Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
Rd16;Nrl−/− mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA (n = 15; ages 7–48) were compared with those of the model.
CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe visual loss); the extent of this island declined slowly with age. The rd16;Nrl−/− model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant Nrl−/− mice, UV- and M-cone ERGs of rd16;Nrl−/− were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age.
The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both CEP290-LCA patients and the rd16;Nrl−/− murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG.
Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.
Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.
To characterize human parafoveal blood flow using adaptive optics scanning laser ophthalmoscopy (AO-SLO).
In 5 normal subjects, erythrocyte aggregate distributions were analyzed on 3 different days. Erythrocyte aggregates were described as a “dark tail” in AO-SLO. The characteristics of the pathways with dark tail flow in the parafovea were measured. Additionally, the tendency for dark tail flow before and after bifurcations was analyzed to study the blood flow in detail.
Average velocity in parent vessels with dark tail flow was 1.30±0.27 mm/s. Average velocity in daughter vessels with dark tail flow was 1.12±0.25 mm/s, and the average velocity of plasma gaps in daughter vessels without dark tail flow was 0.64±0.11 mm/s. Downstream from the bifurcations, the velocity in vessels with dark tail flow was higher than that in those without it (p<0.001), and the branching angles of vessels with dark tail flow were smaller than those of vessels without it (p<0.001).
Images from the AO-SLO noninvasively revealed pathways with and without dark tail flow in the human parafovea. Pathways with dark tail flow in the daughter vessels generally had faster flow and smaller bifurcation angles than daughter vessels without dark tail flow. Thus, AO-SLO is an instructive tool for analyzing retinal microcirculatory hemodynamics.
The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.
Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.
Mutations in the RP2 gene are associated with 10% to 15% of X-linked retinitis pigmentosa (XLRP), a debilitating disorder characterized by the degeneration of retinal rod and cone photoreceptors. The molecular mechanism of pathogenesis of photoreceptor degeneration in XLRP-RP2 has not been elucidated, and no treatment is currently available. This study was undertaken to investigate the pathogenesis of RP2-associated retinal degeneration.
We introduced loxP sites that flank exon 2, a mutational hotspot in XLRP-RP2, in the mouse Rp2 gene. We then produced Rp2-null allele using transgenic mice that expressed Cre-recombinase under control of the ubiquitous CAG promoter. Electroretinography (ERG), histology, light microscopy, transmission electron microscopy, and immunofluorescence microscopy were performed to ascertain the effect of ablation of Rp2 on photoreceptor development, function, and protein trafficking.
Although no gross abnormalities were detected in the Rp2null mice, photopic (cone) and scotopic (rod) function as measured by ERG showed a gradual decline starting as early as 1 month of age. We also detected slow progressive degeneration of the photoreceptor membrane discs in the mutant retina. These defects were associated with mislocalization of cone opsins to the nuclear and synaptic layers and reduced rhodopsin content in the outer segment of mutant retina prior to the onset of photoreceptor degeneration.
Our studies suggest that RP2 contributes to the maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in XLRP caused by RP2 mutations. The Rp2null mice should serve as a useful preclinical model for testing gene- and cell-based therapies.
This study reports generation and characterization of a mouse model of Rp2-mediated retinal degeneration and shows that cone opsin mislocalization is an early step in the pathogenesis of associated disease.
Rp2; retina; photoreceptor
A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.
Age-related macular degeneration (AMD) is a common cause of visual impairment in individuals over 55 years of age worldwide. The varying clinical phenotypes of AMD result from contributions of genetic, epigenetic and non-genetic (environmental) factors. Genetic studies of AMD have come of age as a direct result of tremendous gains from human genome project, genomewide association studies and identification of numerous susceptibility loci. These findings have implicated immune response, high-density lipoprotein cholesterol metabolism, extracellular matrix, and angiogenesis signaling pathways in disease pathophysiology. Here, we address how the wealth of genetic findings in AMD is expected to impact the practice of medicine, providing opportunities for improved risk assessment, molecular diagnosis, preventive and therapeutic intervention. We propose that the potential of using genetic variants for monitoring treatment response (pharmacogenetics) may usher a new era of personalized medicine in the clinical management of AMD.
