Presence of rd8 mutation does not alter the ocular phenotype of late-onset retinal degeneration mouse model.
ABSTRACT: A spontaneous frameshift mutation, c.3481delC, in the Crb1 gene is the underlying cause of dysplasia and retinal degeneration in rd8 mice. The rd8 mutation is found in C57BL/6N but not in C57BL/6J mouse sub-strains. The development of ocular pathology in single knockout Ccl2-/-, Cx3cr1-/- and in double knockout Ccl2-/-, Cx3cr1-/- mice raised on a C57BL/6 background has been reported to depend on the presence of a rd8 mutation. In this study, we investigated the influence of the rd8 mutation on the retinal pathology that we previously described in the late-onset retinal degeneration (L-ORD) mouse model with a heterozygous S163R mutation in the C1q-tumor necrosis factor-related protein-5Ctrp5+/- gene that was generated on a C57BL/6J background.Mouse lines carrying the Ctrp5 S163R and rd8 mutations (Ctrp5+/-;rd8/rd8), corresponding controls without the rd8 mutation (Ctrp5+/-;wt/wt), and wild-type mice with and without the rd8 mutation (Wtrd8/rd8 and Wtwt/wt, respectively) were generated by systematic breeding of mice in our L-ORD mouse colony. Genotyping the mice for the rd8 (del C at nt3481 in Crb1) and Ctrp5 S163R mutations was performed with allelic PCR or sequencing. Retinal morphology was studied with fundus imaging, histology, light microscopy, electron microscopy, and immunohistochemistry.Genotype analysis of the mice in L-ORD mouse colony detected the rd8 mutation in the homozygous and heterozygous state. Fundus imaging of wild-type mice without the rd8 mutation (Wtwt/wt) revealed no autofluorescence (AF) spots up to 6-8 months and few AF spots at 21 months. However, the accumulation of AF lesions accelerated with age in the Ctrp5+/- mice that lack the rd8 mutation (Ctrp5+/-;wt/wt). The number of AF lesions was significantly increased (p<0.001), and they were small and uniformly distributed throughout the retina in the 21-month-old Ctrp5+/-;wt/wt mice when compared to the age-matched controls. Wild-type and Ctrp5+/- mice with the rd8 mutation (Wtrd8/rd8 and Ctrp5+/-;rd8/rd8, respectively) revealed an integrated retinal architecture with well-defined outer segments/inner segments (OS/IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL). The presence of pseudorosette structures reported in the rd8 mice between the ONL and the INL in the ventral quadrant of the retina was not observed in all genotypes studied. Further, the external limiting membrane was continuous in the Ctrp5+/-;rd8/rd8 and Wtrd8/rd8 mice. Evaluation of the retinal phenotype revealed that the Ctrp5+/-;wt/wt mice developed characteristic L-ORD pathology including age-dependent accumulation of AF spots, development of sub-retinal, sub-RPE, and basal laminar deposits, and Bruch's membrane abnormalities at older age, while these changes were not observed in the age-matched littermate WTwt/wt mice.The Wtrd8/rd8 and Ctrp5+/-;rd8/rd8 mice raised on C57BL/6J did not develop early onset retinal changes that are characteristic of the rd8 phenotype, supporting the hypothesis that manifestation of rd8-associated pathology depends on the genetic background. The retinal pathology observed in mice with the Ctrp5+/-;wt/wt genotype is consistent with the L-ORD phenotype observed in patients and with the phenotype we described previously. The lack of rd8-associated retinal pathology in the Ctrp5+/-;wt/wt mouse model raised on the C57BL/6J background and the development of the L-ORD phenotype in these mice in the presence and absence of the rd8 mutation suggests that the pathology observed in the Ctrp5+/-;wt/wt mice is primarily associated with the S163R mutation in the Ctrp5 gene.
Project description:Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology.
Project description:Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.
