
- P6: Characterization of the ciliary flow sensor and its role in epithelial cell polarity (W. Kühn)
- P7: The role of mTOR dependent and independent signalling pathways for renal tubular cystogenesis (F. Grahammer, T. Huber)
- P8: NPHP-related polycystic kidney disease in man and mice (H. Omran)
- P9: Regulation of ciliogenesis and ciliary disassembly by nephrocystins (M. Jung, G. Walz)
P1: Polycystin-2 signalling in Drosophila melanogaster (M. Köttgen)
Autosomal dominant polycystic kidney disease is caused by mutations in PKD1 or PKD2. The respective gene products, polycystin-1 and polycystin-2 (TRPP2), function in a common signalling pathway employing Ca2+ as a second messenger. TRPP2 is a Ca2+ -permeable cation channel that forms a complex with polycystin-1. However, it is poorly understood how TRPP2-mediated Ca2+ signals are translated into cellular functions regulating tubular morphology. Notably, the signalling molecules upstream and downstream of TRPP2 are unknown. Therefore, this proposal focuses on: (1) the identification of upstream and downstream components in the polycystin signalling pathway, and (2) the role of TRPP2 in the spatiotemporal regulation of Ca2+ dynamics. We are using Drosophila as a model to genetically dissect the polycystin signalling pathway. Drosophila TRPP2 is a ciliary protein that is expressed in the sperm tail. We found that TRPP2 is required for directed sperm movement in the female reproductive tract. TRPP2 mutant males are sterile because their sperm fail to reach the female sperm storage organs. Since the fundamental mechanisms of polycystin signalling are likely to be evolutionary conserved from flies to humans, we have performed an unbiased forward genetic screen for mutants that phenocopy the TRPP2 mutant phenotype. This screen has revealed novel candidates in the polycystin signalling network in vivo. We will investigate the function of these proteins using a multidisciplinary approach that combines genetic, biochemical, cell biological and live imaging approaches.
P2: Identification and functional characterization of new components of TOR signalling in C. elegans (E. Neumann-Häfelin)
Upregulation of mammalian target of rapamycin (mTOR) activity has been implicated in polycystic kidney disease and inhibition of the mTOR pathway has been shown to decrease cyst size in animal models and humans. The TOR protein complex 1 (TORC1) has a central role in cystogenesis, but the upstream pathways linking ciliary signalling to mTOR activation are poorly understood. The TORC1 pathway promotes growth and cell proliferation in response to amino acids and hormone-dependent signalling in all higher eukaryotes. Inhibition of TORC1 in C. elegans results in developmental arrest as dauer-like larvae and increases lifespan in the adult worm. We have established proteome-wide screening methods and identified potential new interactors of Rheb. Preliminary results suggest that TSC/Rheb does not only control TORC1 signalling but may also impinge on signalling pathways important for control of cell cycle and proliferation. Here, we propose to employ the strength of C. elegans to perform genetic analyses, and to characterize newly identified components that regulate or effect TORC1 signalling at the genetic level. This proposal will contribute to a better understanding of the complexity and cross-talks of TOR signalling during development and its role in cystogenesis.
P3: Cyst formation in zebrafish (A. Kramer-Zucker)
Polycystic kidney disease is a systemic disease and regarded as ciliopathy. How ciliary dysfunction is causing the different organ manifestations is still not fully understood. Increased cell proliferation and also apoptosis, disturbed cell polarity and tissue repair mechanisms seem to lead to disrupted tubular geometry. We will investigate ELMO1 (Engulfment and cell motility protein 1), a new cyst candidate protein, that was originally discovered in C. elegans in regulating apoptosis and cell migration and that functions together with Dock180 as a Rac-GEF. Our preliminary results show that ELMO1 and Dock180 share localization to the cilium and basal body with other known cyst proteins. Knockdown of ELMO1 by morpholino injection led to cyst formation in zebrafish pronephros. This raises the hypothesis that ELMO1/Dock180 are involved in signalling pathways that are implicated in cyst formation. To test this hypothesis the function of ELMO1 will be studied in vivo by overexpression and morpholino-knockdown experiments. The resulting phenotypes will be evaluated for cilia structure and motility, cell polarity, and fluid flow in the pronephric duct. In addition, transgenic fish lines expressing GFP fusion proteins under the control of pronephros specific promoters will be utilized to try to understand how tubular geometry is disrupted in cystic kidney disease. Studies on kidney regeneration after photoablation of pronephric epithelial tube cells in wild-type and cystic kidney background will be performed to gain further insight into maintenance of tubular geometry.
P4: The role of nephronophthisis proteins in motile cilia and renal tubules of Xenopus laevis (S. Lienkamp)
The renal manifestations of nephronophthisis (NPH) are often part of a complex syndrome, affecting multiple organs and tissues. Most gene products mutated in nephronophthisis (nephrocystins, NPHPs) localize to the cilium, a microtubular organelle attached to most body cells. Hence it has been postulated that a dysfunction of the cilium causes the cystic kidney disease and extrarenal manifestations of NPHP. NPHPs are also required for planar cell polarity signalling (PCP); however the link between cilia and the PCP signalling pathway is still poorly understood. Cilia are positioned in a highly polarized manner in some tissues. This proposal will elucidate the role of NPHPs in ciliary polarization and PCP signalling, using the Xenopus laevis animal model. Focusing on the polarization of multi-ciliated Xenopus epidermal cells, the structural integrity, motility, and anchoring of cilia at the apical membrane as well as the crosstalk with the PCP pathway will be analyzed in detail. Since renal tubules are specified in a proximal-to-distal fashion, the Xenopus pronephros model will be utilized to determine, if single tubular epithelial cells exhibit features of an anterior-to-posterior polarization, and if the planar polarity of tubule cells is mediated by NPHPs. The results will provide further insight in the molecular function of NPHPs, and a framework to understand their role in mammalian organ development and homeostasis.
