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 2

DNA damage is enhanced in CEP290-depleted cells.

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DNA damage is enhanced in CEP290-depleted cells. (A) Western blot of Cep...
(A) Western blot of Cep290LacZ/LacZ and WT primary mouse kidney cell lysates. Increased H2AX phosphorylation was detected in the CEP290-depleted cells. H2AX was used as loading control. (B) Immunofluorescent staining of γH2AX in control cells treated with DMSO and in cells treated with 400 nM APH for 18 hours. Scale bar: 10 μm. (C) Quantification of γH2AX staining intensity per nucleus in control cells treated with DMSO and in cells treated with 400 nM APH for 18 hours (n = 3; 100 cells scored per condition; 2-way ANOVA, *P < 0.05). (D) Quantification of comet tail moments of cells treated with 400 nM APH for 18 hours normalized to control DMSO-treated WT cells (n = 3; 50 cells scored per condition; 2-way ANOVA, *P < 0.05). (E) Images of SYBR Gold–stained DNA in comet tail assays. Scale bar: 10 μm. (F) Immunofluorescent staining of WT and Cep290LacZ/LacZ cells for pericentrin (white) reveals supernumerary centrioles (arrows) in Cep290LacZ/LacZ cells. The inset shows multipolar (>2) spindles. Scale bars: 10 μm; original magnification ×2 (inset). (G) Quantification of centriole number in WT and Cep290LacZ/LacZ cells (84 cells scored per cell line; n = 3). (H) BrdU FACS of WT and Cep290LacZ/LacZ cells shows normal DNA content and cell cycle for WT cells; however, Cep290LacZ/LacZ cells have irregular DNA content (10,000 events measured; n = 3).