Photoreceptor Outer Nuclear Layer Research Articles

AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

Macular outer nuclear layer, ellipsoid zone and outer photoreceptor segment band thickness, axial length and other determinants

The study aims to assess the thickness of the retinal outer nuclear layer (ONL), ellipsoid zone (EZ) and photoreceptor outer segment (POS) band in various macular regions and its associations with axial length and other parameters. Participants of the Beijing Eye Study 2011 underwent a series of examinations including spectral-domain optical coherence tomography of the macula. The current study included 2213 participants without retinal or optic nerve diseases (age: 61.7 ± 8.4 years; range 50–93 years); axial length: 23.15 ± 0.95 mm; range 18.96–29.15 mm). The ONL (fovea: 98.9 ± 8.8 µm), EZ (fovea: 24.1 ± 0.5 µm) and POS band (fovea: 24.3 ± 3.5 µm) were the thickest (P < 0.001) in the fovea (defined as the thinnest central point), followed by the temporal inner, nasal inner, inferior inner, superior inner, inferior outer, temporal outer, nasal outer, and superior outer region. In multivariable analysis, a thicker retinal ONL was associated (correlation coefficient r: 0.40) with shorter axial length (beta: − 0.14; P < 0.001) and shorter disc–fovea distance (beta: − 0.10; P = 0.001), after adjusting for younger age (beta: − 0.26; P < 0.001), male sex (beta: 0.24; P < 0.001), lower serum cholesterol concentration (beta: − 0.05; P = 0.04), and thicker subfoveal choroidal thickness (beta: 0.08; P < 0.001). The POS thickness increased with shorter axial length (beta: − 0.06; P < 0.001) and shorter optic disc–fovea distance (beta: − 0.05; P = 0.03), after adjusting for younger age (beta: − 0.34; P < 0.001), male sex (beta: 0.15; P < 0.001), and thicker subfoveal choroidal thickness (beta: 0.24; P < 0.001). As a conclusion, the photoreceptor ONL, EZ and POS band vary in thickness between different macular regions and differ in their correlations with axial length, disc–fovea distance, age, sex, and subfoveal choroidal thickness. The ONL thickness decrease with longer axial length and longer disc–fovea distance may point to an axial elongation-associated retinal stretching in the macula.

A comparison of optophysiological biomarkers of photoreceptor stress and phototoxicity in BALB/cJ, B6 (Cg)-Tyrc-2J/J, and C57Bl/6J mouse strains.

Ophthalmic imaging instruments, including the confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography system, originally intended for revealing ocular microstructures in the human eye, have been deployed by vision researchers to evaluate the eyes of numerous small and large animal species for more than two decades. In this study, we have used these two instruments to obtain imaging data sequentially from the retinas of three prominent, widely used experimental mouse models to document changes induced by two contrasting vivarium lighting conditions. Mice studied include albino BALB/cJ and B6(Cg)-Tyrc-2J/J and pigmented C57Bl/6J. Mice were reared under dim light conditions until ~8 weeks of age where they underwent baseline imaging. Following, mice were returned to the dim vivarium or relocated to the top rack cage position in a standard vivarium. Mice were then followed for several months by ocular imaging to catalog the retinal dynamics as a function of long-term dim vs. elevated, standard vivarium lighting exposure levels. Upon exposure to elevated light levels, B6(Cg)-Tyrc-2J/J underwent similar changes as BALB/cJ in regard to photoreceptor outer segment shortening, photoreceptor layer proximal aspect hyperreflective changes, and the development of retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate. Noteworthy, however, is that infoldings and infiltrate occurred at a slower rate of progression in B6(Cg)-Tyrc-2J/J vs. BALB/cJ. The photoreceptor outer nuclear layer thickness of BALB/cJ degenerated steadily following elevated light onset. In contrast, B6(Cg)-Tyrc-2J/J degeneration was unremarkable for many weeks before experiencing a noticeable change in the rate of degeneration that was concomitant with a plateau and decreasing trend in number of retinal infoldings and monocyte infiltrate. Pathological changes in C57Bl/6J mice were unremarkable for all imaging biomarkers assessed with exception to autofluorescent sub-retinal inflammatory monocyte infiltrate, which showed significant accumulation in dim vs. elevated light exposed mice following ~1 year of observation. These data were evaluated using Spearman's correlation and Predictive Power Score matrices to determine the best imaging optophysiological biomarkers for indicating vivarium light stress and light-induced photoreceptor degeneration. This study suggests that changes in proximal aspect hyperreflectivity, outer segment shortening, retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate are excellent indicators of light stress and light-induced degeneration in albino B6(Cg)-Tyrc-2J/J and BALB/cJ mouse strains.

