Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration

Abstract

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Figure 1

Light micrographs of wild-type and P23H mouse retinas at postnatal day (P) 8, P10, and P12. Degeneration of photoreceptor morphology in Rho^P23H/P23H mice begins by postnatal day 10. (A–C) At P8, the ROS have not formed, and the thicknesses of the rod inner segment (IS) layer and outer nuclear layer (ONL) are identical between Rho^+/+, Rho^P23H/+ and Rho^P23H/P23H mice. (D–F) Also in P10, nascent ROS can be identified in Rho^+/+ and Rho^P23H/+ mice but are absent in Rho^P23H/P23H mice. By P10, the rod IS layer is thinner in Rho^P23H/P23H retinas compared to those in Rho^+/+ and Rho^P23H/+ mice. By P10, pyknotic nuclei (white arrows) are identified in Rho^P23H/P23H mice at a significantly greater incidence than in Rho^+/+ or Rho^P23H/+ mice. (G–I) By P12, ROS are present in Rho^+/+ and Rho^P23H/+ mice but are absent in Rho^P23H/P23H mice. The rod IS layer and ONL are appreciably thinner in Rho^P23H/P23H mice compared to Rho^+/+ and Rho^P23H/+ mice, and many pyknotic nuclei (white arrows) are identified in the inner aspect of the ONL in Rho^P23H/P23H mice. Scale bar: 20 μM

Figure 2

Light micrographs of wild-type and P23H mouse retinas at P15, P30, and P60. Degeneration of photoreceptor morphology in Rho^P23H/+ mice begins by P15. (A–C) At P15, the rod OS are shorter and more disorganized in Rho^P23H/+ mice (B) compared to Rho^+/+ mice (A), and only short fragments of ROS disc membranes are present in Rho^P23H/P23H mice (C). At P15, the rod inner segments (IS) are shorter in Rho^P23H/+ mice compared to Rho^+/+ and significantly shortened in Rho^P23H/P23H mice. Pyknotic nuclei (white arrows) are evident in Rho^P23H/+ mice. The ONL is significantly thinner in Rho^P23H/P23H mice (C). (D–F) By P30, the OS, IS, and ONL are significantly thinner in Rho^P23H/+ mice (E) compared to Rho^+/+ mice (D). Extensive photoreceptor cell loss is present in Rho^P23H/P23H mice, with less than 1 full row of nuclei present in the ONL (F). (G–I) By P60, the OS, IS, and ONL have progressively thinned in Rho^P23H/+ mice (H) compared to Rho^+/+ mice (G), and even further loss of photoreceptors is evident in the ONL of Rho^P23H/P23H mice (I). Scale bar: 20 μM

Figure 3

Transmission electron microscopy images of photoreceptor cell inner segments at postnatal day 14. (AC) Mitochondria (Mi), rough endoplasmic reticulum (rER), Golgi apparatus (G) and ribosomes (r) were abundant in the inner segment. Golgi apparatus was observed at the proximal part of most inner segments. (A) Image of longitudinal-cut inner segments from Rho^+/+ mouse retinas. Column shaped inner segments were observed between photoreceptor cell outer segments (OS) and outer limiting membranes (OLM). In rare cases, the entire cone photoreceptor outer segment (COS) and inner segment (CIS) of a single cell was observed. (B) Image of tilted-cut inner segments from Rho^P23H/+ mouse retinas. 2–4 rows of oval or column shaped inner segments were observed between OS and OLM. The structure of the rER and Golgi apparatus in the inner segments of Rho^P23H/+ mice was almost identical to Rho^+/+ mouse retinas especially compared to tilted-cut inner segment images from Rho^+/+ mouse retinas. (C) Image of slightly tilted-cut inner segments from Rho^P23H/P23H mouse retinas. 1–3 rows of oval or column shaped inner segments were observed between retinal pigment epithelium microvilli (RM) and OLM. Ciliary protrusions (CP) and cone outer segments (COS) were observed in the space between RM and the distal ends of inner segments. Not all, but some inner segments showed multiple vacuolization (white arrow). Such vacuolization was observed in all four analyzed Rho^P23H/P23H mice, but not in TEM images of Rho^P23H/+ and Rho^+/+ mice (four mice were analyzed for each genotype). Hypothetical diagram of photoreceptor cells are included, indicating the cutting planes with dashed lines. Scale bar, 2 μm.

