lumacaftor and Stargardt-Disease

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

Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment.

Topics: Amino Acid Sequence; Aminopyridines; Anilides; ATP-Binding Cassette Transporters; Benzodioxoles; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Gene Expression; HEK293 Cells; Histone Deacetylase 6; Histone Deacetylases; HSP27 Heat-Shock Proteins; Humans; Hydroxamic Acids; Macrolides; Macular Degeneration; Molecular Sequence Data; Mutation; Protective Agents; Protein Transport; Proteolysis; Sequence Homology, Amino Acid; Signal Transduction; Stargardt Disease; Transgenes