DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome
Gisela G. Slaats, … , Karlene A. Cimprich, Rachel H. Giles
Gisela G. Slaats, … , Karlene A. Cimprich, Rachel H. Giles
Published August 24, 2015
Citation Information: J Clin Invest. 2015;125(9):3657-3666. https://doi.org/10.1172/JCI80657.
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Research Article Nephrology

DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome

Abstract

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

Authors

Gisela G. Slaats, Joshua C. Saldivar, Julien Bacal, Michelle K. Zeman, Andrew C. Kile, Ann Marie Hynes, Shalabh Srivastava, Jekaterina Nazmutdinova, Krista den Ouden, Miriam S. Zagers, Veronica Foletto, Marianne C. Verhaar, Colin Miles, John A. Sayer, Karlene A. Cimprich, Rachel H. Giles

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Figure 1

DDR signaling is enhanced ex vivo, in vitro, and in vivo.

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DDR signaling is enhanced ex vivo, in vitro, and in vivo. (A) Western bl...
(A) Western blot of UREC lysates of a healthy donor (WT) and a patient with JS with compound heterozygous mutations in CEP290, p.Q950Pfs*6, and p.K939N. CEP290 protein expression loss and increased γH2AX levels are detected in the JS protein lysate. H2AX was used as loading control; samples were run on parallel gels contemporaneously. (B) Increased γH2AX levels were detected in siCEP290-transfected WT URECs, 48 hours after siCEP290 transfection. H2AX was used as loading control. (C) RPE and mIMCD3 cells depleted of CEP290 by siRNA show increased γH2AX levels compared with control siRNA transfection (56 hours). H2AX was used as loading control (n = 3). (D) Immunofluorescent staining of γH2AX in WT and JS URECs and RPE and mIMCD3 cells depleted of CEP290 by siRNA and quantification of staining intensity per nucleus (scale bar: 10 μm; n = 3; 100 cells scored per condition; t test, **P < 0.01, ***P < 0.001). (E) Western blot of zebrafish injected with 3.5 or 4 ng cep290 ATG-targeting mo. Loss of CEP290 protein expression and increased γH2AX levels were detected in cep290 mo-injected zebrafish lysates 48 hours after fertilization. Actin was used as loading control (n = 3); samples were run on parallel gels contemporaneously. (F) More γH2AX (brown) staining in kidneys of homozygote Cep290LacZ/LacZ gene trap mice compared with WT mice, which increased with age (n = 13; between 5,000–7,000 cells scored per animal) (linear model, goodness-of-fit test R2 = 0.86; *P < 0.05). (G) Example of WT mouse kidney and Cep290LacZ/LacZ gene trap mouse kidney at age 3 months stained for γH2AX (brown). Insets show high-magnification images of kidneys. Scale bars: 100 μm; original magnification ×2 (inset). Quantification of Western blots is shown in Supplemental Figure 1.