Figure 4

Anatomic distribution of retinal degeneration in Rho^+/+, Rho^P23H/+ and Rho^P23H/P23H mice. (A) Mean ONL thickness of Rho^+/+, Rho^P23H/+ and Rho^P23H/P23H retinas. Each value is the mean ± SEM of 5–7 retinas, one from each mouse; the mean for each retina was determined from 54 measurements around the eye from inferior to superior poles. If error bars are not shown, they fall within the symbol. (B) Retinal “spidergram” showing ONL thickness from the optic nerve head (ONH) to the anterior optic margin (ora serrata) in the inferior and superior hemispheres of Rho^+/+ retinas and Rho^P23H/+ retinas at P12, P20, P30, P60, and P120. The top border of the shaded area represents the Rho^+/+ ONL thickness at P12, and the bottom border shows the Rho^+/+ ONL thickness at P120. Thinning of the retina ONL in Rho^+/+ mice between P12 and P120 is due to growth of the eye, not retinal degeneration. The ONL thickness progressively diminishes in Rho^P23H/+ from P20 to P120 with more pronounced retinal degeneration in the inferior hemisphere. (C) Retinal spidergram showing a rapid decrease in ONL thickness in Rho^P23H/P23H retinas from P12 to P60. In Rho^P23H/P23H retinas, the superior hemisphere shows greater loss of the ONL, the opposite of that seen in the Rho^P23H/+ retinas.

Figure 5

The IRE1 signaling pathway of the UPR is strongly activated in photoreceptors of Rho^P23H/+ERAI^+/− mice. (A–H) Rhodopsin (red) and XBP1-Venus (by anti-GFP, green) were visualized in eyes from Rho^P23H/+ERAI^+/− mice and Rho^+/+ERAI^+/− mice at P30. DAPI (blue) highlights photoreceptor nuclei. Strong XBP1-Venus staining is visualized in the IS and perinuclear region of ONL photoreceptors expressing P23H rhodopsin. (I–L) Peanut agglutinin (PNA) staining of cones (red) and XBP1-Venus (by anti-GFP, green) were visualized in eyes from Rho^P23H/+ERAI^+/− mice and Rho^+/+ERAI^+/− mice at P120. DAPI (blue) highlights photoreceptor nuclei. White arrow in (L) indicated the cone cell overlapping with XBP1-Venus signal. (M) XBP1-Venus (by anti-flag) protein levels were detected in retinal lysates from Rho^P23H/+ERAI^+/− mice and littermate control Rho^+/+ERAI^+/− mice at P30. HSP90 served as a protein loading control. (N) XBP1-Venus protein levels were detected by anti-flag or anti-GFP in retinal lysates from Rho^P23H/+ERAI^+/− mice at indicated ages.

Figure 6

The IRE1 signaling pathway of the UPR is strongly activated in photoreceptors expressing P23H rhodopsin. (A–E) Xbp-1s mRNA level (A) and XBP1s transcriptional target genes, Erdj4 (B), VCP (C), Derl1 (D), and Grp78/BiP (E), were measured in Rho^+/+, Rho^P23H/+, and Rho^P23H/P23H mice at P30 by quantitative PCR. (F–G) Upregulation of Erdj4 was measured in Rho^+/+, Rho^P23H/+, and Rho^P23H/P23H mice at P60 (F) or P90 (G) by quantitative PCR. (H) Retinas from three Rho^+/+, Rho^P23H/+ or Rho^P23H/P23H mice were collected at P15, pooled, and lysed. 40 μg of total retinal lysates were loaded onto the SDS-PAGE. Phosphorylated JNK protein levels were detected by immunoblotting. Total JNK protein levels served as a loading control.