Retinal photoreceptor layer thickness has disease specificity and distinguishes predicted FTLD-Tau from biomarker-determined Alzheimer's disease

While Alzheimer's disease (AD) is associated with inner retina thinning (retinal nerve fiber layer and ganglion cell layer), we have observed photoreceptor outer nuclear layer (ONL) thinning in patients with frontotemporal lobar degeneration tauopathy (FTLD-Tau) compared to normal controls. We hypothesized that ONL thinning may distinguish FTLD-Tau from patients with biomarker evidence of AD neuropathologic change (ADNC) and will correlate with FTLD-Tau disease severity.Predicted FTLD-Tau (pFTLD-Tau; n = 21; 33 eyes) and predicted ADNC (pADNC; n = 24; 46 eyes) patients were consecutively enrolled, underwent optical coherence tomography macula imaging, and disease was categorized (pFTLD-Tau vs. pADNC) with cerebrospinal fluid biomarkers, genetic testing, and autopsy data when available. Adjusting for age, sex, and race, pFTLD-Tau patients had a thinner ONL compared to pADNC, while retinal nerve fiber layer and ganglion cell layer were not significantly different. Reduced ONL thickness correlated with worse performance on Folstein Mini-Mental State Examination and clinical dementia rating plus frontotemporal dementia sum of boxes for pFTLD-Tau but not pADNC. Photoreceptor ONL thickness may serve as an important noninvasive diagnostic marker that distinguishes FTLD-Tau from AD neuropathologic change.

Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2weeks post transplantation as measured by full-field electroretinogram. At 4weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

Gasdermin E mediates photoreceptor damage by all-trans-retinal in the mouse retina

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4−/−Rdh8−/− mice exhibit serious defects in atRAL clearance upon light exposure and serve as an acute model for dry AMD and STGD1. We found that N-terminal fragment of GSDME was distinctly localized in the photoreceptor outer nuclear layer of light-exposed Abca4−/−Rdh8−/− mice. Of note, degeneration and caspase-3 activation in photoreceptors were significantly alleviated in Abca4−/−Rdh8−/−Gsdme−/− mice after exposure to light. The results of this study indicate that GSDME is a common causative factor of photoreceptor pyroptosis and apoptosis arising from atRAL overload, suggesting that repressing GSDME may represent a potential treatment of photoreceptor atrophy in dry AMD and STGD1.

Retina tissue validation of optical coherence tomography determined outer nuclear layer loss in FTLD-tau

Alzheimer’s disease (AD) is associated with inner retina (nerve fiber and ganglion cell layers) thinning. In contrast, we have seen outer retina thinning driven by photoreceptor outer nuclear layer (ONL) thinning with antemortem optical coherence tomography (OCT) among patients considered to have a frontotemporal degeneration tauopathy (FTLD-Tau). Our objective was to determine if postmortem retinal tissue from FTLD-Tau patients demonstrates ONL loss observed antemortem on OCT. Two probable FTLD-Tau patients that were deeply phenotyped by clinical and genetic testing were imaged with OCT and followed to autopsy. Postmortem brain and retinal tissue were evaluated by a neuropathologist and ocular pathologist, respectively, masked to diagnosis. OCT findings were correlated with retinal histology. The two patients had autopsy-confirmed FTLD-Tau neuropathology and had antemortem OCT measurements showing ONL thinning (66.9 μm, patient #1; 74.9 μm, patient #2) below the 95% confidence interval of normal limits (75.1–120.7 μm) in our healthy control cohort. Postmortem, retinal tissue from both patients demonstrated loss of nuclei in the ONL, matching ONL loss visualized on antemortem OCT. Nuclei counts from each area of ONL loss (2 – 3 nuclei per column) seen in patient eyes were below the 95% confidence interval (4 – 8 nuclei per column for ONL) of 3 normal control retinas analyzed at the same location. Our evaluation of retinal tissue from FTLD-Tau patients confirms ONL loss seen antemortem by OCT. Continued investigation of ONL thinning as a biomarker that may distinguish FTLD-Tau from other dementias is warranted.

Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model.

Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in approximately 25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1M30R/M390R;BBS1TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1M300R/M390R;BBS1TG+ mice. The results of this study indicate the male infertility aspect of BBS is an attractive target for gene therapy.

Retinal structure in Leber\u2019s congenital amaurosis caused by RPGRIP1 mutations

This study aimed to evaluate retinal structure in the early stage of Leber’s congenital amaurosis (LCA) caused by RPGRIP1 mutations. Four patients from two families were included. Case 1 was a 13-year-old girl, cases 2 and 3 were 7-year-old monozygotic twin brothers of case 1, and case 4 was a 17-year-old boy. Comprehensive ophthalmic examinations were performed, including visual acuity measurements, perimetry, electroretinography (ERG), and optical coherence tomography (OCT). To identify potential pathogenic mutations, 74 genes known to cause retinitis pigmentosa or LCA were assessed using targeted next-generation sequencing. OCT showed photoreceptor outer nuclear layer (ONL) thinning in all patients. The lamellar structure was retained in all patients, whereas the ellipsoid zone was extinguished in cases 1, 2, and 3. In case 4, the ellipsoid zone was maintained at 9 years of age but became blurred at 17 years of age. In case 1, OCT indicated slight photoreceptor ONL thinning during the period between 7 and 11 years of age. Mutation analysis revealed RPGRIP1 mutations as the cause for autosomal recessive LCA in all patients. Photoreceptor ONL on OCT is relatively well preserved in the early stage of LCA caused by RPGRIP1 mutations.

Blue Autofluorescence Fundus Imaging for Monitoring Retinal Degeneration in Royal College of Surgeons Rats.

PurposeDevelopment of a method for noninvasive longitudinal follow-up of retinal degeneration in the whole retina for Royal College of Surgeons (RCS) rats, a commonly used model of retinitis pigmentosa associated with mutations in the MER-proto-oncogene tyrosine kinase (MERTK) gene.MethodsPigmented RCS rats at postnatal (p) days p28 to p84 were subjected to a biweekly spectral-domain optical coherence tomography (SD-OCT), blue laser fundus autofluorescence (BL-FAF) imaging, and multicolor fundus imaging. Wild-type (WT; Long Evans) rats were tested as control.ResultsHyperautofluorescence developed throughout the fundus at p42, concomitant with a significant increase in SD-OCT thickness and reflectivity of the debris zone (DZ) layer as well as thinning of the photoreceptor outer nuclear layer (ONL). From p56 to p84, discrete hypofluorescent lesions surrounded by hyperfluorescent flecks were demonstrated around the optic disc that gradually spread throughout the retina. The hypofluorescent lesions were associated with loss of ONL and gradual thinning of the DZ layer. No hypofluorescent BL-FAF lesions were observed in WT rats.ConclusionsThis study suggests that BL-FAF imaging may present a new method for noninvasive longitudinal follow-up of retinal degeneration in nearly the whole retina in RCS rats.Translational RelevanceA clinical test was developed that may be implemented in translational studies in the RCS rat model of MERTK-associated retinitis pigmentosa.

Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography.

