Genetic deletion of S-opsin prevents rapid cone degeneration in a mouse model of Leber congenital amaurosis

Abstract

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associated proteins including cone opsins (M- and S-opsins), cone transducin (Gα_t2), G-protein-coupled receptor kinase 1 (GRK1) and guanylate cyclase 1 (GC1) has been suggested to play a role in cone degeneration. However, their precise role in cone degeneration is unclear. Here we investigated the role of S-opsin (Opn1sw) in cone degeneration in Lrat^−/−, a murine model for LCA, by genetic ablation of S-opsin. We show that deletion of just one allele of S-opsin from Lrat^−/− mice is sufficient to prevent the rapid cone degeneration for at least 1 month. Deletion of both alleles of S-opsin prevents cone degeneration for an extended period (at least 12 months). This genetic prevention is accompanied by a reduction of endoplasmic reticulum (ER) stress in Lrat^−/− photoreceptors. Despite cone survival in Opn1sw^−/−Lrat^−/− mice, cone membrane-associated proteins (e.g. Gαt2, GRK1 and GC1) continue to have trafficking problems. Our results suggest that cone opsins are the ‘culprit’ linking 11-cis-retinal deficiency to cone degeneration in LCA. This result has important implications for the current gene therapy strategy that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.

Introduction

Retinoid isomerase (RPE65) and lecithin-retinol acyltransferase (LRAT) are two key enzymes involved in the generation of 11-cis-retinal in the retinal pigment epithelium (RPE). Mutations in either gene lead to Leber congenital amaurosis (LCA), a severe childhood blindness. Three mouse models (Rpe65^−/−, rd12 and Lrat^−/−) and a dog model have been widely used to study the disease mechanism and treatment strategy (1–6), which paved the way for the first successfully treated inherited retinopathy using gene augmentation therapy (7–9). A subsequent 3-year follow-up study found that gene therapy significantly improves vision, but does not slow down the rate of photoreceptor degeneration (10). This study shows the need for combinatorial therapy to both improve vision and slow photoreceptor degeneration. Thus, it is important to understand the mechanism of photoreceptor degeneration in LCA patients to design improved therapy.

In both LCA patients and animal models, both rod and cone function are severely compromised due to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. Early loss of foveal cones was reported in RPE65-deficient patients (11,12). S-cone function is lost earlier than the L/M-cone function (11,13,14). In mouse models, apo-rhodopsin is transported normally to the rod outer segments (ROS) and the rod photoreceptors degenerate slowly (>10 months). In contrast, the cone opsins (S-opsin and M-opsin) fail to traffic from the cone inner segment (CIS) to the cone outer segment (COS) properly, and the cone photoreceptors in the central/ventral regions degenerate rapidly (<4 weeks) (3,15–17). Concomitantly, cone membrane-associated proteins, e.g. cone transducin α-subunit (Gα_t2), cone phosphodiesterase 6α′ (PDE6α′), guanylate cyclase 1 (GC1), G-protein-coupled receptor kinase 1 (GRK1), are not transported to the outer segment properly and are degraded (17). Our previous studies showed that, in the absence of 11-cis-retinal, the short-wavelength (S) opsins are more prone than the medium- and long-wavelength (M and L) opsins to aggregation, which triggers endoplasmic reticulum (ER) stress (18–20). Thus, S-opsin is likely playing an important role in LCA cone degeneration. Because many membrane-associated proteins (e.g. GC1) in cones are also mislocalized and degraded, and could potentially affect cone cell survival, it is important to determine the precise role of S-opsin in cone degeneration in LCA. We have addressed this question by genetically deleting either one or both alleles of S-opsin from Lrat^−/− mice and assessing cone viability in Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice.

