Effect of rapamycin on the fate of P23H opsin associated with retinitis pigmentosa (an American Ophthalmological Society thesis).
OBJECTIVE
To determine the effect of rapamycin on the fate of misfolded opsin associated with retinitis pigmentosa.


METHODS
Stable cell lines separately expressing WT and P23H opsins and WT and DeltaF508 CFTR were used. Cells were incubated with complete media or amino acid-depleted medium or in the presence of rapamycin. At various time points thereafter, quantitative opsin and CFTR immunoblotting was performed. Immunofluorescence and electron microscopy were also performed to observe the expression and colocalization of autophagy specific marker proteins with opsin or CFTR.


RESULTS
Upon incubation with rapamycin, the levels of P23H opsin and DeltaF508 CFTR were reduced more rapidly than in untreated controls while no observable changes in the amounts of WT opsin was seen. The autophagy specific marker proteins, Atg7, Atg8 (LC3), and LAMP-1, which associate with autophagic vacuoles, colocalized with P23H opsin. A dramatic increase in the immunofluorescence signals of Atg7, LC3, and LAMP-1 was observed. All three of these proteins were found to decorate P23H opsin, suggesting that autophagy may be directly responsible for the clearance of this protein. Also, it was determined that neither the unfolded protein response nor the heat shock response was induced upon rapamycin-associated degradation of P23H opsin.


CONCLUSIONS
These data suggest that rapamycin induces the loss of P23H opsin and DeltaF508 CFTR from the cell under the experimental conditions described. Concomitantly, there is increased expression and colocalization of autophagy marker proteins with P23H opsin. Immunogold electron microscopic studies demonstrate autophagic vacuoles clustered in physical proximity to the aggregates of P23H opsin, suggesting that some of the loss of P23H is related to the induction of autophagy. Thus, rapamycin may be useful to clear misfolded proteins associated with retinal degeneration.
Kaushal
Shalesh
S
Departments of Ophthalmology and Anatomy and Cell Biology, University of Florida, Gainesville, Florida, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy; D000071193:Autophagy-Related Protein 7; D002460:Cell Line; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*; D014162:Transfection; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
D000071193:Autophagy-Related Protein 7; D015853:Cysteine Proteinase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D017299:Rod Opsins; D020123:Sirolimus; D044764:Ubiquitin-Activating Enzymes; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C477012:MAP1LC3A protein, human; C509349:ATG7 protein, human