Rd6 Mice Research Articles

Improved gene therapy for MFRP deficiency-mediated retinal degeneration by knocking down endogenous bicistronic Mfrp and Ctrp5 transcript

The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3'UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases.

Drinking hydrogen water improves photoreceptor structure and function in retinal degeneration 6 mice

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H2 gas we have previously demonstrated, we hypothesized that ingestion of H2 water may delay the progression of photoreceptor death in rd6 mice. H2 mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H2 mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H2 group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H2 mice. Our results show that drinking water high in H2 (1.2–1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H2 for the treatment of RP.

Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice.

This study aimed to investigate whether the transplantation of genetically engineered bone marrow-derived mesenchymal stromal cells (MSCs) to overexpress brain-derived neurotrophic factor (BDNF) could rescue the chronic degenerative process of slow retinal degeneration in the rd6 (retinal degeneration 6) mouse model and sought to identify the potential underlying mechanisms. Rd6 mice were subjected to the intravitreal injection of lentivirally modified MSC-BDNF or unmodified MSC or saline. In vivo morphology, electrophysiological retinal function (ERG), and the expression of apoptosis-related genes, as well as BDNF and its receptor (TrkB), were assessed in retinas collected at 28 days and three months after transplantation. We observed that cells survived for at least three months after transplantation. MSC-BDNF preferentially integrated into the outer retinal layers and considerably rescued damaged retinal cells, as evaluated by ERG and immunofluorescence staining. Additionally, compared with controls, the therapy with MSC-BDNF was associated with the induction of molecular changes related to anti-apoptotic signaling. In conclusion, BDNF overexpression observed in retinas after MSC-BDNF treatment could enhance the neuroprotective properties of transplanted autologous MSCs alone in the chronically degenerated retina. This research provides evidence for the long-term efficacy of genetically-modified MSC and may represent a strategy for treating various forms of degenerative retinopathies in the future.

Treatment of Oxidative Stress With Chelation Therapy

mentosa. Hahn et al 1 have documented higher iron levels in eyes of patients with age-related macular degeneration than in eyes of age-matched controls (particularly chelatable iron deposition in the retinal pigment epithelium and Bruch membrane). Because iron catalyzes the Fenton reaction (which produces hydroxyl radical), these results may mean that ironmediated oxidative stress contributes to retinal degeneration in age-related macular degeneration. Furthermore, mice deficient in the ferroxidases ceruloplasmin and hephaestin accumulate iron in the retina and subsequently have retinal degeneration with features of age-related macular degeneration. 2,3 In these mice, iron chelation with the orally absorbed and cellpermeant iron chelator deferiprone (Ferriprox) ameliorated oxidative stress and protected against iron overload–induced retinal degeneration. 4 Because other investigators showed that the iron chelator deferoxamine can protect against retinal light damage and retinal ischemia reperfusion injury, Hadziahmetovicetal 5 decidedtotesttherecentlyUSFoodandDrug Administration–approved drug deferiprone because it is orally absorbed and has not been associated with retinal toxic effects in patients or mice. In the current issue of Translational Vision Science & Technology, Hadziahmetovic and colleagues report the ability of deferiprone to preventretinaldegenerationin2distinctpreclinicalmodels: sodium iodate (NaIO3)–induced retinal degeneration and the rd6 mouse. Sodium iodate is a retinotoxin, anditssystemicadministrationrelativelyselectivelydamagesretinalpigmentepitheliumandphotoreceptors.The rd6mutationaffectstheMfrpgene,afrizzled-relatedgene expressed in the retinal pigment epithelium. Mice homozygous for this mutation have an early-onset, slowly progressive loss of photoreceptors. Theinvestigatorsfoundthatsystemicpretreatmentand concomitant treatment with deferiprone modestly improved photoreceptor survival and markedly improved retinal pigment epithelium survival in the NaIO3model. Deferiprone also modestly improved photoreceptor survivalinrd6mice.Whatisthemechanismbywhichdeferiprone exerts these effects? The investigators found that deferiprone diminished NaIO3-induced upregulation of antioxidantandcomplementgenes(C3)causedbyNaIO3. Deferiprone also protected against NaIO3-induced reduction in the levels of visual cycle genesRhoandRpe65, consistent with retinal protection. These and other findings indicate that deferiprone reduces oxidative stress. The finding that deferiprone treatment ameliorated hereditary retinal degeneration due to the rd6 mutation in B6.C3Ga-Mfrprd6/J mutant mice is consistent with the previous work of Obolensky et al, 6 who found that deferoxamine provides functional and structural retina rescueintherd10mousemodelofretinitispigmentosa.Presumably, amelioration of oxidative stress also reduces photoreceptor damage in these models of human retinitis pigmentosa. 7 The dose of deferiprone used in this study is about 3-fold higher than that given to humans treated for systemic iron overload. In future studies, the investigators will examine both dose response and dosing frequency. The apparent lack of retinal toxic effects of deferiprone combined with the evidence that it can protect the retina against iron overload, NaIO3, the rd6 mutation, and light damage suggest that it merits further investigation for potential treatment of diverse retinal disorders. The results of these deferiprone experiments indicate that iron chelation may serve as a protective agent not only against iron overload–induced retinal degeneration but also against retinal disorders in which iron dysregulation is not the primary cause. In a Research Highlight in Translational Vision Science & Technology, Fliesler 8 notes that “drug repurposing” is a current buzz phrase in translational medicine. Deferiprone has been used for the treatment of thalassemia major and is currently being used in clinical trials in the United States for treatment of contrast-induced acute kidney disease and for slowing the progression of chronic kidney disease. The present article by Hadziahmetovic et al 5 may lead to such repurposing of deferiprone and thus represents yet another potential application of chelator-based therapeutics.