Project description:The mutation S163R in complement C1q tumor necrosis factor-related protein-5 (C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD). In this study, our goal is to evaluate the consequences of mutant S163R C1QTNF5 expression in mouse RPE following its delivery using an adeno-associated viral (AAV) vector.We generated AAV vectors containing either human wild-type C1QTNF5 or mutant S163R C1QTNF5 driven by an RPE-specific BEST1 promoter, and delivered them subretinally into one eye of adult C57BL/6 mice. Transgene expression was detected by immunohistochemistry. Retinal function was assessed by full-field ERG. Pathological changes were further examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT).We show that the AAV-expressed mutant S163R leads to pathological effects similar to some of those found in patients with advanced L-ORD, including RPE thinning, RPE cell loss, and retinal degeneration. In addition, we provide in vivo evidence that mutant S163R C1QTNF5 can form large, transparent, spherical intracellular aggregates throughout the RPE, which are detectable by light microscopy. In contrast to AAV-expressed wild-type C1QTNF5, which is secreted apically from the RPE toward the photoreceptor cells and the outer limiting membrane, the S163R mutant is primarily routed toward the basal side of RPE, where it forms thick, extracellular deposits over time.Adeno-associated viral-targeted expression of mutant S163R in the RPE represents a useful approach for quickly generating animal models that mimic pathological features of L-ORD and offers the potential to understand disease mechanisms and develop therapeutic strategies.
Project description:Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling-features that were stable in the inbred, variable in the second, but virtually absent in the third Crb1(rd8/rd8) line, even at 12 months of age. This suggests that the Crb1(rd8/rd8) mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.
Project description:Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.
Project description:Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant retinal dystrophy, characterised by extensive sub-retinal pigment epithelium (RPE) deposits, RPE atrophy, choroidal neovascularisation and photoreceptor cell death associated with severe visual loss. L-ORD shows striking phenotypic similarities to age-related macular degeneration (AMD), a common and genetically complex disorder, which can lead to misdiagnosis in the early stages. To date, a single missense mutation (S163R) in the C1QTNF5 gene, encoding C1q And Tumor Necrosis Factor Related Protein 5 (C1QTNF5) has been shown to cause L-ORD in a subset of affected families. Here, we describe the identification and characterisation of three novel pathogenic mutations in C1QTNF5 in order to elucidate disease mechanisms. In silico and in vitro characterisation show that these mutations perturb protein folding, assembly or polarity of secretion of C1QTNF5 and, importantly, all appear to destabilise the wildtype protein in co-transfection experiments in a human RPE cell line. This suggests that the heterozygous mutations in L-ORD show a dominant negative, rather than a haploinsufficient, disease mechanism. The function of C1QTNF5 remains unclear but this new insight into the pathogenetic basis of L-ORD has implications for future therapeutic strategies such as gene augmentation therapy.
Project description:Mutations in crumb homologue 1 (CRB1) in humans are associated with Leber's congenital amaurosis (LCA) and retinitis pigmentosa (RP). There is no clear genotype-phenotype correlation for human CRB1 mutations in RP and LCA. The high variability in clinical features observed in CRB1 mutations suggests that environmental factors or genetic modifiers influence severity of CRB1 related retinopathies. Retinal degeneration 8 (rd8) is a spontaneous mutation in the Crb1 gene (Crb1(rdr/rd8)). Crb1(rdr/rd8) mice present with focal disruption in the outer retina manifesting as white spots on fundus examination. Mild retinal dysfunction with decreased b-wave amplitude has been reported in Crb1(rdr/rd8) mice at 18 months. Methylene tetrahydrofolate reductase (MTHFR) is a crucial enzyme of homocysteine metabolism. MTHFR mutations are prevalent in humans and are linked to a broad spectrum of disorders including cardiovascular and neurodegenerative diseases. We recently reported the retinal phenotype in Mthfr-deficient (Mthfr(+/-)) heterozygous mice. At 24 weeks the mice showed decreased RGC function, thinner nerve fiber layer, focal areas of vascular leakage and 20% fewer cells in the ganglion cell layer (GCL). Considering the variability in CRB1-related retinopathies and the high occurrence of human MTHFR mutations we evaluated whether Mthfr deficiency influences rd8 retinal phenotype. Mthfr heterozygous mice with rd8 mutations (Mthfr(+/-)(rd8/rd8)) and Crb(rd8/rd8) mice (Mthfr(+/+rd8/rd8)) mice were subjected to comprehensive retinal evaluation using ERG, fundoscopy, fluorescein angiography (FA), morphometric and retinal flat mount immunostaining analyses of isolectin-B4 at 8-54 wks. Assessment of retinal function revealed a significant decrease in the a-, b- and c-wave amplitudes in Mthfr(+/-)(rd8/rd8) mice at 52 wks. Fundoscopic evaluation demonstrated the presence of signature rd8 spots in Mthfr(+/+rd8/rd8) mice and an increase in the extent of these rd8 spots in Mthfr(+/-)(rd8/rd8) mice at 24 weeks and beyond. FA revealed marked vascular leakage, ischemia and vascular tortuosity in Mthfr(+/-)(rd8/rd8) mice at 24 and 52 weeks. Retinal dysplasia was observed in ?14-33% Mthfr(+/-)(rd8/rd8) mice by morphometric analysis. This was accompanied by a ?20% reduction in cells of the GCL of Mthfr(+/-)(rd8/rd8) mice at 24 and 52 weeks. Retinal flat mount immunostaining with isolectin-B4 showed neovascularization and loss of blood vessel integrity in Mthfr(+/-)(rd8/rd8) mice in contrast to mild vasculopathy in Mthfr(+/+rd8/rd8) mice. Taken together, our data support an earlier onset and worsened retinal phenotype when Mthfr and rd8 mutations coexist. Our study sets the stage for future studies to investigate the role of MTHFR deficiency in human CRB1 retinopathies.
