Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes.

BACKGROUND: Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. METHODS: Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. RESULTS: Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10-9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10-7), and Tenascin X (TNXB) (p = 6.30 × 10-6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. CONCLUSIONS: We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

Presence of rd8 mutation does not alter the ocular phenotype of late-onset retinal degeneration mouse model.

PURPOSE: A spontaneous frameshift mutation, c.3481delC, in the Crb1 gene is the underlying cause of dysplasia and retinal degeneration in rd8 mice. The rd8 mutation is found in C57BL/6N but not in C57BL/6J mouse sub-strains. The development of ocular pathology in single knockout Ccl2-/-, Cx3cr1-/- and in double knockout Ccl2-/-, Cx3cr1-/- mice raised on a C57BL/6 background has been reported to depend on the presence of a rd8 mutation. In this study, we investigated the influence of the rd8 mutation on the retinal pathology that we previously described in the late-onset retinal degeneration (L-ORD) mouse model with a heterozygous S163R mutation in the C1q-tumor necrosis factor-related protein-5Ctrp5+/- gene that was generated on a C57BL/6J background. METHODS: Mouse lines carrying the Ctrp5 S163R and rd8 mutations (Ctrp5+/-;rd8/rd8), corresponding controls without the rd8 mutation (Ctrp5+/-;wt/wt), and wild-type mice with and without the rd8 mutation (Wtrd8/rd8 and Wtwt/wt, respectively) were generated by systematic breeding of mice in our L-ORD mouse colony. Genotyping the mice for the rd8 (del C at nt3481 in Crb1) and Ctrp5 S163R mutations was performed with allelic PCR or sequencing. Retinal morphology was studied with fundus imaging, histology, light microscopy, electron microscopy, and immunohistochemistry. RESULTS: Genotype analysis of the mice in L-ORD mouse colony detected the rd8 mutation in the homozygous and heterozygous state. Fundus imaging of wild-type mice without the rd8 mutation (Wtwt/wt) revealed no autofluorescence (AF) spots up to 6-8 months and few AF spots at 21 months. However, the accumulation of AF lesions accelerated with age in the Ctrp5+/- mice that lack the rd8 mutation (Ctrp5+/-;wt/wt). The number of AF lesions was significantly increased (p<0.001), and they were small and uniformly distributed throughout the retina in the 21-month-old Ctrp5+/-;wt/wt mice when compared to the age-matched controls. Wild-type and Ctrp5+/- mice with the rd8 mutation (Wtrd8/rd8 and Ctrp5+/-;rd8/rd8, respectively) revealed an integrated retinal architecture with well-defined outer segments/inner segments (OS/IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL). The presence of pseudorosette structures reported in the rd8 mice between the ONL and the INL in the ventral quadrant of the retina was not observed in all genotypes studied. Further, the external limiting membrane was continuous in the Ctrp5+/-;rd8/rd8 and Wtrd8/rd8 mice. Evaluation of the retinal phenotype revealed that the Ctrp5+/-;wt/wt mice developed characteristic L-ORD pathology including age-dependent accumulation of AF spots, development of sub-retinal, sub-RPE, and basal laminar deposits, and Bruch's membrane abnormalities at older age, while these changes were not observed in the age-matched littermate WTwt/wt mice. CONCLUSIONS: The Wtrd8/rd8 and Ctrp5+/-;rd8/rd8 mice raised on C57BL/6J did not develop early onset retinal changes that are characteristic of the rd8 phenotype, supporting the hypothesis that manifestation of rd8-associated pathology depends on the genetic background. The retinal pathology observed in mice with the Ctrp5+/-;wt/wt genotype is consistent with the L-ORD phenotype observed in patients and with the phenotype we described previously. The lack of rd8-associated retinal pathology in the Ctrp5+/-;wt/wt mouse model raised on the C57BL/6J background and the development of the L-ORD phenotype in these mice in the presence and absence of the rd8 mutation suggests that the pathology observed in the Ctrp5+/-;wt/wt mice is primarily associated with the S163R mutation in the Ctrp5 gene.

Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene.

PURPOSE: Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5). METHODS: The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry. RESULTS: As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT-PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes. CONCLUSIONS: The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.

Identification of a promoter for the human C1Q-tumor necrosis factor-related protein-5 gene associated with late-onset retinal degeneration.

PURPOSE: The Complement-1q tumor necrosis factor-related protein 5 (C1QTNF5/CTRP5) gene is located in the 3' untranslated region of the Membrane Frizzled Related Protein (MFRP) gene, and these two genes are reported to be dicistronic. The authors examined the 5' upstream sequence of CTRP5 for the presence of a promoter regulating the expression of this gene. METHODS: The sequence upstream of the translational start site of human CTRP5 (hCTRP5) was analyzed by Promoter Inspector software. A series of plasmids containing segments of hCTRP5 putative promoter sequence (-29 bp to -3.6 kb) upstream of the luciferase gene were generated. Cells were transiently transfected with these plasmids, and luciferase activity was measured. 5' RACE analysis was performed to determine the functional transcription start site. V5 tagged-pig CTRP5 (pCTRP5) gene, cloned downstream of the hCTRP5 putative promoter, was expressed in a human retinal cell line (ARPE-19) and a Chinese hamster ovary cell line (CHO-K1) to study the functionality of the putative promoter. RESULTS: Bioinformatic analysis identified a putative promoter region between nt -1322 and +1 sequence of hCTRP5. 5' RACE analysis revealed the presence of the transcriptional start site (TSS) at 62 bp upstream of the start codon in the CTRP5. The 1.3-kb sequence of the hCTRP5 predicted promoter produced higher levels of luciferase activity, indicating the strength of the cloned CTRP5 promoter. The promoter sequence between nt -1322 bp to -29 bp upstream of the first ATG of CTRP5 was found to be essential for this promoter activity. The predicted hCTRP5 promoter was found to control the expression of V5-tagged pCTRP5 and nuclear GFP, indicating that the promoter was functional. CONCLUSIONS: This study revealed the presence of a functional promoter for the CTRP5 gene located 5' of its start site. Understanding the regulation of CTRP5 gene transcription may provide insights into the possible role of CTRP5 in the retina and the pathology underlying late-onset retinal degeneration caused by mutations in this gene. In addition, these studies will determine whether CTRP5 and MFRP are functionally dicistronic.

Age-related retinal degeneration (arrd2) in a novel mouse model due to a nonsense mutation in the Mdm1 gene.

We observed that a naturally occurring mouse strain developed age-related retinal degeneration (arrd2). These mice had normal fundi, electroretinograms (ERGs) and retinal histology at 6 months of age; vessel attenuation, RPE atrophy and pigmentary abnormalities at 14 months, which progressed to complete loss of photoreceptors and extinguished ERG by 22 months. Genetic analysis revealed that the retinal degeneration in arrd2 segregates in an autosomal recessive manner and the disease gene localizes to mouse chromosome 10. A positional candidate cloning approach detected a nonsense mutation in the mouse double minute-1 gene (Mdm1), which results in the truncation of the putative protein from 718 amino acids to 398. We have identified a novel transcript of the Mdm1 gene, which is the predominant transcript in the retina. The Mdm1 transcript is localized to the nuclear layers of neural retina. Expression of Mdm1 in the retina increases steadily from post-natal day 30 to 1 year, and a high level of Mdm1 are subsequently maintained. The Mdm1 transcript was found to be significantly depleted in the retina of arrd2 mice and the transcript was observed to degrade by nonsense-mediated decay. These results indicate that the depletion of the Mdm1 transcript may underlie the mechanism leading to late-onset progressive retinal degeneration in arrd2 mice. Analysis of a cohort of patients with age-related macular degeneration (AMD) wherein the susceptibility locus maps to chromosome 12q, a region bearing the human ortholog to MDM1, did not reveal association between human MDM1 and AMD.

Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity.

The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.