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1.  Retinal Pigment Epithelium Defects Accelerate Photoreceptor Degeneration in Cell Type–Specific Knockout Mouse Models of Choroideremia 
In this study, the authors provide insight into the pathogenesis of choroideremia, which is caused by the disruption of intracellular vesicular transport. They also touch on other issues, such as the photoreceptor-RPE relationship and aging of the RPE.
Purpose.
Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers (photoreceptors, retinal pigment epithelium, and choroid), with a complex and still largely unclear pathogenesis. To investigate the pathophysiology of CHM, the authors engineered mice with a cell type–specific Chm/Rep1 knockout (KO).
Methods.
A mouse line carrying a conditional allele ChmFlox was crossed with the transgenic line IRBP-Cre to achieve Chm KO, specifically in the photoreceptor layer, and Tyr-Cre to produce Chm KO, specifically in the retinal pigment epithelial and other pigmented cells. ChmFlox, Tyr-Cre+ and ChmFlox, IRBP-Cre+ mice were mated to produce mice with Chm KO in both layers. All mouse lines were studied by histology, electron microscopy, electroretinography (ERG), scanning laser ophthalmoscopy (SLO), and biochemical methods.
Results.
In ChmFlox, IRBP-Cre+ mice the authors observed the progressive degeneration of photoreceptors in the presence of normal retinal pigment epithelium (RPE). ChmFlox, Tyr-Cre+ mice exhibited coat color dilution and pigment abnormalities of the RPE in the presence of an intact outer nuclear layer. In 6- to 8-month-old ChmFlox, Tyr-Cre+, IRBP-Cre+ mice, the degeneration of photoreceptors was accelerated compared with ChmFlox, IRBP-Cre+ mice but became leveled with age, such that it was comparable at 12 to 14 months. Detailed ERG and SLO analysis supported the histopathologic findings.
Conclusions.
Defects in photoreceptors and RPE can arise because of intrinsic defects caused cell autonomously by the Chm KO. However, when both photoreceptors and RPE are diseased, the dynamics of the degenerative process are altered. Photoreceptor functional deficit and cell death manifest much earlier, suggesting that the diseased RPE accelerates photoreceptor degeneration.
doi:10.1167/iovs.09-4892
PMCID: PMC3066613  PMID: 20445111
2.  Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium 
PLoS ONE  2013;8(2):e57769.
The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (ChmFlox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the ChmFlox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.
doi:10.1371/journal.pone.0057769
PMCID: PMC3584022  PMID: 23460904
3.  Silencing of the CHM Gene Alters Phagocytic and Secretory Pathways in the Retinal Pigment Epithelium 
The pathogenesis of choroideremia (CHM), an X-linked retinopathy, remains poorly defined. Silencing of the CHM gene in the retinal pigment epithelium in vitro alters phagocytic and secretory pathways and may indicate how the disorder leads to retinal degeneration.
Purpose.
Choroideremia (CHM) is an X-linked progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid caused by mutations in the CHM gene, which encodes Rab escort-protein-1 (REP-1). REP-1 enables posttranslational isoprenyl modification of Rab GTPases, proteins that control vesicle formation, movement, docking, and fusion. The aim of this study was to determine the effect of REP-1 depletion on vesicular trafficking in phagocytic and secretory pathways of human RPE.
Methods.
In vitro, REP-1 expression was inhibited in human fetal RPE (hfRPE) cells by siRNA knockdown and its effects measured on the uptake of bovine photoreceptor outer segments (POS), proteolysis of POS rhodopsin, phagosomal pH, phagosome fusion with early and late endosomes/lysosomes, and polarized secretion of cytokines.
Results.
Depletion of REP-1 in human RPE cells did not affect POS internalization but reduced phagosomal acidification and delayed POS protein clearance. REP-1 depletion also caused a decrease in the association of POS-containing phagosomes with late endosomal markers (Rab7, LAMP-1) and increases in the secretion of monocyte chemotactic protein (MCP-1) and interleukin (IL)-8 by hfRPE cells.
Conclusions.
