Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

et al. (2012) Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2. PLoS ONE 7(12): e52894. doi:10.1371/journal.pone.0052894 Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2 Chun-hong Xia 0 Bo Chang 0 Adam M. DeRosa 0 Catherine Cheng 0 Thomas W. White 0 Xiaohua Gong 0 K. Krishna Sharma, University of Missouri-Columbia, United States of America 0 1 School of Optometry and Vision Science Program, University of California, Berkeley, California, United States of America, 2 The Jackson Laboratory, Bar Harbor, Maine, United States of America, 3 Physiology and Biophysics, State University of New York - Stony Brook , Stony Brook, New York , United States of America The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8R205G point mutation in the second extracellular loop of the Cx50 (or a8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or a3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8R205G mutation occurs at the same conserved residue as the human GJA8R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans. - Funding: This work was supported by the National Institutes of Health (EY13849 to XG and EY13163 to TW, and EY019943 to BC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Cataracts, defined as any opacity in the eye lens, remain the leading cause of blindness worldwide. Genetic studies of gene mutations are important for understanding the molecular bases of cataract formation [1,2,3]. The lens is comprised of a bulk of elongated fiber cells covered by a monolayer of epithelial cells on the anterior hemisphere. Intercellular gap junction channels connect lens fiber cells and epithelial cells, and provide vital pathways for the transport of important metabolites, ions and fluid needed for lens growth and transparency [4,5]. Gap junction channels are composed of transmembrane protein subunits known as connexins [6]. Each connexin subunit can be divided into four transmembrane domains, three intracellular domains (amino terminal, carboxy terminal and cytoplasmic loop) and two extracellular loops [7]. Six connexin proteins oligomerize to form a connexon (or hemichannel) [8]. Connexons can be of uniform (homomeric) or varying (heteromeric) connexin composition. Gap junctions are formed when the extracellular domains of two heteromeric or homomeric connexons from adjacent cells dock, creating an intercellular passage for the diffusion of small molecules between the cytoplasm of neighboring cells [9]. Gap junctions can be homotypic channels (two identical connexons consisting of one type of connexin subunits), heteromeric channels (connexons consisting of different types of connexin subunits) or heterotypic channels (connexons each containing a different connexin subunit) [6]. Altering connexin subunit composition affects both the permeability and electrophysiological properties of gap junctions. Members of connexin gene family are utilized in almost all organs and cell types [10]. Mutations of connexin gene family members cause various types of diseases in the cardiovascular system, nervous system, skin and eyes in animals and humans [11,12,13,14]. Lens gap junction channels can be formed by at least three types of connexin subunits encoded by three different genes, Cx43 or a1 connexin encoded by the Gja1 gene [15], Cx46 or a3 connexin by the Gja3 gene and Cx50 or a8 connexin by the Gja8 gene. These connexins have distinct and redundant expression in the lens [16,17]. In this manuscript, we have selected standard genetic nomenclature Gja8 and Gja3 for describing genes, and will use Cx50 and Cx46 for proteins. The Cx43 protein is predominantly expressed in lens epithelial cells. The Cx46 protein is mainly expressed in lens fiber cells, while Cx50 is expressed in both epithelial and fiber cells. In addition, the Cx23 protein, encoded by Gje1 or Gjf1, is only expressed in lens primary fiber cells. A Gjf1 mutation affects early lens development and causes a variable small-eye phenotype in mice [18]. However, it is unclear whether Cx23 can form gap junction channels [19]. Figure 1. Identification of the Gja8R205G mutation in Nm2249 mice. (A) Photos of eyes and lenses of Nm2249 heterozygous (Nm/+) and Nm2249 homozygous (Nm/Nm) mutant mice in the CWXS/Agl strain background at the age of 3 weeks. Scale bars, 1 mm. (B) Genome-wide screening data of the phenotypes and genotypes of 105 backcrossed mice, generated between mutant Nm2249 and wild-type CAST/Ei mice. The Nm2249 mutation was mapped to a region near D3Mit11 and D3Mit122 on chromosome 3. (C) The Gja8 gene is near the region where the Nm2249 mutation was mapped. (D) DNA sequencing data confirmed a missense mutation (C to G) in the Gja8 gene of Nm2249 mutant (Nm-Gja8), which resulted in the arginine at codon 205 of wild-type Gja8 (WT-Gja8) being replaced by a glycine (R205G). (E) Homozygous Gja8Nm2249/Nm2249 mutant mice were bred with the Gja82/2 knockout mice for a Gja8 allelic test. Phenotypic comparison of eyes and lenses of Gja8Nm2249/Nm2249, Gja8Nm2249/2 and Gja82/2 mice at the age of 3 weeks was shown. The similarity of small lens size in these mutant mice further indicated that Gja8R205G is the causative mutation in Nm2249 mice. Scale bars, 1 mm. doi:10.1371/journal.pone.0052894.g001 Molecular and cellular mechanisms for the function and regulation of gap junction communication in lens growth and transparency are still far from fully understood. It has been hypothesized that the gap junction network maintains lens homeostasis by providing the outflow pathway in a lens circulation model [4]. Thus, a disruption of these intercellular pathways leads to physiol (...truncated)