Article In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations Graphical Abstract Authors Control CEP290-JSRD Hiroko Shimada, Quanlong Lu, Christine Insinna-Kettenhofen, ..., Cilium GPR161 Cilium ADCY3 Tiziana Cogliati, Christopher J. Westlake, Smo ARL13B F CEP290 Cell membrane Anand Swaroop I Gli2 B Cytoplasm R Other proteins O Correspondence Axoneme B Axoneme [email protected] (C.J.W.), L A [email protected] (A.S.) S Cell membrane Cell membrane T Vesicles In Brief Cytoplasm Cytoplasm Using fibroblasts and iPSC-derived optic Control CEP290-LCA cups from patients with distinct CEP290 mutations, Shimada et al. show a P H Cell membrane concordance between ciliogenesis O T ? defects in different cell types and clinical Axoneme ? O ? Axoneme ? Cytoplasm severity in Leber congenital amaurosis R ? Connecting Connecting ? E Cilium Cilium ? and Joubert syndrome. These studies Cell membrane C Cell membrane E establish CEP290 as gatekeeper of P Docked mother centriole T ?? ? ? signaling molecules in and out of the O primary cilium. R Cytoplasm Cytoplasm Highlights d Normal cilia in fibroblasts, but not in photoreceptors of CEP290-LCA organoids d No CEP290 protein and defective ciliogenesis in CEP290- JSRD fibroblasts d Selective reduction in ciliary localization of ARL13B and ADCY3 in JSRD fibroblasts d Cilia defects in JSRD fibroblasts affect Smo and GPR161 transport in Hh signaling Shimada et al., 2017, Cell Reports 20, 384–396 July 11, 2017 http://dx.doi.org/10.1016/j.celrep.2017.06.045 Cell Reports Article In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations Hiroko Shimada,1 Quanlong Lu,2 Christine Insinna-Kettenhofen,2 Kunio Nagashima,3 Milton A. English,1 Elizabeth M. Semler,2 Jacklyn Mahgerefteh,1 Artur V. Cideciyan,4 Tiansen Li,1 Brian P. Brooks,5 Meral Gunay-Aygun,6,7 Samuel G. Jacobson,4 Tiziana Cogliati,1 Christopher J. Westlake,2,* and Anand Swaroop1,8,* 1Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA 2Laboratory of Cell and Developmental Signaling 3Electron Microscope Laboratory, Leidos Biomedical Research, Inc. National Cancer Institute - Frederick, Frederick, MD 21702, USA 4Department of Ophthalmology, Scheie Eye Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 5Pediatric, Developmental, and Genetic Eye Disease Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA 6National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA 7Department of Pediatrics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 8Lead Contact *Correspondence: [email protected] (C.J.W.), [email protected] (A.S.) http://dx.doi.org/10.1016/j.celrep.2017.06.045 SUMMARY 2012). In non-dividing quiescent or post-mitotic differentiated cells, the mother centriole docks at the apical plasma mem- Mutations in CEP290, a transition zone protein in brane and forms the basal body (Ishikawa and Marshall, primary cilia, cause diverse ciliopathies, including 2011). The primary cilium originates from the basal body by spe- Leber congenital amaurosis (LCA) and Joubert-syn- cific targeting of hundreds of proteins along the ciliary axoneme drome and related disorders (JSRD). We examined via intraflagellar transport (IFT) (Pazour and Bloodgood, 2008; cilia biogenesis and function in cells derived from Boldt et al., 2016; Ishikawa et al., 2012; Sang et al., 2011). Not CEP290-LCA and CEP290-JSRD patients. CEP290 surprisingly, mutations in genes encoding ciliary proteins can cause a plethora of divergent yet overlapping clinical pheno- protein was reduced in LCA fibroblasts with no types (ciliopathies) (Hildebrandt et al., 2011; Nigg and Raff, detectable impact on cilia; however, optic cups 2009; Novarino et al., 2011). Clinical findings in Joubert syn- derived from induced pluripotent stem cells (iPSCs) drome (MIM: 213300) and related disorders (JSRD) include of CEP290-LCA patients displayed less developed fibrocystic disease of kidneys, retinal abnormalities, oculomo- photoreceptor cilia. Lack of CEP290 in JSRD fibro- tor apraxia, congenital hepatic fibrosis, and mental retardation blasts resulted in abnormal cilia and decreased cil- with malformation of cerebellar vermis and brainstem (Sattar iogenesis. We observed selectively reduced locali- and Gleeson, 2011). Retinopathy is commonly observed in syn- zation of ADCY3 and ARL13B. Notably, Hedgehog dromic ciliopathies, and photoreceptor degeneration caused by signaling was augmented in CEP290-JSRD because cilia defects in non-syndromic Leber congenital amaurosis of enhanced ciliary transport of Smoothened and (LCA) (MIM: 204000) represents the least severe phenotype