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Valeria Marigo, Antonella Comitato, Clara La Marca, Preeti Subramanian, Angelo Felline, Francesca Fanelli, S Patricia Becerra; Novel therapeutic approaches for Retinitis Pigmentosa caused by mutations in Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4552.
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© ARVO (1962-2015); The Authors (2016-present)
About 140 point mutations have been identified in the rhodopsin gene (RHO) that cause autosomal dominant Retinitis Pigmentosa (adRP), a genetic degenerative disease causing blindness in later life. 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. We characterized cell death pathways activated by mutations in RHO. The high heterogeneity of the mutations calls for targeting its common cell death mechanisms. Given that therapies for adRP are still not available, approaches to develop novel therapies are explored here.
Wild type RHO and 33 RHO mutants were analyzed in silico by the Protein Structure Network (PSN) approach and in vitro in COS-7 cells by analyses of plasma membrane localization using an antibody (RetP1) without permeabilization. Eight structures of the retinal binding site, central in the structural stability as highlighted by the PSN analysis, were targeted by virtual screening of compound libraries. In vivo treatment of RhoP23H/+ mice by intravitreal injection of the Pigment Epithelium-derived Factor (PEDF) protein was followed by western blotting and immunofluorescence analyses of cell death pathways.
First, targeting mutations causing protein misfolding was based on a new screening paradigm starting in silico and followed by in vitro validation. In silico screening led to a selection of 250 hit compounds. The pharmacological chaperone potential of the commercially available compounds from such list was assessed in vitro analyzing plasma membrane localization. Compounds showing chaperone activities for different misfolded and mislocalized mutants will be presented. Second, targeting common molecular mechanisms using short-term treatments with PEDF of the RhoP23H/+ mutant retina protected 40% of photoreceptors from cell death. PEDF was found to reduce activation of calpains and of the apoptosis inducing factor (Aif) involved in photoreceptor cell death.
Two different approaches to develop novel therapies for adRP caused by mutations in RHO are investigated:1) pharmacological chaperone screening, and 2) targeting common cell death mechanisms using the neurotrophic factor PEDF.Funding from Fondazione Roma (call for proposal 2013 sulla Retinite Pigmentosa), Italian Telethon Foundation GGP14180 and the Intramural Research Program of the NEI-NIH
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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