Retinal neurodegeneration; Complex disease; Genomewide association; Risk factors; Genetic diagnosis; Pharmacogenetics; Clinical management
A quantitative analysis of photoreceptor properties was performed in the retina of the nocturnal deer mouse, Peromyscus maniculatus, using pigmented (wildtype) and albino animals. The aim was to establish whether the deer mouse is a more suitable model species than the house mouse for photoreceptor studies, and whether oculocutaneous albinism affects its photoreceptor properties. In retinal flatmounts, cone photoreceptors were identified by opsin immunostaining, and their numbers, spectral types, and distributions across the retina were determined. Rod photoreceptors were counted using differential interference contrast microscopy. Pigmented P. maniculatus have a rod-dominated retina with rod densities of about 450.000/mm2 and cone densities of 3000 - 6500/mm2. Two cone opsins, shortwave sensitive (S) and middle-to-longwave sensitive (M), are present and expressed in distinct cone types. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the S cone visual pigment. The S cones constitute a 5-15% minority of the cones. Different from house mouse, S and M cone distributions do not have dorsoventral gradients, and coexpression of both opsins in single cones is exceptional (<2% of the cones). In albino P. maniculatus, rod densities are reduced by approximately 40% (270.000/mm2). Overall, cone density and the density of cones exclusively expressing S opsin are not significantly different from pigmented P. maniculatus. However, in albino retinas S opsin is coexpressed with M opsin in 60-90% of the cones and therefore the population of cones expressing only M opsin is significantly reduced to 5-25%. In conclusion, deer mouse cone properties largely conform to the general mammalian pattern, hence the deer mouse may be better suited than the house mouse for the study of certain basic cone properties, including the effects of albinism on cone opsin expression.
The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.
Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.
To determine if plasma metabolic profiles can detect differences between patients with neovascular age-related macular degeneration (NVAMD) and similarly-aged controls.
Metabolomic analysis using liquid chromatography with Fourier-transform mass spectrometry (LC-FTMS) was performed on plasma samples from 26 NVAMD patients and 19 controls. Data were collected from mass/charge ratio (m/z) 85 to 850 on a Thermo LTQ-FT mass spectrometer, and metabolic features were extracted using an adaptive processing software package. Both non-transformed and log2 transformed data were corrected using Benjamini and Hochberg False Discovery Rate (FDR) to account for multiple testing. Orthogonal Partial Least Squares-Discriminant Analysis was performed to determine metabolic features that distinguished NVAMD patients from controls. Individual m/z features were matched to the Kyoto Encyclopedia of Genes and Genomes database and the Metlin metabolomics database, and metabolic pathways associated with NVAMD were identified using MetScape.
Of the 1680 total m/z features detected by LC-FTMS, 94 unique m/z features were significantly different between NVAMD patients and controls using FDR (q = 0.05). A comparison of these features to those found with log2 transformed data (n = 132, q = 0.2) revealed 40 features in common, reaffirming the involvement of certain metabolites. Such metabolites included di- and tripeptides, covalently modified amino acids, bile acids, and vitamin D-related metabolites. Correlation analysis revealed associations among certain significant features, and pathway analysis demonstrated broader changes in tyrosine metabolism, sulfur amino acid metabolism, and amino acids related to urea metabolism.
These data suggest that metabolomic analysis can identify a panel of individual metabolites that differ between NVAMD cases and controls. Pathway analysis can assess the involvement of certain metabolic pathways, such as tyrosine and urea metabolism, and can provide further insight into the pathophysiology of AMD.
Amacrine and horizontal interneurons integrate visual information as it is relayed through the retina from the photoreceptors to the ganglion cells. The early steps that generate these interneuron networks remain unclear. Here we show that a distinct RORβ1 isoform encoded by the retinoid-related orphan nuclear receptor β gene (Rorb) is critical for both amacrine and horizontal cell differentiation in mice. A fluorescent protein cassette targeted into Rorb revealed RORβ1 as a novel marker of immature amacrine and horizontal cells and of undifferentiated, dividing progenitor cells. RORβ1-deficient mice lose expression of pancreas-specific transcription factor 1a (Ptf1a) but retain forkhead box n4 factor (Foxn4), two early-acting factors necessary for amacrine and horizontal cell generation. RORβ1 and Foxn4 synergistically induce Ptf1a expression, suggesting a central role for RORβ1 in a transcriptional hierarchy that directs this interneuron differentiation pathway. Moreover, ectopic RORβ1 expression in neonatal retina promotes amacrine cell differentiation.
orphan nuclear receptor; forkhead box transcription factor Foxn4; basic helix-loop-helix factor Ptf1a; neurogenesis; inhibitory interneuron
To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.
Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA.
We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region).
This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.
Identification of mutations in 15% of the screened patients has important implications for guiding clinicians who are ordering genetic testing and provides a strong argument for screening the RPGR gene in simplex cases of retinal degenerative diseases.
Nineteen Wnt ligands and 10 Frizzled (Fz) receptors mediate multiple distinct cellular events during neuronal development. However, their precise roles in cell-type specification and organogenesis are poorly delineated because of overlapping functions and expression profiles. Here, we have explored the role of two closely related Frizzled receptors, Fz5 and Fz8, in mouse retinal development. We previously showed that Fz5−/− mice exhibit mild coloboma and microphthalmia at ∼50% penetrance. Fz8 expression overlaps with Fz5 in the neural retina and optic fissure/disc. Mice lacking Fz8 show minimal eye and retinal defects. The embryos lacking both Fz5 and Fz8 die early in development, but a majority of triallelic Fz5−/−;Fz8+/− mutants survive until birth. The triallelic mutant develops severe retinal coloboma and microphthalmia with full penetrance. At the cellular level, impaired neurogenesis is indicated by increased early-born retinal neurons that result from accelerated cell cycle exit of progenitors. Deficiency of apical retinal neuroepithelium is indicated by altered localization of apical junction markers, such as atypical protein kinase C, RhoA and β-catenin. Hes1 expression, which is critical for retinal progenitor expansion, is down-regulated in the triallelic mutant mouse. Furthermore, blocking Frizzled receptors in cultured retinal explants led to basally shifted divisions of retinal progenitors. Together, our studies suggest a dose-dependent regulation of signaling by Fz5 and Fz8 in optic fissure/disc formation and progenitor expansion.
Dominant mutations occurring in the high-affinity Ca2+-binding sites (EF-hands) of the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) cause slowly progressing cone-rod dystrophy (CORD) in a dozen families worldwide. We developed a nonallele-specific adeno-associated virus (AAV)-based RNAi knockdown strategy to rescue the retina degeneration caused by GCAP1 mutations. We generated three genomic transgenic mouse lines expressing wildtype (WT) and L151F mutant mouse GCAP1 with or without a C-terminal GFP fusion. Under control of endogenous regulatory elements, the transgenes were expressed specifically in mouse photoreceptors. GCAP1(L151F) and GCAP1(L151F)-GFP transgenic mice presented with a late onset and slowly progressive photoreceptor degeneration, similar to that observed in human GCAP1-CORD patients. Transgenic expression of WT GCAP1-EGFP in photoreceptors had no adverse effect. Toward therapy development, a highly effective anti-mGCAP1 shRNA, mG1hp4, was selected from four candidate shRNAs using an in-vitro screening assay. Subsequently a self-complementary (sc) AAV serotype 2/8 expressing mG1hp4 was delivered subretinally to GCAP1(L151F)-GFP transgenic mice. Knockdown of the GCAP1(L151F)-GFP transgene product was visualized by fluorescence live imaging in the scAAV2/8-mG1hp4-treated retinas. Concomitant with the mutant GCAP1-GFP fusion protein, endogenous GCAP1 decreased as well in treated retinas. We propose nonallele-specific RNAi knockdown of GCAP1 as a general therapeutic strategy to rescue any GCAP1-based dominant cone-rod dystrophy in human patients.
Among peripheral regulatory T cells, CD8+ T cells also play an important role in the maintenance of immune homeostasis. A subset of CD8+ Treg that express αβTCR and CD8αα homodimers can recognize TCR-derived peptides in the context of the class Ib MHC molecule Qa-1. To gain a better understanding of the nature and phenotype of CD8αα+TCRαβ+ Treg, a global gene expression profiling using microarray, real-time quantitative PCR, and flowcytometry analysis was performed using functional Treg clones and lines. Our data show that CD8+ Treg shared gene profile expressed by innate-like lymphocytes, including murine intraepithelial lymphocytes and thymic CD8αα+TCRαβ+ T cell populations. Additionally, this subset displays differential expression of several key regulatory molecules, including CD200. CD8αα+ Treg expressed higher levels of a number of NK cell related receptors and molecules belonging to the TNF superfamily. Collectively, peripheral class Ib-reactive CD8αα+TCRαβ+ T cells represent a unique regulatory population different from class Ia MHC-restricted conventional T cells. These studies have important implications for the regulatory mechanisms mediated by the CD8+ Treg population in general.