GPR161. These results demonstrate a direct corre- (Rachel et al., 2012; Zaghloul and Katsanis, 2009). Initially identified for JSRD (Ben-Salem et al., 2014; Sayer lation between the extent of ciliogenesis defects et al., 2006; Valente et al., 2006), the centrosome-cilia gene in fibroblasts and photoreceptors with pheno- CEP290 is associated with multiple ciliopathy phenotypes (Cop- typic severity in JSRD and LCA, respectively, and pieters et al., 2010; Zaghloul and Katsanis, 2009). Subsequent to strengthen the role of CEP290 as a selective ciliary findings in a mouse retinal degeneration mutant (rd16) with hypo- gatekeeper for transport of signaling molecules in morphic Cep290 defect (Chang et al., 2006), mutations in and out of the cilium. CEP290 were discovered and now account for 20%–25% of LCA (den Hollander et al., 2006, 2008; Perrault et al., 2007). INTRODUCTION CEP290-LCA patients also exhibit additional sensory defects such as anosmia caused by olfactory cilia dysfunction (McEwen Primary cilia are highly conserved, non-motile microtubule- et al., 2007). based organelles that protrude from the surface of most verte- The CEP290 gene encodes a large multi-domain 290-kDa pro- brate cells and coordinate diverse sensory and developmental tein involved in cilia biogenesis and transport. The CEP290 pro- signaling pathways in different cell types and tissues (Goetz tein constitutes an integral component of the transition zone (TZ) and Anderson, 2010; Singla and Reiter, 2006; Oh and Katsanis, between the basal body and the ciliary axoneme and serves as a 384 Cell Reports 20, 384–396, July 11, 2017 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). diffusion barrier for transport in and out of the cilium (Craige tively). Phenotypes and genotypes of JSRD patients 1–3 are et al., 2010; Rachel et al., 2015). CEP290 is located in the Y- provided in Figure S2 and Table S1. All three JSRD patients shaped linker region of the TZ in photoreceptor cilium (Betleja had reduced visual acuity, moderate to high hyperopia, and Cole, 2010; Craige et al., 2010) and is necessary for the for- nystagmus, and retinal degeneration. JSRD patient 2 (JRSD-2) mation of the outer segment (Rachel et al., 2015), which is critical and JRSD-3 had coloboma. JSRD-3 also carried c.2501A > G; for phototransduction and cell survival (Gilliam et al., 2012; Mars- p.Gln834Arg variation in KIF7 and c.2882G > A; p.Arg961His zalek et al., 2000; Rachel et al., 2012; Tsujikawa and Malicki, change in NPHP4, which can potentially modify the JSRD 2004; Wright et al., 2010). Mutations in CEP290 are suggested phenotype caused by CEP290 mutations. to differentially impact protein interactions, cilia biogenesis, To establish a correlation between CEP290 mutation and the and trafficking in distinct cellular contexts. cilia phenotype, we examined CEP290 protein in fibroblasts by How specific CEP290 mutations generate a spectrum of over- immunoblot analysis. Full-length CEP290 protein was reduced lapping yet discrete pleiotropic effects in ciliopathies is poorly by 50%–80% in CEP290-LCA fibroblasts compared with understood. While LCA mutations associated with sensory controls; however, no protein was detected in CEP290-JSRD dysfunction (Chang et al., 2006; den Hollander et al., 2006; fibroblasts (Figures 1B and 1C). The splice donor site introduced McEwen et al., 2007) are likely hypomorphic, complex pheno- by the IVS26+1655A > G mutation competes with the normal types (such as JSRD) caused by wide-ranging human CEP290 splice donor site, leading to the production of variable amounts mutations are puzzling. Mouse Cep290 mutants can recapitulate of correctly spliced full-length CEP290 protein in LCA-1– several features associated with human disease; for example, an LCA-3. Full-length CEP290 protein levels were higher in LCA-3 in-frame deletion in Cep290 produces early photoreceptor fibroblasts carrying the homozygous IVS26+1655A > G allele degeneration in rd16 mice (Cideciyan et al., 2011), and Cep290 compared with LCA-1 and LCA-2 with the heterozygous gene-trap and knockout mice phenocopy syndromic ciliopathy IVS26+1655A > G allele, consistent with its being a hypomorphic (Hynes et al., 2014; Rachel et al., 2015). However, the precise mutation. All other heterozygous mutations in LCA and JSRD pa- biological impact of distinct human disease-causing CEP290 tients introduce either a premature stop codon (LCA-1, LCA-2, mutations remains unclear. JSRD-1, JSRD-2, and JSRD-3), a frameshift (JSRD-2), or an We took advantage of fibroblasts and induced pluripotent in-frame