Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration

Abstract

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.

Figure 1. Mutations affecting trafficking to the lysosomes result in light-dependent retinal degeneration.

Cross sections (1 µm) of retinas from white-eyed wild type, car, cm, and lt flies. Respective genotypes were dark-reared (A, C, E and G) or exposed to continuous room light for 7 days (B, D, F and H) prior to fixation. Retinal degeneration is only observed in light-exposed flies. Tissues were fixed and embedded as described in Materials and Methods. Scale bar, 20 µm.

Figure 2. The light-dependent retinal degeneration that is observed in car and lt flies is rescued by the removal of rhodopsin or by preventing its endocytosis.

Cross sections (1 µm) of retinas from (A) car;;Rh1Δ356 flies, (B) lt;Rh1Δ356 flies, (C) car flies raised on vitamin A-deficient media and (D) lt flies raised on vitamin A-deficient media. Flies were exposed to 7 days of constant room light. Eyes were fixed and embedded as described in Materials and Methods. Scale bar, 20 µm.

Figure 3. Light-induced endocytosis of rhodopsin and its presence in late endosomes.

(A) Indirect immunofluorescence of whole-mounted retinas stained for Actin, Rhodopsin (Rh1) and Rab7. Wild type control (w), norpA, car and lt flies subjected to 24 hours of continuous light treatment and the retinas were stained as described in Materials and Methods. Light-exposure results in endocytosis of Rh1 from the rhabdomere membrane into the cell body, where a majority of it co-localizes with Rab7. We hypothesize that the poor rab7 staining observed in the granule group mutants is due to rab7GDP being sequestered in a complex with GDI due to the lack of GDP-exchange activity. Scale bar, 5 µm. (B) Quantitation of Rh1-positive endocytic vesicles in white-eyed wild type control (WT), car, lt and norpA flies after treatment with constant light for 24 hours. Number of Rh1 puncta in the cell body were counted in a confocal section as described in Materials and Methods and divided by the total number of corresponding ommatidia ascertained by Actin staining. Rh1-positive vesicles for WT control, 7.5120±0.326; for car flies, 7.4990±0.299; for lt flies, 7.6450±0.311 and for norpA flies, 11.920±0.77 (n = 140–160 ommatidia). Data are represented as Mean±SEM. (C) Quantitation of Rh1-positive late endosomes in wild type (WT), car, lt, and norpA flies. Number of Rh1-Rab7 double positive puncta were counted in confocal sections as above and divided by the total number of Rh1-positive puncta to calculate the percentage of Rh1-positive vesicles that are Rab7-positive. Percentage of Rh1-positive vesicles that are Rab7-positive for WT controls, 68.59±0.01; for car flies, 75.39±0.02; for lt flies, 75.50±0.01; for norpA flies, 72.59±0.01 (n = 981–1606 Rh1-positive vesicles). Data are represented as Mean±SEM.

Figure 4. Rhodopsin accumulates in the late endosomes in norpA and granule group mutants.

Indirect immunofluorescence of whole-mounted retinas stained for Actin, Rh1 and Rab7. Wild type control (w), norpA, car and lt flies were subjected to 48 hours of constant room light-treatment followed by 13 hours in complete darkness. The retinas were dissected and stained as described in Materials and Methods. Endocytosed Rhodopsin is cleared from the cytoplasm of retinas in wild type flies. Rhodopsin persists in Rab7-positive vesicles in norpA, car and lt flies. Scale bar, 5 µm.

Figure 5. Rhodopsin is not degraded in light-treated norpA flies.

(A) Head lysates were prepared from white-eyed control flies exposed to light for indicated time period and subjected to Western and Slot-Blot analysis. For Western blots, head lysates were fractionated by SDS-PAGE and probed with antibodies against Rh1 and Arr2. The slot-blot analysis was carried out as described in Materials and Methods and blots were probed with antibodies directed against Rh1. The steady-state level of Arr2, another photoreceptor-specific protein, is used as a loading control. (B) Head lysates from norpA flies exposed to light were subjected to Western and slot-blot analysis. Western analysis reveals that the Rh1 protein level drastically decreases with increasing light exposure. Contrary to this observation, slot-blot analysis reveals that Rh1 persists in light-exposed norpA flies. The densitometry data are represented as Mean±SEM. Results here show data from three independent experiments.

Figure 6. A model for light-dependent retinal degeneration.

In wild type flies, Rh1 is endocytosed upon light-activation. The endocytosed Rh1 undergoes regular lysosomal turnover. In norpA flies, a large number of stable Rhodopsin-Arrestin complexes are formed which undergo rapid endocytosis. Massive endocytosis of Rhodopsin overwhelms the endocytic machinery and this rhodopsin fails to undergo degradation by the lysosomes and hence accumulates in the late endosomes. Endosomal accumulation of Rhodopsin triggers cell death by unknown mechanisms. This condition is simulated in granule group mutants that are defective in lysosomal delivery of endocytosed cargo. In these mutants, Rh1 internalization is similar to that in wild type but Rh1 accumulates in late endosomes as a result of defective trafficking. This accumulation of Rhodopsin also leads to photoreceptor cell death.