benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Macular-Degeneration

In age-related macular degeneration (AMD), hydroquinone (HQ)-induced oxidative damage in retinal pigment epithelium (RPE) is believed to be an early event contributing to dysregulation of inflammatory cytokines and vascular endothelial growth factor (VEGF) homeostasis. However, the roles of antioxidant mechanisms, such as autophagy and the ubiquitin-proteasome system, in modulating HQ-induced oxidative damage in RPE is not well-understood. This study utilized an in-vitro AMD model involving the incubation of human RPE cells (ARPE-19) with HQ. In comparison to hydrogen peroxide (H

Topics: Autophagy; Cathepsin D; Cells, Cultured; Humans; Hydroquinones; Leupeptins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Macular Degeneration; Mitochondria; Proteasome Endopeptidase Complex; Retinal Pigment Epithelium

Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued.. We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins.. G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold.. Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.

Topics: Aminopyridines; Anilides; ATP-Binding Cassette Transporters; Benzodioxoles; Cell Membrane; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression Regulation; HEK293 Cells; HSP27 Heat-Shock Proteins; Humans; Hydroxamic Acids; Leupeptins; Lysosomes; Macular Degeneration; Mutation; Protein Transport; Stargardt Disease

The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

Topics: Anaphylatoxins; Arginine; Benzhydryl Compounds; Cells, Cultured; Complement Activation; Complement C3a; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Humans; Leupeptins; Macular Degeneration; Matrix Metalloproteinase 2; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Retinal Pigment Epithelium