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms

To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

Take Two Iron Chelators and Call Me in the Morning

In a report by Hadziahmetovic et al,1 oral administration of an iron chelator deferiprone (Ferriprox), which was recently approved by the Food and Drug Administration, showed substantial protection against retinal degeneration both in a chemically induced (NaIO3) mouse model as well as in a rd6 mouse, which exhibits a hereditary, slowly progressing retinal degeneration that mainly affects the RPE and photoreceptor cells. These authors previously demonstrated that this iron chelator can spare the retina from iron overload-induced degeneration, has no appreciable retinal toxicity in humans or mice, and is readily absorbed upon oral administration.2 Deferiprone has been used for the management of thalassemia major3 and is currently in clinical trials in the United States for treatment of contrast-induced acute kidney disease and for slowing the progression of chronic kidney disease (http://www.cormedix.com/pipeline_CRMD001.php). Drug repurposing is a current buzz phrase in translational medicine. The most recent work by Hadziahmetovic et al1 may lead to such repurposing of deferiprone and thus represents another potential application of chelator-based therapeutics. While the mechanism of action of deferiprone in reducing the extent of retinal degeneration is not fully understood and may, in fact, involve multiple mechanisms, it likely involves suppression of free radical oxidation reactions catalyzed non-enzymatically by free iron. Development of drugs that target these kinds of reactions is an area of intensive research, including bifunctional molecules that are both chelators and free radical scavengers.4 The results of such studies are promising and have clear potential clinical implications for management of a broad spectrum of human retinal degeneration, both hereditary and acquired.

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms.

To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

Gene Therapy for Retinitis Pigmentosa Caused by MFRP Mutations: Human Phenotype and Preliminary Proof of Concept

Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

174delG Mutation in Mouse MFRP Causes Photoreceptor Degeneration and RPE Atrophy

The authors have identified a recessive mutation causing progressive retinal degeneration, white fundus flecks, and eventual retinal pigment epithelium (RPE) atrophy. The goal of these studies was to characterize the retinal phenotype, to identify the causative locus, and to examine possible functions of the affected gene. SNP mapping, DNA sequencing, and genetic complementation were used to identify the affected locus. Histology, electroretinography, immunohistochemistry, Western blot analysis, fundus photography, electron microscopy, and in vitro phagocytosis assays were used to characterize the phenotype of the mouse. Gene mapping identified a single base pair deletion in membrane-type frizzled related protein (MFRP), designated Mfrp(174delG). MFRP is normally expressed in the RPE and ciliary body but was undetectable by Western blot in mutants. CTRP5, a binding partner of MFRP, was upregulated at the mRNA level and at the protein level in most patients. Assays designed to test the integrity of retinoid cycling and phagocytic pathways showed no deficits in Mfrp(174delG) or rd6 animals. However, the RPE of both Mfrp(174delG) and rd6 mice exhibited a dramatic increase in the number of apical microvilli. Furthermore, evidence of RPE atrophy was evident in Mfrp(174delG) mice by 21 months. The authors have identified a novel null mutation in mouse Mfrp. This mutation causes photoreceptor degeneration and eventual RPE atrophy, which may be related to alterations in the number of RPE microvilli. These mice will be useful to identify a function of MFRP and to study the pathogenesis of atrophic macular degeneration.

