Abstract
Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials. Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters. Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes. Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.
Highlights
Mutations in the inwardly rectifying potassium channel gene KCNJ13 have been shown to cause both snowflake vitreoretinal degeneration (SVD) (Hejtmancik et al, 2008) and Leber congenital amaurosis (LCA) (Sergouniotis et al, 2011)
In order to investigate further the pathophysiological role of KCNJ13 mutations in snowflake vitreoretinal degeneration (SVD, MIM 193230) and Leber congenital amaurosis (LCA16, MIM 614186) a knockout mouse model was constructed by inserting a gene trap cassette in intron 1, which consisted of a splice accepter (SA) followed by IRES and beta galactosidase sequences
The gene trap cassette is flanked by FRT sites and the gene trap targeting vector is constructed so that exon two of the Kcnj13 gene is flanked by LoxP sites (Figure 1)
Summary
Mutations in the inwardly rectifying potassium channel gene KCNJ13 have been shown to cause both snowflake vitreoretinal degeneration (SVD) (Hejtmancik et al, 2008) and Leber congenital amaurosis (LCA) (Sergouniotis et al, 2011). LCA, the most severe form of retinal degeneration, shows severe visual impairment and retinal dysfunction in the first year of life, with night blindness and constricted visual fields often accompanied by nystagmus While these retinal diseases have some overlapping features, they are distinguished by the relatively preserved vision and the higher frequency of vitreal changes in SVD. Among other tissues including hypothalamic neurons in which it is regulated by the melanocortin-4receptor (Ghamari-Langroudi et al, 2015), brain choroid plexus, lung, renal and intestinal epithelia and thyroid follicular cells (Döring et al, 1998; Ookata et al, 2000; Cornejo et al, 2018), Kir7.1 is present in the apical membrane of retinal pigmented epithelium (RPE) and the choroid, in which the Na+/K+-pump is expressed apically (Hughes and Takahira, 1996; Shimura et al, 2001). It is regulated by membrane phospholipids including phosphatidylinositol 4,5-biphosphate which is cleaved upon binding of oxytocin by the oxytocin receptor, a G proteincoupled receptor (Pattnaik and Hughes, 2009; York et al, 2017)
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