Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology2011-289033 “DYNANO” and BM1202 European Network onMicrovesicles and Exosomes in Health and Disease (ME-HAD).Bence György is a Kerpel-Fronius Ödön Fellows.Program Number: 3149Presentation Time: 11:15 AM - 11:30 AMNephrocystin-5 knockout mice recapitulate retina and kidneypathologies of Senior-Løken SyndromeCecinio Ronquillo, Jeanne M. Frederick, Wolfgang Baehr.Ophthalmology and Visual Sciences, Moran Eye Center, Universityof Utah, Salt Lake City, UT.Purpose: Senior-Løken syndrome is an autosomal recessive diseasecharacterized by retinal degeneration (RP/LCA) andnephronophthisis (kidney fibrosis and presence of cysts). Mutationsin Nephrocystin-5 (NPHP5) have been shown to be the most commoncause of Senior-Løken syndrome in humans; however, the function ofNPHP5 is poorly understood. Here, we describe a novel mammalianmodel of Senior-Løken syndrome generated by deleting NPHP5 inthe mouse.Methods: A gene trap containing a reporter gene (β-gal) was insertedin intron 4 of the mouse Nphp5 gene causing premature terminationof Nephrocystin-5 protein expression. Retina and renal tissue werecharacterized by immunocystochemistry using various antibodies atdevelopmental time points. ERGs were recorded at different timepoints. Ultrastructure of the photoreceptor connecting cilium andbasal body was examined on postnatal days P6 and P10.Results: Global knockout mice are viable and fertile. Retinalfunction in knockout animals was undetectable by ERG at P12 (eyeopening). At P10, knockout animals already showed absence of outersegments and decreased number of cells in the outer nuclear layer.Rhodopsin transport is impaired as early as P6 and rhodopsinaccumulates in rod perinuclear regions (Fig. 1). Apoptosis, fibrosisand presence of cysts are observed in the kidneys of knockout mice(Fig. 2). Knockdown of NPHP5 in a kidney cell line shows decreasednumbers of primary cilia suggesting a role of NPHP5 in ciliogenesisor ciliary maintenance.Conclusions: NPHP5 global knockout mouse is a novel model ofSenior-Løken syndrome recapitulating the retina and renalpathologies observed in humans. This model will be an importanttool to provide insight into detailed molecular mechanisms of NPHP5function in ciliogenesis and primary cilium structure.Figure 1. Rhodopsin mislocalizes to rod perinuclear regions of P10NPHP5 KO retina (red arrow). Rhodopsin (green, 1D4), cone arrestin(red, mCAR), nuclei (blue, DAPI).Figure 2. NPHP5 knockout kidneys have increased cell death byTUNEL stain (green). Also shown is an example of a cyst present inknockout kidneys (red arrow).Commercial Relationships: Cecinio Ronquillo, None; Jeanne M.Frederick, None; Wolfgang Baehr, NoneSupport: National Eye Institute F31 NRSA - F31EY021972-01A1Program Number: 3150Presentation Time: 11:30 AM - 11:45 AMLong-term preservation of immature cone-like photoreceptors ina mouse model of human LCA caused by dominant CRXframeshift mutationJerome E. Roger 1 , Avinash H. Hiriyanna 1 , Debbie F. Cheng 1 ,Norimoto Gotoh 1 , Rinki Ratna Priya 1 , Matthew Brooks 1 , Harsha K.Rajasimha 1 , Bo Chang 2 , Anand Swaroop 1 . 1 Neurobiol-Neurodegnt'nRep Lab, NEI / National Institutes of Health, Bethesda, MD; 2 TheJackson Laboratory, Bar Harbor, ME.Purpose: Leber’s congenital amaurosis (LCA) is a severe form ofchildhood blindness representing 5% of all retinopathies. Mutationsin CRX, a key transcription factor for photoreceptor development, areassociated with retinal dystrophies including LCA. The pathogenicmechanisms of CRX disease have not been elucidated, and thereported Crx-/- mouse model does not reflect the human phenotype.Here, we describe a spontaneous mutant with autosomal dominantcongenital blindness named RIP (Retina with ImmaturePhotoreceptors), caused by a frameshift mutation in Crx leading tothe differentiation of retina with immature cone-like photoreceptors.Methods: Linkage analysis and exome capture sequencing were usedto identify the mutation. Fundus photography, ERG,immunohistochemistry, RNA-seq, luciferase and gel-shift assayswere performed to characterize the RIP mouse and the mutant CRXprotein. CRX and frameshift mutants were transfected in mouseretina by in vivo electroporation. Crxp::Nrl mice were used for someof the rescue experiments.Results: The RIP mutant revealed autosomal dominant pattern ofinheritance with congenital blindness and exhibited pigmentation andattenuated vasculature upon fundus examination at one-month of age.We identified a frameshift mutation in Crx resulting in the deletion ofthe Otx-like domain and an extended C-terminal region. Interestingly,several LCA-causing CRX mutations result in similar proteinmodifications. In the RIP mutant, the retinal photoreceptors hadcone-like nuclei, but no outer segments; however, unlike Crx-/- micethe photoreceptors did not degenerate. RNAseq analysis of P2 andP21 retina revealed a loss of Nrl and Nr2e3 expression and anabsence of most rod visual signaling components only at P21. Wealso showed the lack of transcriptional activity in CrxRIP was due toits inability to bind DNA. By mating RIP mutant with Crxp-Nrl micein which Nrl is expressed in all photoreceptor precursors, we partiallyrescued the phenotype due to the existence of only rods with shortouter segments. Electroporation in wild type mice of LCA-causingCRX frameshift mutations led to an arrest of photoreceptor©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.