Lack of REP-1 protein expression in hfRPE cells leads to reduced degradation of POS most likely because of the inhibition of phagosome-lysosome fusion events and increased constitutive secretion of MCP-1 and IL-8. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the presence of inflammatory cells in the choroid of patients with CHM.
doi:10.1167/iovs.09-4117
PMCID: PMC2868448  PMID: 19741243
4.  Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo 
Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1006-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00109-013-1006-4
PMCID: PMC3695676  PMID: 23756766
Rab escort protein 1; Gene therapy; Choroideremia; Rab GTPase; Retinitis pigmentosa; AAV
5.  High-Resolution Images of Retinal Structure in Patients with Choroideremia 
Purpose.
To study retinal structure in choroideremia patients and carriers using high-resolution imaging techniques.
Methods.
Subjects from four families (six female carriers and five affected males) with choroideremia (CHM) were characterized with best-corrected visual acuity (BCVA), kinetic and static perimetry, full-field electroretinography, and fundus autofluorescence (FAF). High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). Coding regions of the CHM gene were sequenced.
Results.
Molecular analysis of the CHM gene identified a deletion of exons 9 to 15 in family A, a splice site mutation at position 79+1 of exon 1 in family B, deletion of exons 6 to 8 in family C, and a substitution at position 106 causing a premature stop in family D. BCVA ranged from 20/16 to 20/63 in carriers and from 20/25 to 5/63 in affected males. FAF showed abnormalities in all subjects. SD-OCT showed outer retinal layer loss, outer retinal tubulations at the margin of outer retinal loss, and inner retinal microcysts. Patchy cone loss was present in two symptomatic carriers. In two affected males, cone mosaics were disrupted with increased cone spacing near the fovea but more normal cone spacing near the edge of atrophy.
Conclusions.
High-resolution retinal images in CHM carriers and affected males demonstrated RPE and photoreceptor cell degeneration. As both RPE and photoreceptor cells were affected, these cell types may degenerate simultaneously in CHM. These findings provide insight into the effect of CHM mutations on macular retinal structure, with implications for the development of treatments for CHM. (ClinicalTrials.gov number, NCT00254605.)
High-resolution retinal images in choroideremia carriers and affected males demonstrated degeneration of retinal pigment epithelial and photoreceptor cells. The findings illustrate the effect of CHM mutations on macular cone structure, with implications for the development of treatments for CHM.
doi:10.1167/iovs.12-10707
PMCID: PMC3564452  PMID: 23299470
6.  Molecular Basis for Rab Prenylation 
The Journal of Cell Biology  2000;150(1):89-104.
Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.
PMCID: PMC2185574  PMID: 10893259
choroideremia; REP1; CHM; vesicle traffic; MRS6
7.  Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients 
PLoS ONE  2009;4(12):e8402.
Background
Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients.
Methodology/Principal Findings
To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo™ BioParticles® conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ™ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8.
Conclusions/Significance
We demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population.
doi:10.1371/journal.pone.0008402
PMCID: PMC2793004  PMID: 20027300
8.  The functional effect of pathogenic mutations in Rab escort protein 1 
Mutation research  2009;665(1-2):44-50.
Choroideremia (CHM) is a chorioretinal degeneration with an X-linked pattern of inheritance. Affected males experience progressive atrophy of the choroid, retinal pigment epithelium and retina leading to eventual blindness. The CHM gene encodes Rab escort protein 1 (REP-1). REP-1 is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of REP-1 function. Here we report pathogenic mutations: a novel missense mutation, L550P; a truncation c.1542T>A, STOP; and two deletions (c.525_526delAG, c.1646delC) in the CHM gene and their phenotypic effect. To analyze the effect of mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function were modeled using sequence homology with rat proteins. In silico analysis of the missense mutation L550P suggests that the proline residue at position 550 destabilizes the β-structural elements, and the REP-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein-protein interactions as predicted by the analysis of the structure and stability of these protein products. The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts (FB) from CHM patients.
doi:10.1016/j.mrfmmm.2009.02.01
PMCID: PMC2680797  PMID: 19427510
Choroideremia; Rab escort protein 1; missense mutation; structural mechanism; protein destabilization
9.  Choroideremia: New Findings from Ocular Pathology and Review of Recent Literature 
Survey of ophthalmology  2009;54(3):401-407.