Results

Genetic deletion of either one or both alleles of S-opsin from Lrat^−/− mice prevents rapid cone degeneration

To investigate the role of S-opsin in cone degeneration in Lrat^−/− mice, we bred Lrat^−/− with Opn1sw^−/− mice (21) to generate Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice. At 1 month of age, nearly all cones in the ventral and central regions of Lrat^−/− mice were lost and 39% dorsal cones were dead, as shown previously (3,17) (Fig. 1A–C, Supplementary Material, Fig. S1). Deletion of both alleles of S-opsin completely prevented the rapid ventral/central cone degeneration. There was no significant difference between Opn1sw^−/−Lrat^−/− and WT mice in cone viability (Fig. 1C). Deletion of S-opsin also prevented the slower dorsal cone degeneration in the Opn1sw^−/−Lrat^−/− mice, suggesting that S-opsin is responsible not only for the rapid ventral/central cone degeneration, but also for the slower dorsal cone degeneration in the Lrat^−/− mice at 1 month. Surprisingly, deletion of only one copy of S-opsin gene prevented cone degeneration in all retinal regions of Opn1sw^+/−Lrat^−/− mice, similar to that in the double knockout mice Opn1sw^−/−Lrat^−/−. Western blot results confirmed that the protein level of S-opsin was markedly reduced in Opn1sw^+/−Lrat^−/− mice compared with WT at 1 month (Fig. 1D). The protein level of M-opsin was drastically reduced in both Opn1sw^−/−Lrat^−/− and Opn1sw^+/−Lrat^−/− cones, which is consistent with previous studies showing that M-opsin was degraded in the absence of 11-cis-retinal (18,22). The absence of S-opsin and the drastic reduction of M-opsin in Opn1sw^−/−Lrat^−/− cones support the previous observation that, unlike rods, cones can survive with little or no cone opsins (i.e. ventral cones in Opn1sw^−/−) (21). The protein levels of both M and S opsins in Lrat^−/− mice were drastically reduced due to the loss of cone photoreceptors.

Figure 1.

Genetic removal of either one or both copies of S-opsin from Lrat^−/− mice prevents the rapid cone degeneration at 1 month. (A) Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT central retina were stained with antibodies against S-opsin, M-opsin and cone arrestin (in green). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in top two rows (blue). In Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− retina, mistrafficking of M-opsin is indicated by white arrows. COS, cone outer segment; CIS, cone inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. (B) Cone viability in different retinal regions. Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT retina were stained with M-opsin antibody (in green). White arrows indicate labeled cones. D, dorsal; V, ventral. (C) Cone photoreceptors were counted in the ventral, central and dorsal sections of different genotypes (N = 3). Cones were visualized by cone arrestin antibody labeling. Data (mean ± SEM) were normalized to WT levels of the respective region. **P < 0.01, ***P < 0.001, NS, not significant. (D) Western blot analysis of S/M-opsins in the retinas of each genotypes. The bottom band of the middle panel is due to the non-specific cross reactivity of the M-opsin antibody since the intensity of the band does not change despite advanced cone degeneration in 1-month-old Lrat^−/− (compare WT versus Lrat^−/−). β-actin was included as the internal loading control. Scale bar, 20 µm in (A) and 200 µm in (B).

Ultrastructure of Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− cones

Since S-opsin is a major constituent of the COS in the central and ventral retina, deletion of S-opsin is likely to affect cone morphology (21). We examined the ultrastructure of cones from 1-month-old Lrat^−/−, Opn1sw^−/−Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/− and WT mice. In Lrat^−/− mice, it is difficult to find intact cones in the central and ventral retina. Occasionally, we can find a few remaining cones with severely degenerating COS (red circle, Fig. 2Af). Compared with WT, the overall outer segment morphology of Opn1sw^−/−Lrat^−/− mice is similar to that of Opn1sw^−/− with reduced COS size and various degrees of disorganization (Fig. 2Ah and i). Opn1sw^+/−Lrat^−/− cones have larger COS and better morphology than Opn1sw^−/−Lrat^−/− cones, probably due to the presence of one allele of S-opsin (Fig. 2Ag). In the dorsal retina where M-opsin expression dominates, cones from all four genotypes (Lrat^−/−, Opn1sw^−/−Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−) have relatively normal structure compared with WT (Fig. 2A, panels a–e). Other than the COS, the other cone compartments (i.e. inner segment, cell body, axon and synaptic pedicle) of Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice are normal compared with those in WT as revealed by immunohistochemistry (Fig. 1A). On semi-thin plastic sections, rods from Lrat^−/− background (Lrat^−/−, Opn1sw^−/−Lrat^−/− and Opn1sw^+/−Lrat^−/− mice) have shorter ROS compared with those of Opn1sw^−/− and WT (Fig. 2B), consistent with a slow rod degeneration caused by Lrat^−/− (2,3).

