Candidate Genes for Chromosomes 6 and 10: Quantitative Trait Loci for Age-Related Retinal Degeneration in Mice

Purpose: In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes. Methods: DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT-PCR. Tumor necrosis factor, alpha-induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3(+/-) mice were tested for ageRD. Results: Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT-PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3(+/-) and Tnfaip3(+/+) mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age. Conclusions: The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD

Development and Validation of a Canine-Specific Profiling Array to Examine Expression of Pro-Apoptotic and Pro-Survival Genes in Retinal Degenerative Diseases

We developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of the profiling array was performed with RNA extracted from cultured MDCK cells in the presence or absence of treatment with 10 μM staurosporin for 5 or 10 h. The vast majority of the genes showed positive amplifications, and a number of them also had fold change (FC) differences \u3e ±3 between control and staurosporin-treated cells. To conclude, we established a profiling array that will be used to identify differentially expressed genes associated with photoreceptor death or survival in canine models of retinal degenerative diseases with mutations in genes that cause human inherited blindness with comparable phenotypes

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

Differential Effects of Rapamycin on Rods and Cones During Light-Induced Stress in Albino Mice

Rapamycin was tested for its prosurvival role in light-exposed photoreceptors. Rod survival appeared to occur by normalizing cytokine production, removing damaged proteins by autophagic vacuole (AV)–independent autophagy, or both, whereas cone survival was AV dependent

Oxidative stress sensitizes retinal pigmented epithelial (RPE) cells to complement-mediated injury in a natural antibody-, lectin pathway-, and phospholipid epitope-dependent manner.

International audienceUncontrolled activation of the alternative complement pathway (AP) is thought to be associated with age-related macular degeneration. Previously, we have shown that in retinal pigmented epithelial (RPE) monolayers, oxidative stress reduced complement inhibition on the cell surface, resulting in sublytic complement activation and loss of transepithelial resistance (TER), but the potential ligand and pathway involved are unknown. ARPE-19 cells were grown as monolayers on transwell plates, and sublytic complement activation was induced with H2O2 and normal human serum. TER deteriorated rapidly in H2O2-exposed monolayers upon adding normal human serum. Although the effect required AP activation, AP was not sufficient, because elimination of MASP, but not C1q, prevented TER reduction. Reconstitution experiments to unravel essential components of the lectin pathway (LP) showed that both ficolin and mannan-binding lectin can activate the LP through natural IgM antibodies (IgM-C2) that recognize phospholipid cell surface modifications on oxidatively stressed RPE cells. The same epitopes were found on human primary embryonic RPE monolayers. Likewise, mouse laser-induced choroidal neovascularization, an injury that involves LP activation, could be increased in antibody-deficient rag1(-/-) mice using the phospholipid-specific IgM-C2. In summary, using a combination of depletion and reconstitution strategies, we have shown that the LP is required to initiate the complement cascade following natural antibody recognition of neoepitopes, which is then further amplified by the AP. LP activation is triggered by IgM bound to phospholipids. Taken together, we have defined novel mechanisms of complement activation in oxidatively stressed RPE, linking molecular events involved in age-related macular degeneration, including the presence of natural antibodies and neoepitopes