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Renal-Retinal Ciliopathy Gene Sdccag8 Regulates DNA Damage Response Signaling

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Renal-Retinal Ciliopathy Gene Sdccag8 Regulates DNA Damage Response Signaling BASIC RESEARCH www.jasn.org Renal-Retinal Ciliopathy Gene Sdccag8 Regulates DNA Damage Response Signaling † ‡ | Rannar Airik,* Gisela G. Slaats, Zhi Guo, Anna-Carina Weiss,§ Naheed Khan, †† ‡‡ Amiya Ghosh,¶ Toby W. Hurd,** Simon Bekker-Jensen, Jacob M. Schrøder, ‡ ‡‡ Steve J. Elledge, Jens S. Andersen, Andreas Kispert,§ Maddalena Castelli,§§ † || Alessandra Boletta,§§ Rachel H. Giles, and Friedhelm Hildebrandt* *Division of Nephrology, Boston Children’s Hospital, Boston, Massachusetts; †Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands; ‡Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts; §Institute of Molecular Biology, Hannover Medical School, Hannover, Germany; |Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan; ¶Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; **Medical Research Council Human Genetics Unit, Medical Research Council Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, United Kingdom; ††Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; ‡‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; §§Division of Genetics and Cell Biology, Dulbecco Telethon Institute, San Raffaele Scientific Institute, Milan, Italy; and ||Howard Hughes Medical Institute, Chevy Chase, Maryland ABSTRACT Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8gt/gt mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to pro- gressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8gt/gt-derived cells, characterized by elevated levels of gH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8gt/gt mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specificdisease mechanisms. J Am Soc Nephrol 25: 2573–2583, 2014. doi: 10.1681/ASN.2013050565 Nephronophthisis-related ciliopathies (NPHP-RCs) Received May 31, 2013. Accepted February 25, 2014. (Online Mendelian Inheritance in Man [OMIM] Published online ahead of print. Publication date available at 256100) are heterogenetic autosomal recessive dis- www.jasn.org. orders that feature nephronophthisis, a degeneration Correspondence: Dr. Friedhelm Hildebrandt, Division of Ne- 1 . disorder of the kidney. To date, mutations in 20 phrology, Boston Children’s Hospital, 300 Longwood Avenue, NPHP-RC genes have been identified2 that manifest HU319, Boston MA 02115. Email: Friedhelm.Hildebrandt@ nephronophthisis as part of their pathogenesis in the childrens.harvard.edu context of ciliopathy syndromes such as Senior–Loken Copyright © 2014 by the American Society of Nephrology J Am Soc Nephrol 25: 2573–2583, 2014 ISSN : 1046-6673/2511-2573 2573 BASIC RESEARCH www.jasn.org syndrome (OMIM 266900), Bardet–Biedl syndrome (BBS; mouse embryonic fibroblasts (Supplemental Figure 1C). Im- OMIM 209900), Joubert syndrome (OMIM 213300), and orofa- munoblotting (Supplemental Figure 1D) confirmed the ab- ciodigital syndrome (OFD; OMIM 311200). sence of Sdccag8 protein from lung and kidney lysates of We recently showed that mutations in serologically defined Sdccag8gt/gt mice. Two isoforms of the Sdccag8 protein (78 colon cancer antigen 8 (SDCCAG8) cause nephronophthisis kD and 83 kD) were detected in Sdccag8wt/gt kidneys (Supple- type 10, characterized by retinal and renal degeneration, mild mental Figure 1D).3 Sdccag8gt/gt mice were present at Mende- intellectual disability, obesity, hypogonadism, and recurrent re- lian ratios at weaning age, indicating that the Sdccag8 gene-trap spiratory infections in humans.3,4 Because several of the clinical allele does not cause embryonic or early postnatal lethality. features are shared with BBS, with the exception of the absence of polydactyly, individuals with SDCCAG8 mutations are also con- Sdccag8 Is Expressed in Kidney and Lung Epithelia sidered as part of the BBS spectrum.3,4 SDCCAG8 encodes a Mutations in SDCCAG8 were previously reported to affect two coiled-coil domain protein with no additional conserved do- parenchymal organs in humans, the kidneys and the lungs, caus- mains.5 The protein localizes to the centrioles throughout the ing nephronophthisis