Figure 2.

Cone ultrastructure (A) and retinal histology (B) of 1-month Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/−, Opn1sw^−/− and WT mice. In (A) both dorsal (a–e) and ventral (f–j) cones were examined. The red circle in f indicates the COS of a severely degenerating cone cell in the ventral retinal of Lrat^−/− mice. In (B) 1 µm plastic sections were stained with Richardson's stain. ROS, rod outer segment; RIS, rod inner segment; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar, 1 µm in (A) and 20 µm in (B).

Deletion of S-opsin from Lrat^−/− mice prevents cone degeneration for an extended time

To address the question of whether the removal of S-opsin from Lrat^−/− mice protects cones from degeneration for a longer period, we labeled cones from 6- and 12-month Lrat^−/−, Opn1sw^−/−Lrat^−/−, Opn1sw^+/−Lrat^−/− and WT mice with an antibody against the cone cell marker, cone arrestin. We found that the protective effect is most effective in the central and ventral cones where the expression of S-opsin dominates. There is no significant difference between Opn1sw^−/−Lrat^−/− mice and WT controls in the number of viable cones in these regions, whereas virtually no cones were left in the Lrat^−/− mice at both ages (Fig. 3A–C, Supplementary Material, Fig. S2). In the dorsal region, 32 and 68% Opn1sw^−/−Lrat^−/− cones degenerated compared with 89 and 93% Lrat^−/− cone loss at 6 and 12 months (P < 0.001 and P < 0.01), respectively. The preventive effect is much stronger in the central and ventral retina than in the dorsal retina of Opn1sw^−/−Lrat^−/− mice, probably due to continuous degradation of the remaining M-opsin, which dominates in the dorsal retina, causes proteasome overload (22,23).

Figure 3.

Deletion of S-opsin from Lrat^−/− mice prevents cone degeneration at 6- and 12-month of age. Cone photoreceptors from either 6-month (A) or 12-month (B) Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT were counted in the ventral, central and dorsal retinal sections. Data (mean ± SEM) were normalized to WT levels of the respective region. N = 7 for Lrat^−/−; N = 4 for Opn1sw^+/−Lrat^−/−; N = 5 for Opn1sw^−/−Lrat^−/−; N = 5 for WT control in (A). N = 3 for all genotypes in (B). **P < 0.01, ***P < 0.001, NS, not significant. (C) Representative retinal sections from 12-month WT and Opn1sw^−/−Lrat^−/− mice were stained with cone arrestin antibody (in green). Nuclei were stained with DAPI (blue). White arrows indicate dorsal Opn1sw^−/−Lrat^−/− cones with short and swollen inner and outer segment. Scale bar = 20 µm.

Although deletion of one copy of S-opsin can prevent cone degeneration at 1 month, it is insufficient to protect cones at 6 and 12 months. Most cones degenerate in the central and ventral retina of Opn1sw^+/−Lrat^−/− mice, which is similar to what occurred in Lrat^−/− cones (P > 0.05). However, there are 4.3 and 5.2 times more cones surviving at the dorsal retinal in Opn1sw^+/−Lrat^−/− compared with Lrat^−/− mice at 6 and 12 months (P < 0.001 and P < 0.01), respectively, suggesting that significant reduction of S-opsin (one allele deletion plus reduced expression in dorsal cones) offers some long-term protection of even predominantly M-opsin expressing cones.

