Resvega Alleviates Hydroquinone-Induced Oxidative Stress in ARPE-19 Cells

Retinal pigment epithelial (RPE) cells maintain homeostasis at the retina and they are under continuous oxidative stress. Cigarette smoke is a prominent environmental risk factor for age-related macular degeneration (AMD), which further increases the oxidant load in retinal tissues. In this study, we measured oxidative stress and inflammatory markers upon cigarette smoke-derived hydroquinone exposure on human ARPE-19 cells. In addition, we studied the effects of commercial Resvega product on hydroquinone-induced oxidative stress. Previously, it was observed that Resvega induces autophagy during impaired protein clearance in ARPE-19 cells, for which it has the potential to alleviate pro-inflammatory pathways. Cell viability was determined while using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the cytokine levels were measured using the enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production were measured using the 2′,7′-dichlorofluorescin diacetate (H2DCFDA) probe. Hydroquinone compromised the cell viability and increased ROS production in ARPE-19 cells. Resvega significantly improved cell viability upon hydroquinone exposure and reduced the release of interleukin (IL)-8 and monocytic chemoattractant protein (MCP)-1 from RPE cells. Resvega, N-acetyl-cysteine (NAC) and aminopyrrolidine-2,4-dicarboxylic acid (APDC) alleviated hydroquinone-induced ROS production in RPE cells. Collectively, our results indicate that hydroquinone induces cytotoxicity and increases oxidative stress through NADPH oxidase activity in RPE cells, and resveratrol-containing Resvega products prevent those adverse effects.Peer reviewe

Autophagy Genes for Wet Age-Related Macular Degeneration in a Finnish Case-Control Study

Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF (vascular endothelial growth factor) treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs (rs2295080, rs11121704, rs1057079, rs1064261, rs573775, rs11246867, rs3088051, rs10902469, rs73105013, rs10277) in the mTOR (Mechanistic Target of Rapamycin), ATG5 (Autophagy Related 5), ULK1 (Unc-51-Like Autophagy Activating Kinase 1), MAP1LC3A (Microtubule Associated Protein 1 Light Chain 3 α), SQSTM1 (Sequestosome 1) were analyzed with RT-PCR-based genotyping. The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment

Bayesian inference of ancestral recombination graphs

We present a novel algorithm, implemented in the software ARGinfer, for probabilistic inference of the Ancestral Recombination Graph under the Coalescent with Recombination. Our Markov Chain Monte Carlo algorithm takes advantage of the Succinct Tree Sequence data structure that has allowed great advances in simulation and point estimation, but not yet probabilistic inference. Unlike previous methods, which employ the Sequentially Markov Coalescent approximation, ARGinfer uses the Coalescent with Recombination, allowing more accurate inference of key evolutionary parameters. We show using simulations that ARGinfer can accurately estimate many properties of the evolutionary history of the sample, including the topology and branch lengths of the genealogical tree at each sequence site, and the times and locations of mutation and recombination events. ARGinfer approximates posterior probability distributions for these and other quantities, providing interpretable assessments of uncertainty that we show to be well calibrated. ARGinfer is currently limited to tens of DNA sequences of several hundreds of kilobases, but has scope for further computational improvements to increase its applicability

Inhibition of prolyl oligopeptidase : A promising pathway to prevent the progression of age-related macular degeneration

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.Peer reviewe

A power market-based operation support model for sub-daily hydropower regulation practices

Highlights • Investigate the impact of instant energy demand on sub-daily river regime. • Introducing power market impact index. • Introducing system efficiency ratio index. • Provides an efficient tool for sustainable river management. • Assess the interaction of power market and regulation practices.With increasing power production from renewable energy sources, sub-daily variations in energy demand need to be balanced. Today, hydropower is commonly used as balancing power. In this study, we quantified the impact of capacity constraints, in terms of reservoir volume and hydropower capacity, on the potential to comply with instant energy demand. To evaluate the impact, we developed two new metrics, power market impact and system efficiency ratio, which are based on two threshold flow regimes derived from natural flow as lower threshold release and regulated flow (based on hourly energy prices) as upper threshold release. The operation support model comprises 96 different regulation scenarios based on varying combinations of hydropower and reservoir capacities. For each scenario, an hourly water balance was simulated, to obtain the highest complying with upper threshold release based on actual energy demand. We tested the framework on the Kemijoki river with defined thresholds based on the natural flow regime (tributary river Ounasjoki) and the hourly energy price in Finland in 2017, and estimated the impact of regulation on hourly flow regime at the Taivalkoski hydropower station. The annual flow regime impact in 2013, 2014 and 2015 was estimated to be 74%, 84% and 61%, respectively, while the monthly impact varied from 27% to 100%. Our framework for evaluating interactions between the power market and sub-daily regulation practices is a useful novel tool for sustainable river management and can be easily applied to different rivers and regions and evaluated for different timescales

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38 MAPK

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR’s role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant

Expression of VEGFA-regulating miRNAs and mortality in wet AMD

MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age-related macular degeneration (AMD). Repeated injections with anti-VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti-VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR-34-5p, miR-126-3p, miR-145-5p and miR-205-5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti-VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.Peer reviewe

Drug Target Commons : A Community Effort to Build a Consensus Knowledge Base for Drug-Target Interactions

Knowledge of the full target space of bioactive substances, approved and investigational drugs as well as chemical probes, provides important insights into therapeutic potential and possible adverse effects. The existing compound-target bioactivity data resources are often incomparable due to non-standardized and heterogeneous assay types and variability in endpoint measurements. To extract higher value from the existing and future compound target-profiling data, we implemented an open-data web platform, named Drug Target Commons (DTC), which features tools for crowd-sourced compound-target bioactivity data annotation, standardization, curation, and intra-resource integration. We demonstrate the unique value of DTC with several examples related to both drug discovery and drug repurposing applications and invite researchers to join this community effort to increase the reuse and extension of compound bioactivity data.Peer reviewe

Epithelial-Mesenchymal Transition and Senescence in the Retinal Pigment Epithelium of NFE2L2/PGC-1 alpha Double Knock-Out Mice

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. In our previous studies, we found that deficiencies in the nuclear factor, erythroid 2 like 2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha) genes caused AMD-like pathological phenotypes in mice. In the present work, we show hijacked epithelial-mesenchymal transition (EMT) due to the common loss of PGC-1 alpha and NFE2L2 (double knock-out, dKO) genes in aged animals. The implanted area was assessed by histology, immunohistochemistry and transmission electron microscopy. Confocal microscopy revealed altered regions in the filamentous actin ring. This contrasted with hexagonal RPE morphology in wild-type mice. The ultrastructural RPE features here illustrated loss of apical microvilli, alteration of cell-cell contact, loss of basal in-folding with deposits on Bruch's membrane, and excessive lipofuscin deposition in dKO samples. We also found the expression of epithelial-mesenchymal transition transcription factors, such as Snail, Slug, collagen 1, vimentin and OB-cadherin, to be significantly different in dKO RPEs. An increased immunoreactivity of senescence markers p16, DEC1 and HMGB1 was also noted. These findings suggest that EMT and senescence pathways may intersect in the retinas of dKO mice. Both processes can be activated by damage to the RPE, which may be caused by increased oxidative stress resulting from the absence of NFE2L2 and PGC-1 alpha genes, important for antioxidant defense. This dKO model may provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1 alpha in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1 alpha dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1 alpha dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.Peer reviewe