Histopathology of young individuals affected by choroideremia is rarely available to allow correlation with the clinical presentation. A 30-year-old male with choroideremia died in a motor vehicle accident and one eye was subjected to histopathological examination. Immunoblot analysis of protein derived from white blood cells of a living brother, also affected with choroideremia, confirmed the absence of Rab escort protein-1, the normal CHM gene product. Direct sequencing of the coding region and adjacent splice sites of the CHM gene was undertaken on genomic DNA from the living brother and revealed a transition mutation, C to T, in exon 6 (R253X) which resulted in a stop codon and was predicted to truncate the protein product. Histopathological examination of the eye of the deceased brother showed relative independent degeneration of choriocapillaris, retinal pigment epithelium and retina, similar to observations in the mouse model of choroideremia. In addition, mild T-lymphocytic infiltration was found within the choroid. The ophthalmic features and the pathology of choroideremia are discussed in light of new findings in the current case.
doi:10.1016/j.survophthal.2009.02.008
PMCID: PMC2679958  PMID: 19422966
choroideremia; histopathology; mutation analysis; retinal degeneration
10.  Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models 
BMC Cell Biology  2002;3:26.
Background
Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.
Results
To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.
Conclusions
We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.
doi:10.1186/1471-2121-3-26
PMCID: PMC137576  PMID: 12401133
11.  AAV-Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models 
PLoS ONE  2013;8(5):e61396.
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1st or 2nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.
doi:10.1371/journal.pone.0061396
PMCID: PMC3646845  PMID: 23667438
12.  Molecular genetic diagnostic techniques in choroideremia 
Molecular Vision  2014;20:535-544.
Purpose
To optimize and streamline molecular genetics techniques in diagnosing choroideremia (CHM).
Methods
PCR primers were designed for exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15 of the CHM gene. Each PCR protocol was optimized so that all exons could be amplified with the same component ratio and PCR conditions. Sense and antisense primers were tested for their ability to be used as sequencing primers. Fibroblast cells were cultured, and an immunoblot analysis was performed to detect the presence or absence of Rab escort protein 1 (REP-1) in a suspected CHM patient sample when no mutation was detected with sequencing. Multiplex ligation-dependent probe amplification (MLPA) of the CHM gene was performed and used to detect deletions and duplications in affected males and female carriers. RNA analysis using cDNA was used to detect the presence or absence of the CHM transcript and to search for splice defects.
Results
The newly designed PCR primers allow for more efficient PCR preparation and sequencing to detect point mutations in affected males and female carriers. Immunoblot successfully detects the absence of REP-1 in a CHM patient. MLPA identifies deletions and duplications spanning multiple exons in the CHM gene. RNA analysis aids in detecting splice variants.
Conclusions
The development of new molecular biology techniques and ongoing optimization of existing methods allows for an improved integrated approach to confirm CHM diagnosis and carrier status in consideration of patient family history and available patient sample materials. CHM can be confirmed with an immunoblot assay. To detect the molecular cause of CHM, an examination of the genomic DNA or the mRNA must be performed. Presymptomatic carriers with no identifiable fundus signs can be identified only through molecular analysis of genomic DNA or through quantitative assays.
PMCID: PMC4000712  PMID: 24791138
13.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
Background
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
Conclusions
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Background.
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347.
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
doi:10.1371/journal.pmed.0030347
PMCID: PMC1592340  PMID: 17032058
14.  Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial 
Lancet  2014;383(9923):1129-1137.
Summary
Background
Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease.
Methods
In a multicentre clinical trial, six male patients (aged 35–63 years) with choroideremia were administered AAV.REP1 (0·6–1·0×1010 genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213.
Findings
Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8–3·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm2 of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p>0·05) were noted in the control eyes in both maximal sensitivity (–0·8 dB [1·5]) and mean sensitivity (–1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector.
Interpretation
The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning.
Funding
UK Department of Health and Wellcome Trust.
doi:10.1016/S0140-6736(13)62117-0
PMCID: PMC4171740  PMID: 24439297
15.  Transition Zones between Healthy and Diseased Retina in Choroideremia (CHM) and Stargardt Disease (STGD) as Compared to Retinitis Pigmentosa (RP) 
The transition zone between healthy and severely affected regions of the retina differ in structural abnormalities in patients with choroideremia, Stargardt disease, or retinitis pigmentosa.
Purpose.
To describe the structural changes across the transition zone (TZ) in choroideremia (CHM) and Stargardt disease (STGD) and to compare these to the TZ in retinitis pigmentosa (RP).
Methods.
Frequency-domain (Fd)OCT line scans were obtained from seven patients with CHM, 20 with STGD, and 12 with RP and compared with those of 30 previously studied controls. A computer-aided manual segmentation procedure was used to determine the thicknesses of the outer segment (OS) layer, the outer nuclear layer plus outer plexiform layer (ONL+), the retinal pigment epithelium plus Bruch's membrane (RPE+BM), and the outer retina (OR).
Results.
The TZ, while consistent within patient groups, showed differences across disease groups. In particular, (1) OS loss occurred before ONL+ loss in CHM and RP, whereas ONL+ loss occurred before OS loss in STGD; (2) ONL+ was preserved over a wider region of the retina in CHM than in RP; (3) RPE+BM remained normal across the RP TZ, but was typically thinned in CHM. In some CHM patients, it was abnormally thin in regions with normal OS and ONL+ thickness. In STGD, RPE+BM was thinned by the end of the TZ; and (4) the disappearances of the IS/OS and OLM were more abrupt in CHM and STGD than in RP.
Conclusions.
On fdOCT scans, patients with RP, CHM, and STGD all have a TZ between relatively healthy and severely affected retina. The patterns of changes in the receptor layers are similar within a disease category, but different across categories. The findings suggest that the pattern of progression of each disease is distinct and may offer clues for strategies in the development of future therapies.
doi:10.1167/iovs.11-8554
PMCID: PMC3341121  PMID: 22076985
16.  CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents 
PLoS Medicine  2008;5(2):e39.
Background
In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.
Methods and Findings
We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.
Conclusions
CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.
Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium.
Editors' Summary
Background.
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in industrialized countries. The macula is the central region of the retina, the tissue at the back of the eye that detects light and converts it into electrical messages that are sent to the brain. In the commonest form of AMD—“dry” AMD—the light-sensitive cells in the retina (the photoreceptors) gradually die. This degeneration might occur because of damage to the retinal pigment epithelium (RPE). This layer of dark cells lies between the photoreceptors and the choroid, the layer of the eye that contains blood vessels and brings oxygen to the retina. The RPE keeps the retina healthy by transferring the right amount of oxygen and nutrients from the choroid to the retina and by removing worn-out photoreceptor outer segments (the part of the photoreceptor that actually absorbs light) in a process called phagocytosis (engulfment and digestion). In addition to photoreceptor degeneration and RPE shrinkage, a layer of the choroid rich in small blood vessels (the choriocapillaris) also shrinks in dry AMD. For affected individuals, all these changes (which experts describe as retinal atrophy and choroidal involution) mean that the sharp central vision that is needed for reading and driving is destroyed, leaving only dim, burred images or a black hole at the center of the vision.
Why Was This Study Done?
Little is known about the molecular mechanisms that underlie dry AMD and, consequently, there is no cure for it. In this study, the researchers have tested whether a molecule called CD36, which is expressed on the surface of RPE cells, is involved in dry AMD. CD36 is a scavenger receptor—which means it binds many potentially harmful molecules including oxidized fats (which are present in the photoreceptor outer segments) and is involved in their phagocytosis. Phagocytosis itself induces the expression of several proteins in the RPE cells, including COX2, a “proangiogenic” protein that stimulates the growth of blood vessels. Putting this information together, the researchers hypothesized that a defect in CD36 might cause the characteristic retinal atrophy (by preventing the phagocytosis of worn-out photoreceptor outer segments) and choroidal involution (by preventing the induction of COX2 expression and consequently the maintenance of the blood vessels in the choroid) of dry AMD.
What Did the Researchers Do and Find?
The researchers first show that retinal degeneration occurs in rats and mice that express no CD36. This degeneration (which included a reduction in the thickness of the retina, the presence of irregularly shaped photoreceptor outer segments, and the detachment of these structures from the RPE) was seen in old but not young animals. Choroidal involution was also seen in these CD36-deficient animals. This change was present in young mice and rats but increased with age so that by one year old, the choriocapillaris looked moth-eaten. Next, the researchers show that although RPE cells taken from normal animals and grown in dishes were able to make COX2 in response to exposure to purified photoreceptor outer segments, RPE cells from CD36-deficient animals did not. The expression of vascular endothelial growth factor (VEGF; a protein that is needed for normal choroidal development and whose expression is controlled by COX2) showed a similar pattern. Finally, the researchers report that COX2 deficiency in mice caused similar age-dependent choroidal involution and similar effects on VEGF expression in RPE cells as CD36 deficiency.
What Do These Findings Mean?
These findings show that CD36 deficiency leads to progressive, age-related degeneration of photoreceptors and choroidal involution in rats and mice. They also show that CD36 deficiency causes this choroidal involution, the key feature of dry AMD, because it leads to down-regulation of COX2 expression (and subsequently reduced VEGF expression) in the RPE. Researchers now need to find out whether this mechanism for the development of dry AMD holds in people—what happens in animals does not necessarily happen in people. If it does, pharmacological activation of CD36 or restoration of CD36 expression in the RPE might eventually provide a way to treat dry AMD.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050039.
MedlinePlus provides links to information on macular degeneration and an encyclopedia page on macular degeneration (in English and Spanish)
Pages on the US National Institutes of Health NIH SeniorHealth site provides text and spoken information about AMD
The US National Eye Institute and the UK Royal National Institute of Blind People also provide information about AMD
Wikipedia has pages on the retina, photoreceptor cells, retinal pigment epithelium, and choroid (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050039
PMCID: PMC2245984  PMID: 18288886
17.  Molecular analysis of the choroideremia gene related clinical findings in two families with choroideremia 
Molecular Vision  2011;17:2564-2569.
Purpose
To investigate the choroideremia (CHM) gene in two families with CHM and to characterize the related clinical features.
Methods
Two families underwent complete ophthalmic examinations and three males were diagnosed with CHM. Genomic DNA was extracted from the leukocytes of peripheral blood collected from the two families and from 100 unrelated control subjects from the same population. Exons 1–15 of CHM were amplified by PCR and directly sequenced. Ophthalmic examinations included best-corrected visual acuity, slit-lamp examination, fundus examination, visual field, optical coherence tomography, electroretinogram, and Pentacam.
Results
The affected men were hemizygous and had night blindness, chorioretinal atrophy spreading from the posterior pole to the mid-periphery, and bareness of the sclera. A novel c.1488delGinsATAAC mutation was detected in CHM in family 1. Another mutation c.1703 C>G (S558X) within exon 14 of CHM was identified in family 2, which caused the serine 558 codon (TCA) to be changed to a stop codon (TGA).
Conclusions
This study identified a novel mutation in CHM associated with CHM and its related clinical features. Our findings expand the genotypic spectrum of CHM mutations associated with CHM and confirm the role of Rab escort protein-1 in the pathogenesis of CHM.
PMCID: PMC3198496  PMID: 22025891
18.  Clinical and genetic studies in a family with a new splice-site mutation in the choroideremia gene 
Molecular Vision  2014;20:325-333.
Purpose
To describe the clinical and molecular findings of an Italian family with a new mutation in the choroideremia (CHM) gene.
Methods
We performed a comprehensive ophthalmologic examination, fundus photography, macular optical coherence tomography, perimetry, electroretinography, and fluorescein angiography in an Italian family. The clinical diagnosis was supported by western blot analysis of lymphoblastoid cell lines from patients with CHM and carriers, using a monoclonal antibody against the 415 C-terminal amino acids of Rab escort protein-1 (REP-1). Sequencing of the CHM gene was undertaken on genomic DNA from affected men and carriers; the RNA transcript was analyzed with reverse transcriptase-PCR.
