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Abstract Trypanosoma cruzi can modulate a large number of host intracellular responses during its invasion. GTPases such as RhoA, Rac1 and Cdc42 are examples of molecules that could be activated at this moment and trigger changes in the pattern of F-actin cytoskeleton leading to the formation of structures like stress fibers, lamellipodium and fillopodium, respectively. Here we investigate the role of these GTPases in the cytoskeletal rearrangement of MDCK cell transfectants expressing variants of RhoA, Rac1 and Cdc42 during T. cruzi infection. The adhesion, internalization and the survival rate were determined. Rac1 mutants showed the higher adhesion and internalization indexes but the lower survival index after 48 h of infection. Confocal laser scanning microscopy showed changes in the pattern of F-actin distribution and reorganization at the site of trypomastigote invasion. These observations suggest that these GTPases act in the signaling mechanisms that affect the F-actin cytoskeleton during T. cruzi invasion. [ABSTRACT FROM AUTHOR]

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the Polg D257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed in vivo and ex vivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

The sodium iodate (NaIO 3 ) model of increased oxidative stress recapitulates dry AMD features such as patchy RPE loss, secondary photoreceptors, and underlying choriocapillaris death, allowing longitudinal evaluation of the retinal structure. Due to the time- and dose-dependent degeneration observed in diverse animal models, this preclinical model has become one of the most studied models. The events leading to RPE cell death post- NaIO 3 injection have been extensively studied, and here we have reviewed different modalities of cell death, including apoptosis, necroptosis, ferroptosis, and pyroptosis with a particular focus on findings associated with in vivo and in vitro NaIO 3 studies on RPE cell death. Because the fundamental cause of vision loss in patients with dry AMD is the death of these same cells affected by NaIO 3 , studies using NaIO 3 can provide valuable insights into RPE and photoreceptor cell death mechanisms and can help understand mechanisms behind RPE degeneration in AMD., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Heparan-α-glucosaminide N-acetyltransferase (HGSNAT) participates in lysosomal degradation of heparan sulfate. Mutations in the gene encoding this enzyme cause mucopolysaccharidosis IIIC (MPS IIIC) or Sanfilippo syndrome type C. MPS IIIC patients exhibit progressive neurodegeneration, leading to dementia and death in early adulthood. Currently there is no approved treatment for MPS IIIC. Incidences of non-syndromic retinitis pigmentosa and early signs of night blindness are reported in some MPS IIIC patients, however the majority of ocular phenotypes are not well characterized. The goal of this study was to investigate retinal degeneration phenotype in the Hgsnat knockout mouse model of MPS IIIC and a cadaveric human MPS IIIC eye. Cone and rod photoreceptors in the eyes of homozygous 6-month-old Hgsnat knockout mice and their wild-type counterparts were analyzed using cone arrestin, S-opsin, M-opsin and rhodopsin antibodies. Histological observation was performed on the eye from a 35-year-old MPS IIIC donor. We observed a nearly 50% reduction in the rod photoreceptors density in the Hgsnat knockout mice compared to the littermate wild-type controls. Cone photoreceptor density was unaltered at this age. Severe retinal degeneration was also observed in the MPS IIIC donor eye. To our knowledge, this is the first report characterizing ocular phenotypes arising from deleterious variants in the Hgsnat gene associated with MPS IIIC clinical phenotype. Our findings indicate retinal manifestations may be present even before behavioral manifestations. Thus, we speculate that ophthalmological evaluations could be used as diagnostic indicators of early disease, progression, and end-point evaluation for future MPS IIIC therapies., Competing Interests: Declaration of competing interest AVP received research grants and honoraria from Phoenix Nest Inc., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Cub domain-containing protein 1 (CDCP1) is a protein that is highly expressed on the surface of many cancer cells. However, its distribution in normal tissues and its potential roles in nontumor cells are poorly understood. We found that CDCP1 is present on both human and mouse retinal pigment epithelial (RPE) cells. CDCP1-KO mice developed attenuated retinal inflammation in a passive model of autoimmune uveitis, with disrupted tight junctions and infiltrating T cells detected in RPE flat mounts from WT but not CDCP1-KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE cells was upregulated by IFN-γ in vitro and after EAU induction in vivo. CD6 stimulation induced increased RPE barrier permeability of WT but not CDCP1-knockdown (CDCP1-KD) RPE cells, and activated T cells migrated through WT RPE monolayers more efficiently than the CDCP1-KD RPE monolayers. In addition, CD6 stimulation of WT but not the CDCP1-KD RPE cells induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPE cells interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.