Project description:To investigate the impact of photoreceptor oxidative stress on photoreceptor degeneration in mice carrying the rd8 mutation (C57BL/6N). We compared the hyperoxia-induced proliferative retinopathy (HIPR) model in two mouse strains (C57BL/6J and C57BL/6N). Pups were exposed to 75% oxygen, starting at birth and continuing for 14 days (P14). Mice were euthanized at P14, or allowed to recover in room air for one day (P15), seven days (P21), or 14 days (P28). We quantified retinal thickness and the length of residual photoreceptors not affected by rosette formation. In addition we explored differences in retinal immunostaining for NADPH oxidase 4 (NOX4), Rac1, vascular endothelium, and activated M?ller cells. We analyzed photoreceptor oxidative stress using DCF staining in cross sections and quantified NOX4 protein levels using western blotting. C57BL/6N mice in HIPR showed increased oxidative stress, NOX4, and Rac1 in the photoreceptors at P14 and P15 compared to C57BL/6J. In addition, we observed significant progression of photoreceptor degeneration, with significantly accelerated rosette formation in C57BL/6N under HIPR, compared to their room air counterparts. Furthermore, C57BL/6N under HIPR had significantly thinner central retinas than C57BL/6J in HIPR. We did not find a difference in vascular disruption or M?ller cell activation comparing the two strains in hyperoxia. In HIPR, the C57BL/6N strain carrying the rd8 mutation showed significantly accelerated photoreceptor degeneration, mediated via exacerbated photoreceptor oxidative stress, which we believe relates to Rac1-NOX dysregulation in the setting of Crb1 loss-of-function.
Project description:The Age-Related Eye Diseases Study 2 (AREDS2) clinical trial is assessing the effects of higher dietary xanthophyll (lutein and zeaxanthin) and long-chain n3 polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on progression to advanced age-related macular degeneration (AMD). This study's purpose was to examine the retinal effects of the AREDS2 formulation on Chemokine (C-C motif) ligand 2 (Ccl2(-/-))/CX3C chemokine receptor 1 (Cx3cr1(-/-)) mice on Crumbs homolog 1 retinal degeneration phenotype 8 (Crb1(rd8)) background (DKO), which develop focal retinal lesions with certain features similar to AMD. DKO and C57BL/6N rd8 background mice (WT) were bred and randomized into 4 groups. Two groups, WT mice on AREDS2 diet (A-WT) and DKO mice on AREDS2 diet (A-DKO), were supplemented daily with 1.76 ?mol of lutein, 35.1 ?mol of zeaxanthin, 215 ?mol EPA, and 107 ?mol of DHA, and 2 control groups, WT mice on control diet (C-WT) and DKO mice on control diet (C-DKO), were fed an isocaloric diet. All mice had monthly fundus photos and were killed after 3 mo for biochemical and histologic analyses. After 3 mo, 81% of A-DKO mice had lesion regression compared with 25% of C-DKO mice (P < 0.05). Toxic retinal 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E) concentrations were significantly lower in A-DKO compared with C-DKO mice. The outer nuclear layer thickness in A-DKO mice was significantly greater than that in C-DKO mice. Retinal expression of inducible nitric oxide synthase (iNos) tumor necrosis factor-? (Tnf-?), Cyclooxygenase-2 (Cox-2), interleukin1beta (IL-1?), and vascular endothelial growth factor (Vegf) was significantly lower in A-DKO compared with C-DKO mice. Xanthophylls and LCPUFAs have antiinflammatory, neuroprotective, and antiangiogenic properties. Our data provide potential mechanisms by which the AREDS2 formula has a protective effect on retinal lesions in DKO mice.
Project description:The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc-/- mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc(-/-)-retinae. In adult WT and Hdc(-/-)-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc-/- retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc-/- mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdcrd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc-/- mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc-/- compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc-/- retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.