PurposeSpectral-domain optical coherence tomography (SD-OCT) was used to characterize the retinal phenotype, natural history, and treatment responses in a mouse model of X-linked retinoschisis (Rs1-KO) and to identify new structural markers of AAV8-mediated gene therapy outcome.MethodsOptical coherence tomography scans were performed on wild-type and Rs1-KO mouse retinas between 1 and 12 months of age and on Rs1-KO mice after intravitreal injection of AAV8-scRS/IRBPhRS (AAV8-RS1). Cavities and photoreceptor outer nuclear layer (ONL) thickness were measured, and outer retina reflective band (ORRB) morphology was examined with age and after AAV8-RS1 treatment. Outer retina reflective band morphology was compared to immunohistochemical staining of the outer limiting membrane (OLM) and photoreceptor inner segment (IS) mitochondria and to electron microscopy (EM) images of IS.ResultsRetinal cavity size in Rs1-KO mice increased between 1 and 4 months and decreased thereafter, while ONL thickness declined steadily, comparable to previous histologic studies. Wild-type retina had four ORRBs. In Rs1-KO, ORRB1was fragmented from 1 month, but was normal after 8 months; ORRB2 and ORRB3 were merged at all ages. Outer retina reflective band morphology returned to normal after AAV-RS1 therapy, paralleling the recovery of the OLM and IS mitochondria as indicated by anti–β-catenin and anti-COX4 labeling, respectively, and EM.ConclusionsSpectral-domain OCT is a sensitive, noninvasive tool to monitor subtle changes in retinal morphology, disease progression, and effects of therapies in mouse models. The ORRBs may be useful to assess the outcome of gene therapy in the treatment of X-linked retinoschisis patients.

Molecular Heterogeneity Within the Clinical Diagnosis of Pericentral Retinal Degeneration.

To characterize in detail the phenotype and genotype of patients with pericentral retinal degeneration (PRD). Patients were screened for an annular ring scotoma ranging from 3° to 40° (n = 28, ages 24-71) with kinetic perimetry. All patients had pigmentary retinopathy in the region of the dysfunction. Further studies included cross-sectional and en face imaging, static chromatic perimetry, and electroretinography. Molecular screening was performed. Genotypes of 14 of 28 PRD patients were identified: There were mutations in eight different genes previously associated with autosomal dominant or autosomal recessive RDs. Kinetic fields monitored in some patients over years to more than a decade could be stable or show increased extent of the scotoma. Electroretinograms were recordable but with different severities of dysfunction. Patterns of photoreceptor outer nuclear layer (ONL) loss corresponded to the distribution of visual dysfunction. Outer nuclear layer thickness topography and en face imaging indicated that the greatest disease expression was in the area of known highest rod photoreceptor density. Molecular heterogeneity was a feature of the PRD phenotype. Many of the molecular causes were also associated with other phenotypes, such as maculopathies, typical retinitis pigmentosa (RP) and cone-rod dystrophy. The pericentral pattern of retinal degeneration is thus confirmed to be an uncommon phenotype of many different genotypes rather than a distinct disease entity.

Visual Cone Arrestin 4 Contributes to Visual Function and Cone Health.

Visual arrestins (ARR) play a critical role in shutoff of rod and cone phototransduction. When electrophysiological responses are measured for a single mouse cone photoreceptor, ARR1 expression can substitute for ARR4 in cone pigment desensitization; however, each arrestin may also contribute its own, unique role to modulate other cellular functions. A combination of ERG, optokinetic tracking, immunohistochemistry, and immunoblot analysis was used to investigate the retinal phenotypes of Arr4 null mice (Arr4-/-) compared with age-matched control, wild-type mice. When 2-month-old Arr4-/- mice were compared with wild-type mice, they had diminished visual acuity and contrast sensitivity, yet enhanced ERG flicker response and higher photopic ERG b-wave amplitudes. In contrast, in older Arr4-/- mice, all ERG amplitudes were significantly reduced in magnitude compared with age-matched controls. Furthermore, in older Arr4-/- mice, the total cone numbers decreased and cone opsin protein immunoreactive expression levels were significantly reduced, while overall photoreceptor outer nuclear layer thickness was unchanged. Our study demonstrates that Arr4-/- mice display distinct phenotypic differences when compared to controls, suggesting that ARR4 modulates essential functions in high acuity vision and downstream cellular signaling pathways that are not fulfilled or substituted by the coexpression of ARR1, despite its high expression levels in all mouse cones. Without normal ARR4 expression levels, cones slowly degenerate with increasing age, making this a new model to study age-related cone dystrophy.