Cones of aged Opn1sw^−/−Lrat^−/− mice (i.e. 12 months) have two distinctive features. Firstly, central and ventral cones are morphologically healthier than dorsal cones, which showed very short and swollen outer and inner segments (Fig. 3C, white arrows). This does not occur in 6-month-old Opn1sw^−/−Lrat^−/− mice, suggesting that, in the absence of S-opsin, dorsal cone degeneration progresses slowly (Supplementary Material, Fig. S2A). Dorsal Opn1sw^−/−Lrat^−/− cone degeneration is likely caused by M-opsin (see Discussion). Secondly, Opn1sw^−/−Lrat^−/− cones in the central and ventral retina have shorter outer segments than WT cones (Fig. 3C) due to the loss of S-opsin (see Fig. 2A). Dorsal cones in 12-month Opn1sw^+/−Lrat^−/− mice are morphologically similar to Opn1sw^−/−Lrat^−/− cones (Supplementary Material, Fig. S2). Among the four genotypes, dorsal cones in Lrat^−/− mice have the most abnormal morphology. Most remaining cones have disrupted structures. Indeed, some cones have no outer segment (Supplementary Material, Fig. S2, red arrow) while others have abnormal cell bodies (Supplementary Material, Fig. S2, white arrow).

Genetic removal of either one or both alleles of S-opsin from Lrat^−/− mice reduces ER stress

We have shown previously that the central and ventral Lrat^−/− retina have increased ER stress, which is caused by the accumulation and aggregation of S-opsin (18,19,24). Indeed, marked CHOP (C/EBP homology protein, a B-ZIP transcription factor and an ER stress marker associated with apoptosis (25)) activation was observed in the ONL of the ventral retina of P18 (an early stage of cone degeneration) Lrat^−/− mice (Fig. 4). In sharp contrast, there was no CHOP activation in Opn1sw^−/−Lrat^−/− retina (which is similar to that of P18 WT retina) and only low CHOP activation was detected in the ONL of Opn1sw^+/−Lrat^−/− retina (Fig. 4). This result suggests that the accumulated misfolded S-opsin is responsible for the ER stress in ventral and central cones of Lrat^−/− mice, and eventually leads to cone degeneration.

Figure 4.

Deletion of S-opsin from Lrat^−/− mice reduces ER stress. The ventral or central retinal sections of P18 Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT mice were labeled with a CHOP antibody (red) and DAPI (blue). White arrows indicate CHOP signals in the ONL of Lrat^−/− and Opn1sw^+/−Lrat^−/− mice. Red signals in the OPL were due to the labeling of retinal vessels by the Cy3-conjugated goat anti-mouse secondary antibody. Scale bar = 20 µm.

Ubiquitination of cone opsins in cones of Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice

Ubiquitination of proteins that aggregate in the neurons of the central nervous system is a key characteristic of neurodegenerative diseases (26). We have shown previously that S-opsin ubiquitination may contribute to the death of Lrat^−/− ventral/central cones (18). At 1 month, all S-opsin co-localized with ubiquitin in the remaining cones of Lrat^−/− mice (Fig. 5a, e and i, white arrows). In contrast, the ubiquitin signal was either absent (Fig. 5, comparing red arrows in f & j with b) or reduced (Fig. 5b, blue arrows) in cones of Opn1sw^+/−Lrat^−/− mice. In Opn1sw^−/−Lrat^−/− cones with only M-opsin expression, the ubiquitin signal was either absent (Fig. 5, comparing red arrows in g and k with c) or very weak (Fig. 5c, yellow arrows). The ubiquitin signal was barely detectable in WT retina where both M and S opsins were folded correctly and were targeted to the COS. Collectively, cones expressing more S-opsin have a higher level of ubiquitin signal and a faster rate of degeneration in the Lrat^−/− background (i.e. Lrat^−/− > Opn1sw^+/−Lrat^−/− > Opn1sw^−/−Lrat^−/−), suggesting that, (1) most of the ubiquitin signal is associated with S-opsin; (2) ubiquitination of misfolded S-opsin may be an important factor in the pathological process of LCA.

Figure 5.

