Purchase this article with an account.
R. Malhotra, W.A. Dunn, S. Kaushal; Role of Autophagy in the Preferential Degradation of Misfolded P23H Opsin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1655.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study the role of either starvation–induced or rapamycin–induced autophagy in degradation of misfolded P23H opsin aggregates. Methods: HEK293 tetracycline–inducible stable cell lines separately expressing WT and P23H opsins were used. In control samples, opsin expression was induced and the cells were incubated in normal media. Autophagy was induced in cells expressing the WT or P23H opsin by incubating with either amino acid–depleted medium, or in the presence of rapamycin. Cell lysates were prepared at various time points thereafter and quantitative immunoblotting was performed. Immunofluorescence microscopy was also performed to observe the expression and co–localization of autophagy specific marker proteins with opsin. Results: After the addition of tetracycline, control cells expressing P23H opsin developed intracellular aggregates within 24 hrs, unlike WT cells. Upon induction of autophagy, the P23H opsin aggregates were degraded much faster than the untreated controls. However, no observable degradation of WT opsin was observed under autophagic conditions. The autophagy specific marker proteins, Atg7, LC3 and LAMP1, which associate with autophagic vacuoles, colocalized with the opsin aggregates. A dramatic increase in the immunofluorescence signals of Atg7, LC3 and LAMP1 was observed. All three of these proteins were found to decorate the opsin aggregates suggesting that autophagy may be directly responsible for the clearance of these aggregates. We also determined the levels of the ER and cytoplasmic chaperones, calnexin, calreticulin and Hsp70, which remained unchanged upon autophagy–induced degradation of P23H opsin. Conclusions: Using two separate methods of induction, our data suggests that macroautophagy preferentially assists the degradation and removal of P23H opsin. There is increased expression and co–localization of autophagy marker proteins with the P23H opsin, along with the rapid degradation of the misfolded protein under autophagy–induced conditions. This effect does not appear to be associated with either the ER stress response or the cytoplasmic heat shock response. The therapeutic implications of this work will be discussed.
This PDF is available to Subscribers Only