and, infrequently, bronchiectasis.3,4 To cell cycle,3,5 to the basal body of cilia, and also to the spermato- understand the underlying pathogenetic mechanisms, we first cytes in the rat testis.3,6 Immunohistochemical analysis of retina examined the expression pattern of Sdccag8 in these organs by has shown SDCCAG8 colocalization with retinitis pigmentosa taking advantage of the lacZ cassette in the Sdccag8 gene-trap allele. protein 1 (RP1), retinitis pigmentosa GTPase regulator (RPGR), b-Galactosidase activity staining of wild-type and Sdccag8wt/gt and retinitis pigmentosa GTPase regulator interacting protein 1 whole urogenital systems at E16.5 showed strong Sdccag8 (RPGRIP1) in the connecting cilium of the photoreceptors.3,7 expression in the corticomedullary region of the Sdccag8wt/gt Biochemical studies have demonstrated SDCCAG8 homodime- kidneys (Figure 1A) and no staining in the wild-type control rization and direct interaction with two ciliopathy proteins: (1) (Supplemental Figure 2A). Examination of the X-gal–stained OFD1 and (2) family with sequence similarity 161, member A kidney sections at higher resolution showed staining in the (FAM161A).3,5,8 Despite the data on SDCCAG8 protein locali- renal tubule epithelia in a pattern compatible with the distal zation and its interaction partners, the precise molecular func- convoluted tubule (DCT) and cortical collecting ducts (CCDs) tion at centrosomes and cilia remains unknown. (Figure 1B). Sdccag8 expression in the collecting ducts was also Werecentlydemonstrated that mutations inthe gene encoding observed in postnatal P14 and P100 kidneys by in situ hybrid- the centrosomal protein CEP164 cause NPHP-RC whose path- ization (Figure 1, C and D), whereas the sense probe showed no ogenesis involves defects in the DNA damage response (DDR) staining (Supplemental Figure 2, B and C). In the lung, X-gal signaling pathway.9 The same study also implicated SDCCAG8 in staining in Sdccag8wt/gt mice at E16.5 showed Sdccag8 expres- this pathway through its colocalization with CEP164 and Tat- sion in the epithelium of the developing bronchi and bron- interactive protein 60 in the cell nucleus.9 Indeed, there is a wealth chioles (Figure 1E). Examination of lung sections at higher of evidence in the literature that implicates centrosomal protein resolution confirmed this observation and further showed 2 function in the regulation of genome stability, including the that the blue lacZ+ cells were interspersed with lacZ cells in NPHP-RC proteins AHI1,10 NIMA-related kinase 8 (NEK8/ the bronchioles (Figure 1F). No b-galactosidase staining was NPHP9),11 and the SDCCAG8-interacting protein OFD1.12,13 detected in the epithelial cells of alveoli—the terminal ends of To study the role of Sdccag8 in the pathogenesis of NPHP- the airways, which do not have cilia (Figure 1F, delineated with RC, we generated a transgenic Sdccag8gt/gt mouse model. We dashed line). To determine whether the lacZ+ cells in the lung demonstrate that Sdccag8gt/gt mice recapitulate aspects of the epithelium represent the multiciliated cells, we performed im- human disease phenotype. Furthermore, we show that munofluorescence analysis on E16.5 CETN2-GFP mouse lung Sdccag8 is involved in cell cycle S-phase progression and its sections using the cilia marker anti-polyglutamylated tubulin loss leads to replication stress–related DDR activation. antibody and anti-SDCCAG8 antibody. CETN2-GFP fusion protein localizes to centrioles,14 which are presented in hun- dreds of copies in the multiciliated cells of the respiratory RESULTS epithelium.15 Because centriologenesis precedes multiciliogen- esis, almost no cilia were detected in E16.5 distal bronchioles Generation of Sdccag8gt/gt Mice (Supplemental Figure 2, D–F), as previously described.16 To investigate the function of the Sdccag8 gene, the embryonic However, we found that the CETN2-GFP–positive structures stem cell line OST40418 containing the gene-trap cassette (Figure 1G) in the progenitors of the multiciliated cells fully VICTR24 in the intronic region downstream of Sdccag8 exon overlapped with SDCCAG8 antibody staining in E16.5 bron- 1 (Supplemental Figure 1A) was microinjected and founders chioles (Figure 1, H and
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