Synaptic pathology, altered gene expression, and degeneration in photoreceptors impacted by drusen.
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PURPOSE: Drusen are risk factors for age-related macular degeneration and have been shown to negatively impact cells of the RPE and retina. In this study, the effects of drusen on the synaptic machinery of retinal photoreceptors are investigated. METHODS: Human donor eye tissue containing retina, RPE, and choroid was processed for confocal immunofluorescence microscopy, laser capture microdissection, and light and electron microscopy. Tissue sections were immunostained with a panel of antibodies to synapse-associated proteins. Populations of photoreceptors over drusen and normal populations of photoreceptors were microdissected from fresh frozen tissue, RNA was purified, and quantitative PCR was performed to compare relative levels of gene expression. RESULTS: The number of photoreceptor synaptic terminals is reduced in regions of the outer plexiform layer over drusen, synaptic proteins are mislocalized in photoreceptor cells, and synaptic terminals are often observed within the outer nuclear layer. Photoreceptors over drusen also increase expression of the stress response proteins apolipoprotein E and alphaB-crystallin. Abnormal immunolabeling patterns are not restricted to photoreceptors directly over drusen but are also observed in cells flanking drusen. Gene expression analysis confirms reductions in the expression of genes coding for synapse-associated proteins and signal transduction proteins and increases in the expression of apolipoprotein E and alphaB-crystallingene transcripts. Ultrastructural analysis of photoreceptor synaptic terminals over drusen reveals significant abnormalities, and cell counts show a reduction in photoreceptor density directly over, and lateral to, drusen of all sizes. CONCLUSIONS: Photoreceptors overlying and flanking drusen exhibit morphologic and biochemical signs of degeneration. The expression of synapse-associated proteins decreases in photoreceptor synaptic terminals, whereas the expression of stress-response proteins increases. Reductions in photoreceptor cell densities over, and flanking, drusen suggest that these degenerative effects eventually result in the death of photoreceptors. (+info)
Differences in vascular bed disease susceptibility reflect differences in gene expression response to atherogenic stimuli.
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Atherosclerosis occurs predominantly in arteries and only rarely in veins. The goal of this study was to test whether differences in the molecular responses of venous and arterial endothelial cells (ECs) to atherosclerotic stimuli might contribute to vascular bed differences in susceptibility to atherosclerosis. We compared gene expression profiles of primary cultured ECs from human saphenous vein (SVEC) and coronary artery (CAEC) exposed to atherogenic stimuli. In addition to identifying differentially expressed genes, we applied statistical analysis of gene ontology and pathway annotation terms to identify signaling differences related to cell type and stimulus. Differential gene expression of untreated venous and arterial endothelial cells yielded 285 genes more highly expressed in untreated SVEC (P<0.005 and fold change >1.5). These genes represented various atherosclerosis-related pathways including responses to proliferation, oxidoreductase activity, antiinflammatory responses, cell growth, and hemostasis functions. Moreover, stimulation with oxidized LDL induced dramatically greater gene expression responses in CAEC compared with SVEC, relating to adhesion, proliferation, and apoptosis pathways. In contrast, interleukin 1beta and tumor necrosis factor alpha activated similar gene expression responses in both CAEC and SVEC. The differences in functional response and gene expression were further validated by an in vitro proliferation assay and in vivo immunostaining of alphabeta-crystallin protein. Our results strongly suggest that different inherent gene expression programs in arterial versus venous endothelial cells contribute to differences in atherosclerotic disease susceptibility. (+info)
CRYBA4, a novel human cataract gene, is also involved in microphthalmia.
