sirolimus and Retinitis-Pigmentosa

Understanding the mechanisms that contribute to secondary cone photoreceptor loss in retinitis pigmentosa (RP) is critical to devise strategies to prolong vision in this neurodegenerative disease. We previously showed that constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1), by loss of its negative regulator the tuberous sclerosis complex protein 1 (Tsc1; also known as Hamartin), was sufficient to promote robust survival of nutrient-stressed cones in two mouse models of RP by improving glucose uptake and utilization. However, while cone protection remained initially stable for several weeks, eventually cone loss resumed. Here we show that loss of Tsc1 in the cones of RP mice causes a defect in autophagy, leading to the accumulation of ubiquitinated aggregates. We demonstrate that this defect was not due to an inhibition of autophagy initiation, but due to an accumulation of autolysosomes, suggesting a defect in the end-stage of the process causing an amino-acid shortage in cones, thereby hampering long-term cone survival. Because cells with TSC loss fail to completely inhibit mTORC1 and properly activate autophagy in the absence of amino acids, we sporadically administered the mTORC1 inhibitor rapamycin, which was sufficient to correct the defects seen in cones, further enhancing the efficiency of cone survival mediated by Tsc1 loss. Concordantly, activation of mTORC1 by loss of the phosphatase and tensin homolog (Pten) did not affect autophagy and amino-acid metabolism, leading to a more sustained long-term protection of cones. As loss of Pten, which in cones results in less robust mTORC1 activation when compared with loss of Tsc1, still affords long-term cone survival, therapeutic interventions with mTORC1 activators or gene therapy with selected mTORC1 targets that improve glucose metabolism are potential strategies to delay vision loss in patients with RP.

Topics: Amino Acids; Animals; Autophagy; Cell Survival; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; PTEN Phosphohydrolase; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

To determine the effect of rapamycin on the fate of misfolded opsin associated with retinitis pigmentosa.. 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.. 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.. 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.

Topics: Amino Acid Sequence; Animals; Autophagy; Autophagy-Related Protein 7; Cell Line; Cricetinae; Cysteine Proteinase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique, Indirect; Humans; Immunoblotting; Kidney; Lysosomal Membrane Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Microtubule-Associated Proteins; Molecular Sequence Data; Protein Conformation; Protein Folding; Retinitis Pigmentosa; Rod Opsins; Sirolimus; Transfection; Ubiquitin-Activating Enzymes