Purpose: The purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection., Methods: Eyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for Epon embedding and immunocytochemistry., Results: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the controls. OCT-based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes; however, the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples., Conclusions: In situ analysis of the retinal tissue suggests that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus., (© 2021. The Author(s).)

Long interspersed nuclear element-1 (L1)–mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA–induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA–induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

Best disease (BD), also known as vitelliform macular dystrophy, is an inherited disease of the central retina caused by more than 300 pathogenic variants in the BEST1 gene. The phenotype of BD is variable, and there are just a few reports on the histopathology of eyes from donors with BD. Here, we describe the histopathological comparison of donor's eyes from two patients with BD. Eyes obtained from 85-year-old (donor 1) and 65-year-old (donor 2) donors were fixed within 25 h postmortem. Perifoveal and peripheral retinal regions were processed for histology and immunocytochemistry using retinal-specific and retinal pigment epithelium (RPE)-specific antibodies. Three age-matched normal eyes were used as controls. DNA was obtained from donor blood samples. Sequence analysis of the entire BEST1 coding region was performed and identified a c.886A > C (p.Asn296His) variant in donor 1 and a c.602T > C (p.Ile201Thr) variant in donor 2; both mutations were heterozygous. Fundus examination showed that donor 1 displayed a macular lesion with considerable scarring while donor 2 displayed close to normal macular morphology. Our studies of histology and molecular pathology in the perifovea and periphery of these two BD donor eyes revealed panretinal abnormalities in both photoreceptors and RPE cellular levels in the periphery; donor 1 also displayed macular lesion. Our findings confirm the phenotypic variability of BD associated with BEST1 variants., (Copyright © 2020 Bonilha, Bell, DeBenedictis, Hagstrom, Fishman and Hollyfield.)

Sorsby Fundus Dystrophy (SFD) is a rare inherited autosomal dominant macular degeneration caused by specific mutations in TIMP3. Patients with SFD present with pathophysiology similar to the more common Age-related Macular Degeneration (AMD) and loss of vision due to both choroidal neovascularization and geographic atrophy. Previously, it has been shown that RPE degeneration in AMD is due in part to oxidative stress. We hypothesized that similar mechanisms may be at play in SFD. The objective of this study was to evaluate whether mice carrying the S179C-Timp3 mutation, a variant commonly observed in SFD, showed increased sensitivity to oxidative stress. Antioxidant genes are increased at baseline in the RPE in SFD mouse models, but not in the retina. This suggests the presence of a pro-oxidant environment in the RPE in the presence of Timp3 mutations. To determine if the RPE of Timp3 mutant mice is more susceptible to degeneration when exposed to low levels of oxidative stress, mice were injected with low doses of sodium iodate. The RPE and photoreceptors in Timp3 mutant mice degenerated at low doses of sodium iodate, which had no effect in wildtype control mice. These studies suggest that TIMP3 mutations may result in a dysregulation of pro-oxidant-antioxidant homeostasis in the RPE, leading to RPE degeneration in SFD., (Copyright © 2020. Published by Elsevier B.V.)

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4 -/- . These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype., (Published by Elsevier B.V.)

High levels of oxidative radicals generated by daily light exposure and high metabolic rate suggest that the antioxidant machinery of the retina and retinal pigment epithelium (RPE) is crucial for their survival. DJ-1 is a redox-sensitive protein that has been shown to have neuroprotective function in the brain in Parkinson's disease and other neurodegenerative diseases. Here, we analyzed the role of DJ-1 in the retina during oxidative stress and aging. We induced low-level oxidative stress in young (3-month-old) and old (15-month-old) C57BL/6J (WT) and DJ-1 knockout (KO) mice and evaluated effects in the RPE and retina. Absence of DJ-1 resulted in increased retinal dysfunction in response to low levels of oxidative stress. Our findings suggest that loss of DJ-1 affects the RPE antioxidant machinery, rendering it unable to combat and neutralize low-level oxidative stress, irrespective of age. Moreover, they draw a parallel to the retinal degeneration observed in AMD, where the occurrence of genetic variants may leave the retina and RPE unable to fight sustained, low-levels of oxidative stress., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Choroidal neovascularization (CNV) is the abnormal growth of blood vessels that sprout from the choroid vasculature and grow beneath and into the retina. The newly formed blood vessels in CNV often leak blood and fluid which deteriorates vision over time, eventually leading to blindness. In the present study, we examined the efficacy of intravenously injected gold nanoparticles in the laser-induced CNV animal model. Using optical coherence tomography (OCT) and fluorescein angiography, we evaluated CNV lesions longitudinally, over a period of 21 days, with and without nanoparticle treatment. Intravenously injected low concentration of bare gold nanoparticles showed significant anti-angiogenic properties by suppressing CNV development and progression. The treatment group showed significantly decreased fluorescein leakage at the CNV site compared to vehicle injected control mice. OCT assisted CNV volume measurement at all time points showed a significant reduction in lesion size in the treatment group compared with controls., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion., Methods: Enucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD., Results: Around the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells., Conclusions: Fluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Purpose: The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD)., Methods: Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis., Results: Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Müller cells formed pre- and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss., Conclusions: Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Müller cell membranes correlating with RPE loss. Müller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