Drusen and Photoreceptor Abnormalities in African-Americans with Intermediate Non-neovascular Age-related Macular Degeneration

Purpose/Aim: To investigate the relationship of drusen and photoreceptor abnormalities in African-American (AA) patients with intermediate non-neovascular age-related macular degeneration (AMD).Materials and methods: AA patients with intermediate AMD (n = 11; age 52–77 years) were studied with spectral-domain optical coherence tomography. Macular location and characteristics of large drusen (≥125 µm) were determined. Thickness of photoreceptor laminae was quantified overlying drusen and in other macular regions. A patient with advanced AMD (age 87) was included to illustrate the disease spectrum.Results: In this AA patient cohort, the spectrum of changes known to occur in AMD, including large drusen, sub-retinal drusenoid deposits and geographic atrophy, were identified. In intermediate AMD eyes (n = 17), there were 183 large drusen, the majority of which were pericentral in location. Overlying the drusen there was significant thinning of the photoreceptor outer nuclear layer (termed ONL+) as well as the inner and outer segments (IS + OS). The reductions in IS + OS thickness were directly related to ONL+ thickness. In a fraction (∼8%) of paradrusen locations with normal lamination sampled within ∼280 µm of peak drusen height, ONL+ was significantly thickened compared to age and retinal-location-matched normal values. Topographical maps of the macula confirmed ONL thickening in regions neighboring and distant to large drusen.Conclusions: We confirm there is a pericentral distribution of drusen across AA-AMD maculae rather than the central localization in Caucasian AMD. Reductions in the photoreceptor laminae overlying drusen are evident. ONL+ thickening in some macular areas of AA-AMD eyes may be an early phenotypic marker for photoreceptor stress.

Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild-type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca(2 +) responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih ) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild-type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2 +) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within 2 weeks after transplantation into the retina of Crx-knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter-driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell-replacement therapy.

Abnormal Thickening as well as Thinning of the Photoreceptor Layer in Intermediate Age-Related Macular Degeneration

To investigate the relationship between photoreceptor layers overlying and adjacent to large drusen in intermediate nonneovascular AMD. Patients with AMD (n = 41; aged 53-83 years) and elderly control subjects without eye disease (n = 10; aged 51-79 years) were studied with spectral-domain optical coherence tomography. Characteristics of large drusen (≥125 μm) were measured and the thickness of photoreceptor laminae overlying drusen and in retinal regions neighboring the drusen were quantified. There were 750 large drusen in 63 intermediate AMD eyes studied. The width of the drusen sampled averaged 352 μm (SD = 153) and the height averaged 78 μm (SD = 31). There was significant reduction of the photoreceptor outer nuclear layer (ONL) thickness overlying 92% of the drusen. The thickness of the layer corresponding to photoreceptor inner and outer segments above drusen was also reduced, and the reduction was proportional to ONL thickness. In a substantial fraction (~20%) of normally laminated paradrusen locations sampled within ~300 μm of peak drusen height, ONL thickness was significantly increased compared with age and retinal location-matched normal values. Topographical analyses of the macula showed ONL thickening occurring in paradrusen regions as well as retinal locations distant from drusen. Reductions in the photoreceptor laminae overlying drusen were detectable and this is consistent with histological studies revealing neuronal degeneration in AMD. ONL thickening in some macular areas of AMD eyes has not been previously reported and may be an early phenotypic marker for photoreceptor stress, as it has been speculated to be in hereditary retinal degenerations.