Ubiquitination of cone opsins in Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT mice. One month mouse retinal sections were double-labeled with antibodies against S-opsin (e, f and h) or M-opsin (g) and ubiquitin (a–d). Merged pictures were shown at the bottom row (i–l). Nuclei were stained with DAPI (blue). Scale bar = 20 µm.

Mistargeting of membrane-associated proteins in Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− cones

Several membrane-associated proteins (e.g. M/S opsins, GRK1, Gα_t2, GC1) involved in cone phototransduction fail to traffic to the outer segment of Lrat^−/− or Rpe65^−/− cones properly (17). The mistrafficked proteins are degraded through a posttranslational mechanism (17), except that S-opsin aggregates and resists proteasome degradation (18). Although all cones survived in 1-month Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice, M-opsin was mislocalized in the inner regions of cones (e.g. inner segment, ONL, synaptic pedicle) (Fig. 1A, white arrows). The signal of mislocalized M-opsin was weak due to protein degradation (Fig. 1D, middle row). Another transmembrane protein, GC1, was abundantly expressed in WT COS (Fig. 6l, white arrows), but markedly reduced in Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− cones (Fig. 6j and k, white arrows). We also examined two peripheral membrane-associated proteins GRK1 and Gα_t2 in Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− COS. We were not able to detect GRK1 (Fig. 6b and c) while the signal of Gα_t2 was markedly reduced (Fig. 6f and g, white arrows) compared with the robust signals in WT COS for both proteins (Fig. 6d and h). The signals for GRK1, Gα_t2 and GC1 were reduced in Lrat^−/− retina compared with either Opn1sw^−/−Lrat^−/− or Opn1sw^+/−Lrat^−/− cones due to the significant loss of cones in addition to protein degradation (Fig. 6a, e and i). These results suggest that mislocalization and degradation of many cone membrane-associated proteins other than cone opsins do not play a significant role in cone viability (see Discussion).

Figure 6.

Immunolocalization of cone membrane-associated proteins, GRK1 (a–d), Gα_t2 (e–h), and GC1 (i–l), in Lrat^−/−, Opn1sw^+/−Lrat^−/−, Opn1sw^−/−Lrat^−/− and WT mice. One month mouse retinal sections were stained with antibodies against GRK1, Gα_t2, and GC1. GRK1 signals were only detected in WT COS (d, white arrows). Both Gα_t2 and GC1 signals were greatly reduced in Lrat^−/−, Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− cones compared with that in WT cones (e–l, white arrows). Nuclei were stained with DAPI (blue). Scale bar = 20 µm.

Discussion

The main finding of this work is that S-opsin plays a major pathological role in the rapid cone degeneration in the LCA mouse model Lrat^−/−. After we genetically deleted both alleles of S-opsin (i.e. in Opn1sw^−/−Lrat^−/−), both ventral and central cones (where S-opsin expression dominates) survived for at least 12 months, which is in sharp contrast to the rapid cone degeneration (<1 month) in the presence of S-opsin (i.e. in Lrat^−/− 3,17). Another interesting finding is that deletion of one allele of S-opsin is sufficient to prevent cones from degenerating at 1 month, but insufficient at 6 and 12 months. This dosage dependent ‘rescue’ of cones provides additional support for the critical role of S-opsin in cone degeneration.

We have shown previously that a Phe-rich region in S-opsins, but not in M/L-opsins, is responsible for the aggregation-prone property of S-opsins in the absence of 11-cis-retinal (18,19). Aggregated S-opsin is resistant to the ER-associated protein degradation pathway (ERAD) and triggers endoplasmic reticulum (ER) stress and apoptosis. Deletion of both alleles of S-opsin relieves cone cells from this toxic buildup and reduces ER stress (Fig. 4), which completely prevents S-opsin enriched cones from degeneration. On the other hand, deletion of one allele of S-opsin provides partial relief of cone stress. It is interesting that the removal of S-opsin leads to a dramatic reversal of the region-dependent cone degeneration pattern at 6 and 12 months (i.e. dorsal cones degenerated faster than ventral and central cones in Opn1sw^−/−Lrat^−/− mice) (Fig. 3A–C and Supplementary Material, Fig. S2). The degeneration of dorsal Opn1sw^−/−Lrat^−/− cones is likely due to proteasome overload caused by the continuous degradation of the remaining M-opsin (23), which dominates in the dorsal retina (27). In other words, in the absence of the ‘bad’ aggregation-prone S-opsin, M-opsin takes the lead role in determining cone viability in Lrat^−/− mice. Based on the work presented here and in our previous study (18,19), we proposed a model to explain the different roles of S-opsin and M/L-opsins in the degeneration of cone photoreceptors in LCA due to RPE65 and LRAT mutations (Fig. 7). This model explains the region-dependent cone degeneration pattern in the retina of LCA mouse models (3,15–17). It also explains why S-cone function is lost earlier than the L/M-cone function in LCA patients (11,13,14). Moreover, this model may represent a general mechanism for the faster cone degeneration in the ventral and central retina that occurs in a number of mouse models of cone dystrophy with cone opsin mislocalization (e.g. cone cyclic nucleotide-gated channel A subunit (Cnga3) knockout (28), GC1 (Gucy2e) knockout (29,30), and kinesin 3A (Kif3a) knockout in cones (31)).

Figure 7.

Different roles of S-opsin and M/L-opsins in the degeneration of cone photoreceptors caused by Rpe65 (or Lrat) knockout or mutations. Mislocalized S-opsin aggregates/accumulates and causes intense ER stress, which leads to the rapid cone degeneration. In contrast, mislocalized M/L-opsins are degraded, which relieves ER stress and leads to slower cone degeneration. Deletion of S-opsin prevents S-opsin enriched central/ventral cone degeneration. Two different strategies were proposed to protect S-cones and M/L-cones (see details in Discussion).

Despite the survival of Opn1sw^−/−Lrat^−/− cones, membrane-associated proteins (e.g. GRK1, Gα_t2, GC1) are mislocalized and degraded as in Lrat^−/− cones (Fig. 6) (17). One possible mechanism is that COS-associated proteins rely on cone opsins, which are the most abundant membrane proteins in COS, to be co-transported to the COS (17,32). When cone opsins are not trafficked properly (i.e. in Lrat^−/− cones) or absent (i.e. in Opn1sw^−/−Lrat^−/− cones), many COS-associated proteins do not traffic properly to the COS, having apparently lost their means of trafficking via opsin-laden vesicles. Our results suggest that the mistrafficking of COS proteins other than S-opsin does not play a significant role in cone viability. This could be due to two reasons: (1) Similar to M-opsin, these proteins are largely degraded (17), likely via the ERAD pathway, and therefore pose less of a problem for cone cells (Fig. 6). (2) The expression of these proteins is much less than that of either M- or S-opsin, so that their loss or mistrafficking should, in theory, have even a ‘milder’ effect than that of mistrafficked M-opsin for cone viability. Collectively, we conclude that cone opsins are the ‘second messenger’ linking 11-cis-retinal deficiency to cone degeneration in LCA due to opsin misfolding (33), mistrafficking and aggregation (for S-opsin). Thus, RPE65 (or LRAT) LCA shares the etiology of a large diversity of disorders (i.e. conformational diseases) such as Alzheimer's disease, Parkinson's disease and type 2 diabetes, caused by improper protein folding (misfolding) as well as the accrual of unfolded proteins. However, different cone opsins have very different roles in cone degeneration (Fig. 7). The aggregation-prone S-opsin causes intense ER stress and rapid cone degeneration (i.e. ventral and central cones in Lrat^−/−). On the other hand, misfolded and mislocalized M/L-opsins are largely degraded and cause fewer problems for the cell (i.e. dorsal cones in Opn1sw^−/−Lrat^−/−). This has important implications for therapeutic strategies for LCA. The vision loss in LCA is caused by a combination of 11-cis-retinal deficiency and progressive photoreceptor (rods and cones) degeneration (1,34). A recent study suggests that gene therapy by delivering AAV2-RPE65 treats the 11-cis-retinal deficiency, but not photoreceptor degeneration (10). This study highlights the importance of slowing photoreceptor degeneration in LCA patients regardless of the patients' age in future clinical gene therapy trials. Furthermore, since photoreceptor degeneration occurs early in human RPE65-LCA, it is necessary to test new therapies in animal models that exhibit both vision dysfunction and photoreceptor degeneration. The prominent role of cone opsins in cone degeneration suggests that, in addition to restoring the 11-cis-retinal supply, research needs to focus on the folding, trafficking and aggregation (for S-opsin or degradation for M-opsin) properties of cone opsins to design an effective prevention and treatment strategy. Based on the different degeneration mechanisms in S-cones and M/L-cones, we propose two different strategies to slow S-cone and M/L-cone degeneration (Fig. 7). For S-cones, new drugs need to be designed to reduce the aggregated S-opsin, to promote folding and targeting of newly synthesized S-opsin, and to enhance proteasome activity in removing misfolded S-opsin. For M/L-cones, we only need to design drugs to help the folding and targeting of M/L-opsins (i.e. pharmacological chaperones) and to enhance proteasome activity.

Materials and Methods

Animals

Lrat^−/− and Opn1sw^−/− mice were generated previously (2,21). Opn1sw^+/−Lrat^−/− and Opn1sw^−/−Lrat^−/− mice were produced by crossing the Opn1sw^−/− and Lrat^−/− lines. WT (C57BL/6J) mice were purchased from Jackson Laboratory. All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Utah and were performed in accordance with the ARVO Statement for the Use of Animal in Ophthalmic and Vision Research. Mice were reared under cyclic light (12 h light/12 h dark).

Immunohistochemistry

The immunohistochemical procedure was performed as previously described (18,35). Cryostat sections (10–15 µm) were incubated with primary antibodies as indicated, and were visualized with Alexa 488- or Cy3-conjugated secondary antibodies. Sections were examined by Olympus FV1000 confocal microscope. Primary antibodies against S-opsin, M-opsin, cone arrestin, ubiquitin, CHOP, GRK1, GC1 and Gα_t2 were described in our previous publications (18,19,24). For experiments in Figs. 4 and 5, an antigen retrieval step (10 mm sodium citrate, pH 6.0, 95°C, 5 min) was added before immunolabeling.

Histology and electron microscopy

Mouse eyes were immersion-fixed overnight in a fixative containing 2.5% glutaraldehyde/1% formaldehyde and resin embedded as previously described (36,37). Samples were sectioned at 1 µm and stained with Richardson's stain. For electron microscopy, fixed tissues were osmicated 45–60 min in 0.5–1% OsO₄ in 0.1M cacodylate buffer, processed in maleate buffer for staining with uranyl acetate, and resin-embedded as described (38). Ultrathin sections were cut at 90 nm with a PT-X ultramicrotome (RMC) and imaged at 80 KeV in JEOL JEM 1400 electron microscope.

Western blot

Retinas from both eyes of the mouse were sonicated in 150 µl of radioimmunoprecipitation assay buffer (150 mm NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) plus protease inhibitors (Sigma). Protein concentration was measured by BCA assay (Bio-Rad). Fifteen microgram of protein was loaded for each sample for SDS-PAGE. The primary antibodies were detected by incubation with goat anti-rabbit or anti-mouse secondary antibodies conjugated with Horseradish Peroxidase. The primary antibodies were the same as used in immunohistochemistry. In addition, anti-β-actin (AC-15, Sigma-Aldrich) was used as a loading control.

Statistics

Data were presented as mean ± SEM, and the differences were analyzed with unpaired two-sample Student's t-test. P-values < 0.05 were considered statistically significant.

Supplementary Material

Supplementary Materials is available at HMG online.

Funding

This work was supported by National Institute of Health (EY022614 to Y.F. and EY014800 to core grant to the Department of Ophthalmology, University of Utah); E. Matilda Ziegler Foundation for the Blind to Y.F.; Knights Templar Eye Foundation to T.Z.; and Research to Prevent Blindness (an unrestricted grant to the Department of Ophthalmology at the University of Utah).