P6: Characterization of the ciliary flow sensor and its role in epithelial cell polarity (W. Kühn)
Disruption of primary cilia in the kidney leads to cyst formation, involving the loss of planar polarity, increased proliferation and deregulated mTOR signalling. Exactly how this function is orchestrated by the ciliary flow sensor is difficult to examine in vivo and has not been clarified. Published data from our group using an in vitro approach suggests that two independent signalling pathways exist downstream of cilia, calcium and mTOR. Shear stress through fluid flow results in calcium transients involving the Pkd2 gene product TRPP2. Ciliary calcium signalling is required for the flow dependent orientation of centriole movements in the direction of flow. Independently of calcium, cilia under flow down-regulate the mTOR pathway specifically through the basal body. To improve the understanding of ciliary dysfunction in the pathogenesis of PKD, we aim to further dissect the control of these pathways downstream of cilia with the aim of gaining insight into the molecular control of polarity, proliferation and mTOR. Specifically we will study the role of the PKD1 gene product polycystin 1 in flow dependent mTOR regulation and analyze the role of other upstream mTOR regulators that have been associated with cystic disease such as TSC and Wnt. We will investigate the role of candidate proteins in flow induced polarity and analyze cell proliferation under flow with respect to cilia, the polycystins and components of the mTOR pathway.
P7: The role of mTOR dependent and independent signalling pathways for renal tubular cystogenesis (F. Grahammer, T. Huber)
Recent experimental and clinical data suggest the mammalian target of rapamycin (mTOR) kinase as a molecular switch balancing tubular proliferation, tubular maintenance and cystogenesis. However, most data have been derived from pharmacological inhibition of the pathway by rapamycin making it impossible to differentiate the precise and cell specific role of mTOR activation. We have now established a set of complementary transgenic mouse models that identify tubular cell autonomous mTOR signalling events as mediators of cyst initiation and cyst progression. Strikingly, while genetic deletion of mTORC1 delays the onset and progression of cysts, there is an mTORC1 independent cyst formation suggesting a synergistic action of multiple signalling pathways in cyst initiation and progression. Based on these mouse models we hypothesize that an efficient drug therapy of ADPKD will need to target multiple signalling pathways synchronously or sequentially. The overall goal of this study is to understand the precise timing and the molecular interplay of mTORC1 dependent and mTORC1 independent signalling events regulating cystogenesis to identify a novel and innovative multi-target based treatment approach for ADPKD.
P8: NPHP-related polycystic kidney disease in man and mice (H. Omran)
Our recent demonstration, that mutations of orthologous genes result in cystic kidney disease in mice and man, gives now a unique opportunity to study pathogenesis and therapy. We have shown that recessive hypomorphic mutations of NPHP3 cause cystic kidney disease (adolescent nephronophthisis) in children and adults, whereas more severe NPHP3 mutations result in either lethal congenital disease (heterotaxia) and/or renal-hepatic-pancreatic dysplasia syndrome. Likewise we demonstrated that orthologous mutations in mice result in similar renal phenotypes. We recapitulated these distinct renal disorders and generated three distinct mouse models with stable expression of phenotypes: i) hypomorphic Nphp3pcy/pcy mutant mice (adult onset); ii) Nphp3ko/ko deficient mice (congenital disease); iii) compound mutant Nphp3ko/pcy mice (early onset). In addition we have contributed to the understanding that NPHP proteins function as gatekeepers at the ciliary base. Now we will utilize our mouse models as well as patient material to decipher the specific function of NPHP proteins on the cellular level. For that purpose we will perform immuno- EM and high resolution IF to study the NPHP protein network at the ciliary gate and analyze the functional sequelae of NPHP mutations for the ultrastructure of the ciliary necklace as well as the y-connectors using transmission and freeze fracture EM. In addition we will analyze in mutant and control cilia to determine which ciliary proteins are affected by altered NPHP gate function in man and mice.
Nephronophthisis is an autosomal recessive cystic kidney disease caused by mutations of nephrocystins. Preliminary results revealed that three nephrocystins (NPHP1, 2, 4) regulate the activity of Hef1/Aurora A, a complex that triggers ciliary disassembly through activation of histone deacytelases (HDACs). Since HDAC inhibitors rescue the ciliogenesis defect induced by the loss of NPHP2/Inversin, these findings suggest that some nephrocystins control the balance between ciliary assembly and disassembly. This project will therefore focus on the role of nephrocystins in the regulation of ciliogenesis and ciliary disassembly. The specific aims will 1) elucidate the mechanisms through which nephrocystins regulate ciliogenesis and ciliary disassembly, 2) test which ciliary defects are sensitive to HDACi, and 3) establish an in vitro model to screen for compounds that facilitate ciliogenesis, and exploit the potential of HDACi to suppress cyst formation in animal models of polycystic kidney disease.