Temporal requirement of RPE‐derived VEGF in the development of choroidal vasculature

Vascular endothelial growth factor (VEGF-A or VEGF) is a potent growth factor for the development of retinal and choroidal vasculatures. To define the temporal requirement of the retinal pigmented epithelium (RPE)-derived VEGF in choroidal vascular development, we generated conditional VEGF knockout mice using an inducible Cre/lox system. The loss of the RPE-derived VEGF was confirmed with immunoblotting and immunohistochemistry. Retinal function and structure were assessed with electroretinography and histology, respectively. Choroidal vascular density was analyzed with computer-assisted semi-quantitative assay using fluorescently labeled choroidal flat-mounts. Induction of RPE-specific VEGF disruption at embryonic day 10 (E10) or E13 for 2 days caused regulatable decreases in choroidal vascular density, photoreceptor function, and photoreceptor outer nuclear layer thickness. The loss of the RPE-produced VEGF after E15 did not cause detectable defects in choroidal vasculatures and photoreceptor function and morphology. These results suggest that the RPE-derived VEGF plays a critical role in choroidal vascular development during organogenesis before E15.

SOD2 Knockdown Mouse Model of Early AMD

To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD). A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas. The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin. These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.

Gene Expression Analysis of Photoreceptor Cell Loss inBbs4-Knockout Mice Reveals an Early Stress Gene Response and Photoreceptor Cell Damage

To identify and characterize gene expression changes associated with photoreceptor cell loss in a Bbs4-knockout mouse model of retinal degeneration. Differential gene expression in the eyes of 5-month-old Bbs4(-/-) mice undergoing retinal degeneration were analyzed using gene microarrays (Affymetrix, Santa Clara, CA). Elevated ocular transcripts were confirmed by Northern blotting of RNA from Bbs4(-/-) and three additional mouse models of Bardet-Biedl Syndrome (BBS). TUNEL assays and transmission electron microscopy were used to study cell death and photoreceptor morphology in these mice. Three hundred fifty-four probes were differentially expressed in Bbs4(-/-) eyes compared with controls using a twofold cutoff. Numerous vision-related transcripts decreased because of photoreceptor cell loss. Increased expression of the stress response genes Edn2, Lcn2, Serpina3n, and Socs3 was noted at 5 months of age and as early as postnatal week 4 in the eyes of four BBS mouse model strains. A burst of apoptotic activity in the photoreceptor outer nuclear layer at postnatal week 2 and highly disorganized outer segments by postnatal weeks 4 to 6 was observed in all four strains. The specific loss of photoreceptors in Bbs4(-)(/)(-) mice allows us to identify a set of genes that are preferentially expressed in photoreceptors compared with other cell types found in the eye and is a valuable resource in the continuing search for genes involved in retinal disease. The molecular and morphologic changes observed in young BBS animal model eyes implies that BBS proteins play a critical, early role in establishing the correct structure and function of photoreceptors.

Loss of BCL-XLin Rod Photoreceptors: Increased Susceptibility to Bright Light Stress

BCL-X(L), an anti-apoptotic member of the BCL-2 family proteins and a cell death/survival checkpoint regulator, was shown to be upregulated in bright light-stressed mouse photoreceptors during an investigation of bright light-induced protein expression. To investigate the significance of BCL-X(L) upregulation in the bright light damage model, the Bcl-x gene was disrupted specifically in mouse rod photoreceptors, and the effect of Bcl-x disruption was characterized on retinal apoptosis, function, and morphology. Rod-specific Bcl-x knockout mice, generated by mating mouse opsin promoter-controlled Cre mice with floxed Bcl-x mice, were subjected to bright light stress. Retinal apoptosis in the bright light-stressed conditional Bcl-x knockout mice was characterized with TUNEL, DNA fragmentation, and nuclear staining assays. Photoreceptor structural and functional integrity in the bright light-stressed conditional Bcl-x knockout mice was determined by measuring photoreceptor outer nuclear layer (ONL) thickness and electroretinography amplitudes. Disruption of Bcl-x in rod photoreceptors caused increased photoreceptor apoptosis, decreased retinal function, and decreased ONL thickness in bright light-stressed mice. The loss of BCL-X(L) increased rod photoreceptor susceptibility to bright light stress. Although the biochemical mechanism(s) of BCL-X(L) in photoreceptor death or survival has not been investigated extensively, results of the present study suggest that BCL-X(L), a cell survival/death checkpoint regulator, is involved in photoreceptor survival under bright light stress.