Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA virus infection in Drosophila.

Intrinsic immune responses autonomously inhibit viral replication and spread. One pathway that restricts viral infection in plants and insects is RNA interference (RNAi), which targets and degrades viral RNA to limit infection. To identify additional genes involved in intrinsic antiviral immunity, we screened Drosophila cells for modulators of viral infection using an RNAi library. We identified Ars2 as a key component of Drosophila antiviral immunity. Loss of Ars2 in cells, or in flies, increases susceptibility to RNA viruses. Consistent with its antiviral properties, we found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, we show that Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing pri-miRNAs. The identification of Ars2 as a player in these small RNA pathways provides new insight into the biogenesis of small RNAs that may be extended to other systems.

Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus.

Intrinsic innate immune mechanisms are the first line of defense against pathogens and exist to control infection autonomously in infected cells. Here, we showed that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, played a direct antiviral role against the mammalian viral pathogen vesicular stomatitis virus (VSV) in the model organism Drosophila. We found that the surface glycoprotein, VSV-G, was likely the pathogen-associated molecular pattern (PAMP) that initiated this cell-autonomous response. Once activated, autophagy decreased viral replication, and repression of autophagy led to increased viral replication and pathogenesis in cells and animals. Lastly, we showed that the antiviral response was controlled by the phosphatidylinositol 3-kinase (PI3K)-Akt-signaling pathway, which normally regulates autophagy in response to nutrient availability. Altogether, these data uncover an intrinsic antiviral program that links viral recognition to the evolutionarily conserved nutrient-signaling and autophagy pathways.

Effect of gamma irradiation on the structural stability and functional activity of plasma-derived IgG.

Plasma-originated commercial intravenous immunoglobulin, which is used for a variety of clinical purposes, has been studied to determine the effect of virus-inactivating doses of gamma irradiation on the structural-functional characteristics of the protein. A detailed analysis has been performed in response to a concern that the use of conventional gamma irradiation may damage biologically active proteins. The results demonstrate that although gamma irradiation of the IgG may have some impact on protein structure, the damage can be reduced or even prevented by appropriate irradiation conditions. At the virucidal dose of gamma irradiation (50 kGy) and a temperature of -80 °C, the integrity of the polypeptide chain of immunoglobulin and the secondary structure of IgG can be completely protected, while conformational changes in tertiary structure are significantly minimized to a level that preserves functional activity. The irradiated IgG retains specific antigen-binding properties and F(c)-binding activity, indicating that the conformational integrity of the most important structural regions is not affected by γ-irradiation. These results present strong evidence that gamma irradiation treatment can be effectively implemented for inactivation of pathogens in IgG solutions that are used for intravenous injection.

Retinal localization and copper-dependent relocalization of the Wilson and Menkes disease proteins.

PURPOSE: Menkes and Wilson diseases are associated with retinal degeneration. The Menkes and Wilson genes are homologous copper transporters, but differences in their expression pattern lead to different disease manifestations. To determine whether the Wilson and Menkes genes may act locally in the retina, this study was undertaken to assess retinal Wilson and Menkes expression and localization. METHODS: RT/PCR was used to test for the presence of Wilson and Menkes mRNAs in mouse and human retinas and retinal pigment epithelial cell lines. The Menkes and Wilson proteins were immunolocalized in human and mouse retinas and in the ARPE-19 cell line. RESULTS: The Menkes mRNA and protein were present in the RPE and neurosensory retina whereas the Wilson mRNA and protein were limited to the RPE. In the RPE, both proteins localized to the Golgi. Increased copper concentration led to relocalization of the Wilson protein to a diffuse cytoplasmic distribution. CONCLUSIONS: Both the Menkes and Wilson proteins are present in the RPE. Since the RPE is a blood-brain barrier, these proteins most likely regulate not only their own copper levels but also copper levels of the overlying photoreceptors. Because the Wilson protein delivers copper to the ferroxidase ceruloplasmin in the liver, it is likely that the Wilson and/or Menkes proteins provide copper to ceruloplasmin made in the RPE. Retinopathy in Wilson and Menkes diseases may result not only from abnormal systemic copper levels but also from loss of retinal Wilson or Menkes protein.

Generation of Cre transgenic mice with postnatal RPE-specific ocular expression.

PURPOSE: To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes. METHODS: A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography. RESULTS: The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity. CONCLUSIONS: This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

Iron chelation protects the retinal pigment epithelial cell line ARPE-19 against cell death triggered by diverse stimuli.

PURPOSE: Cell death can be induced by exogenous reactive oxygen species (ROS). Endogenous ROS can also play a role in cell death triggered by agents that are not themselves ROS. One of the most potent ROS-generating systems is the iron-catalyzed Fenton reaction. Herein, the authors tested whether iron plays an important role in cell death induced by diverse stimuli in retinal pigment epithelial (RPE) cells. METHODS: The ability of the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) to chelate intracellular labile iron was tested in the human cell line ARPE-19. The ability of SIH to protect against RPE cell death induced by hydrogen peroxide, staurosporine, anti-Fas, and exposure to A2E plus blue light was determined. ROS production by staurosporine was assessed in the presence and absence of SIH. The protective activity of SIH was compared with that of other iron chelators and an antioxidant. RESULTS: Acute exposure to SIH was nontoxic and at least partially protective against cell death induced by all tested agents. On a molar basis, SIH was more protective against hydrogen peroxide than other iron chelators and an antioxidant. SIH decreased levels of staurosporine-induced ROS. CONCLUSIONS: Iron chelation with SIH can decrease levels of ROS and protect RPE cells against cell death induced by diverse stimuli. These results suggest a central role for iron in cell death pathways, potentially involving the generation of oxidative stress. SIH or related iron chelators may prove useful for protection against diseases involving RPE death, such as AMD.

Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide.

Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinson's, Alzheimer's, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection.