Michael T. Matthes

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning andin vivo genetic complementation to demonstrate thatMertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.

mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

Abstract. C57BL/6J-c2J (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J × BALB/c)F1× c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c.When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

The relationship of photoreceptor degeneration to retinal vascular development and loss in mutant rhodopsin transgenic and RCS rats.

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60-180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium

Aims: Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI). Methods: ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting of human RPE cell extracts. Lipid transport assays were performed using radiolabelled photoreceptor outer segments (POS). ABCA1 and SR-BI expression was examined in normal mouse eyes by immunofluorescence staining. BrMs of ABCA1 and SR-BI heterozygous mice were examined microscopically. Results: Human RPE cells expressed ABCA1 mRNA and protein. The ABCA1 and SR-BI inhibitor glyburide (also known as glibenclamide) abolished basal transport of POS-derived lipids in RPE cells in the presence of high-density lipoprotein. Mouse retina and RPE expressed ABCA1 and SR-BI. SR-BI was highly expressed in RPE. BrMs were significantly thickened in SR-BI heterozygous mice, but not in ABCA1 heterozygous mice. Conclusion: RPE cells express ABCA1 and SR-BI. This implies a significant role for SR-BI and ABCA1 in lipid transport and RCT in the retina and RPE.

Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.

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.

Induction of Endoplasmic Reticulum Stress Genes, BiP and Chop, in Genetic and Environmental Models of Retinal Degeneration

PURPOSE Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes. METHODS Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses. RESULTS S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss. CONCLUSIONS Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Tyrosine-Mutant AAV8 Delivery of Human MERTK Provides Long-Term Retinal Preservation in RCS Rats

PURPOSE The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported. METHODS An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively. RESULTS Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months. CONCLUSIONS This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Stanniocalcin-1 Rescued Photoreceptor Degeneration in Two Rat Models of Inherited Retinal Degeneration

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).