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.
View: Text | PDF
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

×

Figure 5

CDK1/2i rescues DNA damage and primary cilia.

Options: View larger image (or click on image) Download as PowerPoint
CDK1/2i rescues DNA damage and primary cilia. (A) Western blot of Cep290...
(A) Western blot of Cep290LacZ/LacZ and WT cell lysates showing increased levels of CDK1, CKD2, cyclin A, and cyclin B1 in CEP290-depleted cells. GAPDH was used as loading control; samples were run on parallel gels contemporaneously (B) In vitro CDK kinase assay of Cep290LacZ/LacZ and WT cell lysates after precipitation of cyclin A–CDK and cyclin B1–CDK complexes (n = 4). (C) CDK kinase assay of WT URECs and URECs from a patient with JS (n = 1). Histone 1 represents substrate level control. (D) DNA breaks are rescued by CDKi in comet tail moments of cells treated with 200 nM CDK1/2i (18 hours) normalized to control DMSO-treated WT cells (n = 3; 50 cells scored per condition). (E) Western blot of Cep290LacZ/LacZ and WT cells treated with 400 nM APH and/or 200 nM CDKi (18 hours). Higher phosphorylation levels of KAP1, H2AX, and CHK1 in Cep290LacZ/LacZ cells with or without APH treatment, rescued by CDK1/2i. Unphosphorylated protein and GAPDH are loading controls; samples were run on parallel gels contemporaneously. (F) 3D spheroids of WT and Cep290LacZ/LacZ cells, stained for γH2AX (red) and ciliation (acetylated tubulin; green), treated with 400 nM APH and/or 200 nM CDKi (18 hours). Scale bars: 5 μm. (G) CDKi rescues ciliation in Cep290LacZ/LacZ and APH-treated spheroids. (35 spheroids scored per condition) (n = 3; data shown for single experiment; 3-way ANOVA, *P < 0.05, **P < 0.01). (H) Cell cycle distribution of WT cells treated with DMSO or 200 nM CDKi (18 hours) reveals increased cell number in G2 (black) and decreased cell number in G1 (gray) after CDKi treatment (10,000 events measured; n = 3).