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Genetic analysis of a large Indian family with an autosomal dominant cataract phenotype allowed us to identify a novel cataract gene, CRYBA4. After a genomewide screen, linkage analysis identified a maximum LOD score of 3.20 (recombination fraction [theta] 0.001) with marker D22S1167 of the beta -crystallin gene cluster on chromosome 22. To date, CRYBA4 was the only gene in this cluster not associated with either human or murine cataracts. A pathogenic mutation was identified in exon 4 that segregated with the disease status. The c.317T-->C sequence change is predicted to replace the highly conserved hydrophobic amino acid phenylalanine94 with the hydrophilic amino acid serine. Modeling suggests that this substitution would significantly reduce the intrinsic stability of the crystalline monomer, which would impair its ability to form the association modes critical for lens transparency. Considering that CRYBA4 associates with CRYBB2 and that the latter protein has been implicated in microphthalmia, mutational analysis of CRYBA4 was performed in 32 patients affected with microphthalmia (small eye). We identified a c.242T-->C (Leu69Pro) sequence change in exon 4 in one patient, which is predicted here to disrupt the beta -sheet structure in CRYBA4. Protein folding would consequently be impaired, most probably leading to a structure with reduced stability in the mutant. This is the first report linking mutations in CRYBA4 to cataractogenesis and microphthalmia. (+info)
Deamidation alters the structure and decreases the stability of human lens betaA3-crystallin.
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According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the betaA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type betaA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the betaA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions. (+info)
Two Chinese families with pulverulent congenital cataracts and deltaG91 CRYBA1 mutations.
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PURPOSE: To characterize the disease-causing mutations and related phenotypes in two Chinese families with autosomal dominant congenital cataract. METHODS: Family members were clinically characterized by a complete eye examination. Genome-wide linkage screening was performed in Family 1 using a 10K single nucleotide polymorphism approach followed by genotyping of microsatellite markers from the regions with highest support for linkage. The candidate gene, betaA1-crystallin (CRYBA1), was sequenced in both families. RESULTS: Lens examinations in three affected phakic members showed bilateral pulverulent nuclear cataracts in two subjects of Family 1 while another subject of Family 2 displayed bilateral pulverulent lamellar cataract. Linkage analysis in 14 individuals (eight affected, three unaffected and three of their spouses) of Family 1 gave a maximum logarithm of odds score of 2.41 for D17S1294 in chromosomal region 17q11.12 that includes the CRYBA1 gene. In both families in-frame deletions of three bp were detected in exon 4 of CRYBA1 leading to loss of a guanine residue (deltaG91). The mutations cosegregated completely with the cataract phenotype in both families but were associated with distinct haplotypes suggesting that they had occurred independently. CONCLUSIONS: The previously described CRYBA1 mutation deltaG91 was demonstrated in two Chinese families with distinct phenotypes of congenital cataract, suggesting a lack of genotype-phenotype correlation. The findings also raise the possibility that the delta91 mutation arise in a relatively mutation-prone sequence of the CRYBA1 gene. (+info)
Nonsense mutation in the CRYBB2 gene causing autosomal dominant progressive polymorphic congenital coronary cataracts.
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PURPOSE: We sought to identify the genetic defect in a large, five-generation Chinese family with autosomal dominant progressive polymorphic congenital coronary cataracts and to examine the clinical features in detail. METHODS: Clinical and ophthalmologic examinations were conducted on family members. All members were genotyped with microsatellite markers at loci previously associated with cataracts. Two-point LOD scores were calculated using a linkage package after genotyping. A mutation was detected by direct sequencing and verified by denaturing high-performance liquid chromatography (DHPLC). RESULTS: Clinical observations showed that all affected family members had progressive polymorphic coronary cataracts. Linkage analysis was obtained at markers, D22S303 (LOD score [Z]=2.11, recombination fraction [theta]=0.0) and D22S1167 (Z=1.20, theta=0.0). Haplotype analysis indicated that the cataract gene was closely linked with these two markers. Sequencing the betaB-crystallin gene (CRYBB2) revealed a C --> T transition in exon 6, which changed a codon from Gln to a stop codon (P.Q155X). This mutation cosegregated with all affected individuals and was not observed in any unaffected family member or 100 normal, unrelated individuals. CONCLUSIONS: This study identified a mutation in CRYBB2 in a large Chinese family with autosomal dominant progressive polymorphic congenital coronary cataracts. These results provide evidence that CRYBB2 is a pathogenic gene for congenital cataracts; at the same time, congenital cataracts are a clinically and genetically heterogeneous lens condition. (+info)
Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin.
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