March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
The Hsp90 Inhibitor 17-AAG Prevents Aberrant Endocytosis Mediated By The Rhodopsin Retinitis Pigmentosa R135L Mutation
Author Affiliations & Notes
  • Monica Aguila
    Ocular Biology and Therapeutics, Institute of Ophthalmology, UCL, London, United Kingdom
    Departament d'Enginyeria Quimica, Centre de Biotecnologia Molecular, Universitat Politecnica de Catalunya, Terrassa, Spain
  • Caroline McCulley
    Ocular Biology and Therapeutics, Institute of Ophthalmology, UCL, London, United Kingdom
  • Nele Schwarz
    Ocular Biology and Therapeutics, Institute of Ophthalmology, UCL, London, United Kingdom
  • Pere Garriga
    Departament d'Enginyeria Quimica, Centre de Biotecnologia Molecular, Universitat Politecnica de Catalunya, Terrassa, Spain
  • Michael E. Cheetham
    Ocular Biology and Therapeutics, Institute of Ophthalmology, UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships  Monica Aguila, None; Caroline McCulley, None; Nele Schwarz, None; Pere Garriga, None; Michael E. Cheetham, None
  • Footnotes
    Support  The Wellcome Trust, RP Fighting Blindness and Fight for Sight
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1623. doi:
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      Monica Aguila, Caroline McCulley, Nele Schwarz, Pere Garriga, Michael E. Cheetham; The Hsp90 Inhibitor 17-AAG Prevents Aberrant Endocytosis Mediated By The Rhodopsin Retinitis Pigmentosa R135L Mutation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1623.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Mutations in rhodopsin are the most common cause of autosomal dominant Retinitis pigmentosa (RP) and over 200 mutations lead to RP. Amino acid substitutions at the highly conserved Arginine 135 residue (e.g. R135L) lead to a mutant rod opsin protein that is constitutively phosphorylated and binds to visual arrestin, leading to disturbances in endocytic pathways (Chuang et al., 2004). It has been previously shown that pharmacological intervention can improve folding and improve traffic, or reduce protein aggregation of a rod opsin misfolding mutant P23H. Here, we investigate the ability of these drugs to modulate R135L rod opsin phenotype.

Methods: : SK-N-SH neuroblastoma cells were transiently transfected with wild-type (WT)-GFP, P23H-GFP, R135L-GFP, Arrestin-FLAG or GRK1-FLAG. The transfected cells were treated with a variety drugs (9-cis-retinal, 4-PBA and 17-AAG) for 24 hours. Immunofluorescence was used to determine the incidence of opsin intracellular vesicles or inclusions. The processing and expression of opsin within the cell was analysed by immunofluorescence and western blotting.

Results: : R135L-GFP behaved as previously described, recruiting arrestin and disrupt endocytosis causing and accumulation of R135L positive intracellular vesicles. Furthermore, R135L acted as a dominant negative to recruit WT rod opsin to intracellular vesicles. Interestingly, pharmacological chaperones (9-cis-retinal) enhanced R135L mediated endocytosis. 4-PBA had no effect on R135L. In contrast, the Hsp90 inhibitor and molecular chaperone inducer, 17-AAG, reduced the intracellular accumulation of R135L and abolished arrestin binding. Importantly, this was due to a requirement for Hsp90 in rhodopsin kinase (GRK1) stability function.

Conclusions: : These data suggest that 17-AAG can protect against both class II and class III rod opsin mutants, but by different mechanisms. Therefore, Hsp90 inhibition could be used as a potential therapeutic target for different types of rhodopsin RP, although it may also affect visual function through the need for Hsp90 chaperone function by components of the phototransduction machinery.

Keywords: opsins • proteins encoded by disease genes • chaperones 
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