Spatial and Temporal Expression of MFRP and Its Interaction with CTRP5

Mutations in the membrane frizzled-related protein (MFRP) gene cause nanophthalmos in humans, and a splice site mutation causes recessive retinal degeneration in the rd6 mouse. In human and mouse genomes, the MFRP gene lies adjoining to the complement 1q tumor necrosis factor-related protein 5 (CTRP5/C1QTNF5) gene involved in causing retinal degeneration and abnormal lens zonules in human. The purpose of this study was to characterize the spatial and temporal expression of the mouse Mfrp gene, determine tissue and subcellular localization of MFRP protein, and study its interaction with CTRP5. Expression of the Mfrp gene in the mouse was studied by quantitative (q)RT-PCR. MFRP protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Interaction with CTRP5 was studied by immunoprecipitation and immunoblot analysis, using mouse eye and human retinal pigmented epithelium (RPE) choroid extracts and by expressing full-length CTRP5 and MFRP in a heterologous system. The Mfrp gene is specifically expressed in RPE and ciliary body (CB), and its expression starts during early stages of embryogenesis. In the albino mouse eye, MFRP is localized to the apical and basal membranes of RPE and ciliary epithelium (CE). In addition, MFRP and CTRP5 were found to colocalize in RPE, CE, and MDCK cells, a general model of polarized epithelia. These proteins interact with each other in ocular tissues and also in a heterologous system. MFRP is localized to the plasma membrane of CE and RPE, and colocalizes and interacts with CTRP5 indicating a functional relationship between these two proteins.

Mutation Screen of the Membrane-Type Frizzled-Related Protein (MFRP) Gene in Patients with Inherited Retinal Degenerations

MFRP is a member of the frizzled-related protein family and contains a cysteine-rich domain essential for Wnt binding and signaling. MFRP is highly expressed in the retinal pigment epithelial cells of the eye. A splice donor mutation in the mouse ortholog of Mfrp is responsible for photoreceptor degeneration in the rd6 mouse. For these reasons, we investigated MFRP as a candidate gene for a phenotype associated with mutations. We screened 152 patients with inherited retinal degenerations including retinitis pigmentosa, Leber congenital amaurosis and Stargardt macular dystrophy. We identified five polymorphisms in the 5′ untranslated region, four missense changes, six isocoding variants and four intronic changes. None of the sequence variants were interpreted as pathogenic.

Mfrp, a gene encoding a frizzled related protein, is mutated in the mouse retinal degeneration 6

The autosomal recessive mouse mutation retinal degeneration 6 (rd6) causes small, white retinal spots and progressive photoreceptor degeneration similar to that observed in human flecked retinal diseases. Using a positional cloning approach, we determined that rd6 mice carry a splice donor mutation in the mouse homolog of the human membrane-type frizzled-related protein (Mfrp) gene that results in the skipping of exon 4. We found that mRNA of Mfrp is predominantly expressed in the eye, and at a lower level in the brain. To determine where in the eye Mfrp is expressed, in situ hybridization was done and showed that Mfrp is expressed specifically in the retinal pigment epithelium (RPE) and ciliary epithelium of the eye. The deduced amino acid sequence of MFRP contains a region with similarities to the cysteine-rich domain (CRD) of frizzled, a gene originally found in Drosophila that controls tissue polarity. The CRD is essential for Wnt binding and signaling. Wnt signaling has been shown to be involved in the control of gene expression, cell adhesion, planar polarity, proliferation and apoptosis. We also observed the localization of Wnt family proteins in the apical membrane of the RPE. Our results provide genetic evidence for an involvement of the Mfrp gene expressed by RPE in the degeneration of photoreceptors.