Results
The affected men showed a variability in the rate of visual change and in the degree of clinical and functional ophthalmologic involvement, mainly age-related, while the women displayed aspecific areas of chorioretinal degeneration. Western blot did not show a detectable amount of normal REP-1 protein in affected men who were hemizygous for a novel mutation, c.819+2T>A at the donor splicing site of intron 6 of the CHM gene; the mutation was confirmed in heterozygosity in the carriers.
Conclusions
Western blot of the REP-1 protein confirmed the clinical diagnosis, and molecular analysis showed the new in-frame mutation, c.819+2T>A, leading to loss of function of the REP-1 protein. These results emphasize the value of a diagnostic approach that correlates genetic and ophthalmologic data for identifying carriers in families with CHM. An early diagnosis might be crucial for genetic counseling of this type of progressive and still untreatable disease.
PMCID: PMC3962727  PMID: 24672218
19.  Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1 
eLife  2013;2:e00324.
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life.
DOI: http://dx.doi.org/10.7554/eLife.00324.001
eLife digest
The inner surface of the vertebrate eye is lined with a multilayered structure known as the retina. The bottom layer of the retina is composed of rods and cones—neurons that are directly sensitive to light—and is called the photoreceptor layer. Rods function primarily in dim light and provide black-and-white vision, while cones support daytime vision and are responsible for colour perception. Unlike the upper layers of the retina, the photoreceptor layer does not contain blood vessels: oxygen and nutrients are instead provided by a structure just underneath the retina called the choroid.
The eye relies on the rods and cones converting light into electrical signals, and the photoreceptor layer must remain free of blood vessels for this process to work properly. If blood vessels extend into the photoreceptor layer from rest of the retina (which is above it) or the choroid (below), they can disrupt the retina and give rise to a condition called age-related macular degeneration, which is a leading cause of irreversible blindness in adults.
Within the eye, the development of new blood vessels from pre-existing vessels is stimulated by a protein known as vascular endothelial growth factor A (VEGF-A), while an inhibitor protein called sFLT-1 prevents the growth of new blood vessels in the other tissues of the eye like the cornea. However, it has not been clear what keeps the photoreceptor layer (and also the cells that support the photoreceptor layer) free of blood vessels, and what happens to disrupt this process of vascular demarcation in age-related macular degeneration.
Now, Luo et al. reveal that cells in the photoreceptor layer produce sFLT-1, and that the levels of this protein are indeed reduced in people with age-related macular degeneration. Using genetic and pharmacological methods, they show that a reduction in sFLT-1 triggers blood vessels to grow into the photoreceptor layer from above or below. Luo et al. also report two new genetic mouse models in which blood vessels form spontaneously in the photoreceptor layer at an early age, which should prove useful for further research into age-related macular degeneration.
DOI: http://dx.doi.org/10.7554/eLife.00324.002
doi:10.7554/eLife.00324
PMCID: PMC3687373  PMID: 23795287
age-related macular degeneration; photoreceptor metabolism; retinal vasculature; soluble VEGF receptor-1; vascular demarcation; transgenic model; Human; Mouse
20.  Otx2 Gene Deletion in Adult Mouse Retina Induces Rapid RPE Dystrophy and Slow Photoreceptor Degeneration 
PLoS ONE  2010;5(7):e11673.
Background
Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells.
Methodology/Principal Findings
Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease.
Conclusions
Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.
doi:10.1371/journal.pone.0011673
PMCID: PMC2908139  PMID: 20657788
21.  X-Box Binding Protein 1 Is Essential for the Anti-Oxidant Defense and Cell Survival in the Retinal Pigment Epithelium 
PLoS ONE  2012;7(6):e38616.
Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.
doi:10.1371/journal.pone.0038616
PMCID: PMC3371004  PMID: 22715395
22.  Mitochondrial Oxidative Stress in the Retinal Pigment Epithelium Leads to Localized Retinal Degeneration 
Purpose.
Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer.
Methods.
Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2flox/floxVMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2flox/flox mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology.
Results.
Following doxycycline induction of cre, Sod2flox/flox cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2flox/flox cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes.
Conclusions.
Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.
The RPE-specific deletion of mitochondrial superoxide dismutase in mice leads to oxidative stress in the RPE, accumulation of autofluorescent material, a decline in the ERG response, and localized death of photoreceptors. This model may be useful in understanding the mechanism of geographic atrophy.
doi:10.1167/iovs.14-14633
PMCID: PMC4112607  PMID: 24985474
retinal degeneration; mouse model; reactive oxygen species; manganese superoxide dismutase; retinal pigment epithelium
23.  Genetic and phenotypic characteristics of three Mainland Chinese families with choroideremia 
Molecular Vision  2012;18:309-316.
Purpose
To describe the phenotype and genotype of three Mainland Chinese families affected by choroideremia (CHM).
Methods
Complete ophthalmic examinations were conducted in three unrelated Chinese families with CHM. Peripheral blood samples were collected from the families for genetic and immunoblot analysis. All exons and flanking intronic regions of the gene encoding Rab escort protein-1 (Rep-1) were amplified with PCR and screened for mutations with Sanger sequencing. The three-dimensional structure of mutated Rep-1 was modeled using sequence homology with rat proteins to analyze the effect of the mutation detected in one family.
Results
All affected males had characteristic signs and symptoms of CHM; however, central visual acuity impairment occurred earlier than expected. All female carriers older than 45 years had pigmentary changes, and one female carrier was symptomatic with vision loss. Three different mutations in Rep-1, c.1801–1G>A, c.1130 T>A, and c.612delAG, were detected in the three families.
Conclusions
In Mainland Chinese families, the central visual acuity of male patients with CHM can be affected at an early age (second decade), whereas female CHM carriers may manifest signs and symptoms at a later age (≥45 years). One previously reported and two novel Rep-1 mutations were detected in three Chinese patients with CHM.
PMCID: PMC3283217  PMID: 22355242
24.  Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration 
PLoS ONE  2009;4(8):e6616.
Pathogenesis in the Rpe65−/− mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65−/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.
Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65−/−/Gnat1−/− mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65−/−/Bax−/− mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65−/− mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65−/−/Bax−/− mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.
This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.
doi:10.1371/journal.pone.0006616
PMCID: PMC2720534  PMID: 19672311
25.  Choroideremia: Analysis of the retina from a Female Symptomatic Carrier 
Ophthalmic genetics  2008;29(3):99-110.
Purpose
To define the retinal pathology in a 91 year-old affected matriarch of a three-generation choroideremia family with multiple manifesting carriers.
Methods
Tissue from three different retinal areas was processed for immunohistochemistry. The macular area was processed for transmission electron microscopy. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to cone cytoplasm, rhodopsin and cone opsins. The affected donor eyes were compared to a postmortem matched normal eye.
Results
The retina displayed areas of severe degeneration, with no photoreceptor outer segments, photoreceptor nuclear atrophy, and atrophy of the inner retina. Other retinal areas were near to normal. The RPE was severely degenerated, with thinning, pigment clumping and sub-epithelial debris deposition in all the areas examined. The choroid displayed depigmentation. Labeling with cone opsin antibodies revealed that cones were drastically affected: blue opsin was almost completely absent, while red/green opsins were distributed along the entire plasma membrane of the cell. Rhodopsin was also distributed along the entire rod plasma membrane. Ultrastructural analysis of the affected macula revealed the absence of RPE apical microvilli and basal infoldings. Instead, RPE’s basal surface and choroid displayed the presence of banded fibers composed of clumps of wide-spacing collagen. Bruch’s membrane was filled with vesicular structures, some smooth and others with bristle-like projections.
Conclusions
The histological data suggests that the clinical manifestation in this donor is related to degenerative changes in the retina, RPE and choroid.
doi:10.1080/13816810802206499
PMCID: PMC3652314  PMID: 18766988
Choroideremia; carrier state; immunohistochemistry; cone opsins; rhodopsin

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