Figure 7

P23H rhodopsin protein co-immunoprecipitates with ERAD components and is strongly ubiquitinated in photoreceptor cells. (A) ERAD-related proteins that co-immunoprecipitated with P23H rhodopsin were identified by mass spectrometry. Total spectra counts of the proteins co-immunoprecipitated with WT or P23H rhodopsin from three experimental mass spectrometry analyses are shown. Immunoprecipitation of MEF cell samples served as experimental control. (B) Rhodopsin was immunoprecipitated from retinal lysates of Rho^+/+ and Rho^P23H/P23H mice at P15. The elutions were concentrated by chloroform/methanol precipitation. Rhodopsin, VCP, and calnexin proteins were detected by immunoblotting. (C) Rhodopsin was immunoprecipitated from retinas of Rho^+/+, Rho^P23H/+ and Rho^P23H/P23H mice at P15, and rhodopsin (top panel) and ubiquitin (bottom panel) levels were detected by immunoblotting.

Figure 8

Rhodopsin protein, but not mRNA, levels are significantly diminished in P23H mice. (A) Retinal protein lysates were collected from Rho^+/+, Rho^P23H/+, and Rho^P23H/P23H mice at P15. Rhodopsin was detected by immunoblotting. GAPDH protein levels served as a loading control (B) Rhodopsin mRNA levels from Rho^+/+, Rho^P23H/+, and Rho^P23H/P23H mice at P12 were measured by quantitative PCR. Filled circles represent rhodopsin mRNA levels in individual mouse. Horizontal red bars represent mean rhodopsin mRNA levels from each experimental cohort of mice. NS: statistically not significant. (C) Retinas from Rho^+/+ and Rho^P23H/P23H mice at P10 were sectioned and rhodopsin (red) and calnexin (green) were visualized by confocal microscopy. DAPI counterstain highlighted nuclei. Under equivalent confocal microscopy detection settings, strong rhodopsin protein staining was evident in the developing OS of Rho^+/+ but only a scant amount of rhodopsin protein staining was visualized in the presumptive OS layer in Rho^P23H/P23H photoreceptors. Rhodopsin protein did not colocalize with calnexin in either Rho^+/+ or Rho^P23H/P23H photoreceptors. (D) Retinal protein lysates were collected from Rho^+/+, Rho^P23H/+, and Rho^P23H/P23H mice at P5. Rhodopsin and GNAT1 were detected by immunoblotting. HSP90 protein levels served as a loading control. (E) P23H rhodopsin was expressed in HEK293 cells. The cells were treated with 10 μM kifunensine for 20 hours. Cells were also treated with bortezomib 10 μM as a control for proteasomal-mediated degradation (F) HEK293 cells expressing P23H rhodopsin were treated with 1.25, 2.5, 3.75, 5, or 7.5 μM of eeyarestatin I for 20 hours (Lane 2 to 6). (E and F) Rhodopsin protein was detected by immunoblotting. GAPDH protein levels served as a loading control. Significant cell toxicity was observed with some drug treatment conditions (indicated with *).

Figure 9

Chop is not induced in retinas of P23H rhodopsin mice, and loss of Chop does not affect P23H-induced retinal degeneration. (A–B) Chop mRNA levels were quantified in the retinas of Rho^P23H/+ and Rho^P23H/P23H mice and are shown relative to levels in age-matched wild-type mice at indicated ages. (C) PERK downstream targets, phospho-eIF2α and ATF4, were analyzed by immunoblotting. Retinas from three Rho^+/+, Rho^P23H/+ or Rho^P23H/P23H mice were collected at P15, pooled, and lysed. MEF cells were treated with thapsigargin (500 nM) for indicated times. Then, 10 μg of MEF total lysates or 40 μg of total retinal lysates were loaded onto the SDS-PAGE. HSP90 protein levels served as a loading control. (D) Mean ONL thicknesses of wild-type Rho^+/+Chop^+/+, Rho^P23H/+Chop^+/+, Rho^+/+Chop^−/−, Rho^P23H/+Chop^+/−, and Rho^P23H/+Chop^−/− from perfused enucleations were quantified at P95. Error bars represent SDs based on measurements from at least three mice of each genotype. For Rho^P23H/+Chop^−/−, only two mice were analyzed.