Lens ER-stress response during cataract development in Mip-mutant mice

AbstractMajor intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=<0.05) when compared with wild-type. A pathway analysis of up-regulated genes in the Lop/+ lens (P7) was consistent with endoplasmic reticulum (ER)-stress and activation of the unfolded protein response (UPR). The most up-regulated UPR genes (>4-fold) in the Lop/+ lens included Chac1>Ddit3>Atf3>Trib3>Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress

Germ-line and somatic EPHA2 coding variants in lens aging and cataract

Rare germ-line mutations in the coding regions of the human EPHA2 gene (EPHA2) have been associated with inherited forms of pediatric cataract, whereas, frequent, non-coding, single nucleotide variants (SNVs) have been associated with age-related cataract. Here we sought to determine if germ-line EPHA2 coding SNVs were associated with age-related cataract in a case-control DNA panel (> 50 years) and if somatic EPHA2 coding SNVs were associated with lens aging and/or cataract in a post-mortem lens DNA panel (> 48 years). Micro-fluidic PCR amplification followed by targeted amplicon (exon) next-generation (deep) sequencing of EPHA2 (17-exons) afforded high read-depth coverage (1000x) for > 82% of reads in the cataract case-control panel (161 cases, 64 controls) and > 70% of reads in the post-mortem lens panel (35 clear lens pairs, 22 cataract lens pairs). Novel and reference (known) missense SNVs in EPHA2 that were predicted in silico to be functionally damaging were found in both cases and controls from the age-related cataract panel at variant allele frequencies (VAFs) consistent with germ-line transmission (VAF > 20%). Similarly, both novel and reference missense SNVs in EPHA2 were found in the post-mortem lens panel at VAFs consistent with a somatic origin (VAF > 3%). The majority of SNVs found in the cataract case-control panel and post-mortem lens panel were transitions and many occurred at di-pyrimidine sites that are susceptible to ultraviolet (UV) radiation induced mutation. These data suggest that novel germ-line (blood) and somatic (lens) coding SNVs in EPHA2 that are predicted to be functionally deleterious occur in adults over 50 years of age. However, both types of EPHA2 coding variants were present at comparable levels in individuals with or without age-related cataract making simple genotype-phenotype correlations inconclusive

cis-6-Methoxycarbonyl-2,10-dioxa-1-phosphabicyclo[4.4.0]decane 1-Oxide

The crystal structure of the title compound, C9H1505P, is described. The molecule consists of two cis-fused six-membered rings, both in the chair conformation.

Surface and interstitial Ti diffusion at the rutile TiO2(110) surface

Diffusion of Ti through the TiO2 (110) rutile surface plays a key role in the growth and reactivity of TiO2. To understand the fundamental aspects of this important process, we present an analysis of the diffusion of Ti adspecies at the stoichiometric TiO2(110) surface using complementary computational methodologies of density functional theory corrected for on-site Coulomb interactions (DFT+U) and a charge equilibration (QEq) atomistic potential to identify minimum energy pathways. We find that diffusion of Ti from the surface to subsurface (and vice versa) follows an intersticialcy exchange mechanism, involving exchange of surface Ti with the 6-fold coordinated Ti below the bridging oxygen rows. Diffusion in the subsurface between layers also follows an interstitialcy mechanism. The diffusion of Ti is discussed in light of continued attempts to understand the re-oxidation of non-stoichiometric TiO2(110) surfaces

Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with ball-and-socket double-membrane junctions during lens fiber cell differentiation

Exome sequencing identifies a missense variant in EFEMP1 co-segregating in a family with autosomal dominant primary open-angle glaucoma

Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic

Cat-Map: putting cataract on the map

Lens opacities, or cataract(s), may be inherited as a classic Mendelian disorder usually with early-onset or, more commonly, acquired with age as a multi-factorial or complex trait. Many genetic forms of cataract have been described in mice and other animal models. Considerable progress has been made in mapping and identifying the genes and mutations responsible for inherited forms of cataract, and genetic determinants of age-related cataract are beginning to be discovered. To provide a convenient and accurate summary of current information focused on the increasing genetic complexity of Mendelian and age-related cataract we have created an online chromosome map and reference database for cataract in humans and mice (Cat-Map)