CD8+ Treg; EAE; Microarray; Innate cells; Qa-1/HLA-E
Carriers of blue cone monochromacy have fewer cone photoreceptors than normal. Here we examine how this disruption at the level of the retina affects visual function and cortical organization in these individuals. Visual resolution and contrast sensitivity was measured at the preferred retinal locus of fixation and visual resolution was tested at two eccentric locations (2.5° and 8°) with spectacle correction only. Adaptive optics corrected resolution acuity and cone spacing were simultaneously measured at several locations within the central fovea with adaptive optics scanning laser ophthalmoscopy (AOSLO). Fixation stability was assessed by extracting eye motion data from AOSLO videos. Retinotopic mapping using fMRI was carried out to estimate the area of early cortical regions, including that of the foveal confluence. Without adaptive optics correction, BCM carriers appeared to have normal visual function, with normal contrast sensitivity and visual resolution, but with AO-correction, visual resolution was significantly worse than normal. This resolution deficit is not explained by cone loss alone and is suggestive of an associated loss of retinal ganglion cells. However, despite evidence suggesting a reduction in the number of retinal ganglion cells, retinotopic mapping showed no reduction in the cortical area of the foveal confluence. These results suggest that ganglion cell density may not govern the foveal overrepresentation in the cortex. We propose that it is not the number of afferents, but rather the content of the information relayed to the cortex from the retina across the visual field that governs cortical magnification, as under normal viewing conditions this information is similar in both BCM carriers and normal controls.
Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl (Nrl−/−) in mice results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl−/− retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl−/− mouse illustrates the long-term viability of cones in the absence of rods and RPE defects in a rodless retina. We propose that Nrl−/− retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.
Background Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case–control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking.
Methods To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, ‘Y402H’) with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26 494 individuals, including 14 174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene–smoking interaction; and 16 published studies from non-European ancestry.
Results In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10–2.45; P = 1.1 x 10−161]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies.
Conclusion The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.
Age-related macular degeneration (AMD); Complement factor H gene; meta-ananlysis
We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model.
XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5–72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset.
Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration.
RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy.
Progress in treating canine RPGR disease prompted us to characterize patients with RPGR-ORF15 mutations and provide a detailed natural history of a novel Rpgr-mutant mouse for further proof-of-concept experiments.
The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration.
Using a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking.
We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.
Retina; Photoreceptor; Mouse; Retinal degeneration; Photoreceptor development; Rabac1; Prenylated Rab Acceptor 1; Rab6; Vesicular trafficking
Ciliopathies encompass a broad array of clinical findings associated with genetic defects in biogenesis and/or function of the primary cilium, a ubiquitous organelle involved in the transduction of diverse biological signals. Degeneration or dysfunction of retinal photoreceptors is frequently observed in diverse ciliopathies. The sensory cilium in a photoreceptor elaborates into unique outer segment discs that provide extensive surface area for maximal photon capture and efficient visual transduction. The daily renewal of approximately 10% of outer segments requires a precise control of ciliary transport. Here, we review the ciliopathies with associated retinal degeneration, describe the distinctive structure of the photoreceptor cilium, and discuss mouse models that allow investigations into molecular mechanisms of cilia biogenesis and defects. We have specifically focused on two ciliary proteins – CEP290 and RPGR – that underlie photoreceptor degeneration and syndromic ciliopathies. Mouse models of CEP290 and RPGR disease, and of their multiple interacting partners, have helped unravel new functional insights into cell type-specific phenotypic defects in distinct ciliary proteins. Elucidation of multifaceted ciliary functions and associated protein complexes will require concerted efforts to assimilate diverse datasets from in vivo and in vitro studies. We therefore discuss a possible framework for investigating genetic networks associated with photoreceptor cilia biogenesis and pathology.
Ciliopathy; Retinal degeneration; Primary cilium; Sensory cilia; CEP290; RPGR; Bardet–Biedl syndrome; Leber congenital amaurosis; Joubert syndrome; Nephronophthisis
Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.