Inherited retinal degenerations (IRDs) are characterized by the progressive loss of photoreceptors and represent one of the most prevalent causes of blindness among working-age populations. Cyclic nucleotide dysregulation is a common pathological feature linked to numerous forms of IRD, yet the precise mechanisms through which this contributes to photoreceptor death remain elusive. Here we demonstrate that cAMP induced upregulation of the dependence receptor neogenin in the retina. Neogenin levels were also elevated in both human and murine degenerating photoreceptors. We found that overexpressing neogenin in mouse photoreceptors was sufficient to induce cell death, whereas silencing neogenin in degenerating murine photoreceptors promoted survival, thus identifying a pro-death signal in IRDs. A possible treatment strategy is modeled whereby peptide neutralization of neogenin in Rd1, Rd10, and Rho P23H-knockin mice promotes rod and cone survival and rescues visual function as measured by light-evoked retinal ganglion cell recordings, scotopic/photopic electroretinogram recordings, and visual acuity tests. These results expose neogenin as a critical link between cAMP and photoreceptor death, and identify a druggable target for the treatment of retinal degeneration.

Sorsby's fundus dystrophy (SFD) is an inherited blinding disorder caused by mutations in the tissue inhibitor of metalloproteinase-3 ( TIMP3 ) gene. The SFD pathology of macular degeneration with subretinal deposits and choroidal neovascularization (CNV) closely resembles that of the more common age-related macular degeneration (AMD). The objective of this study was to gain further insight into the molecular mechanism(s) by which mutant TIMP3 induces CNV. In this study we demonstrate that hyaluronan (HA), a large glycosaminoglycan, is elevated in the plasma and retinal pigment epithelium (RPE)/choroid of patients with AMD. Mice carrying the S179C-TIMP3 mutation also showed increased plasma levels of HA as well as accumulation of HA around the RPE in the retina. Human RPE cells expressing the S179C-TIMP3 mutation accumulated HA apically, intracellularly and basally when cultured long-term compared with cells expressing wildtype TIMP3 . We recently reported that RPE cells carrying the S179C-TIMP3 mutation have the propensity to induce angiogenesis via basic fibroblast growth factor (FGF-2). We now demonstrate that FGF-2 induces accumulation of HA in RPE cells. These results suggest that the TIMP3-MMP-FGF-2-HA axis may have an important role in the pathogenesis of CNV in SFD and possibly AMD.

Retinal lesions in the posterior pole of laboratory mice occur due to native, developmental abnormalities or as a consequence of environmental or experimental conditions. In this study, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Following experimental preparation induction procedures (EPIP) involving general anesthesia, mydriasis/cycloplegia, and topical anesthesia to the cornea, two ocular recovery conditions (protected and unprotected) were tested within two different animal recovery chambers (open or closed). The anterior and posterior poles were evaluated for the development of retinal lesions using digital color photography, scanning laser ophthalmoscopy, and spectral-domain optical coherence during anesthesia recovery and up to 2.5 months thereafter. In some mice, electroretinograms, histological and immunohistological evaluations were performed to assess functional and structural changes that accompanied the retinal lesions detected by in vivo imaging. Our data suggests that prolonged ocular surface exposure to circulating ambient room air leads to significant anterior and posterior segment ocular complications. The most abundant, semi-reversible complication observed was the development of lesions in the outer retina, which had a 90% probability of occurring after 45 min of exposure. The lesions mostly resolved short-term, but functional and imaging evidence suggest that some perturbations to the outer retina may persist one or more months following initial development., (Copyright © 2019. Published by Elsevier Ltd.)

Hypothesis: Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina., Materials and Methods: The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of streptozotocin-induced diabetes in rats., Results: In the nondiabetic eye, Ang II was localized to the endfeet of Müller cells, extending into the cellular processes of the inner plexiform layer and inner nuclear layer; Ang-(1-7) showed a wider distribution, extending from the foot plates of the Müller cells to the photoreceptor layer. Eyes from diabetic animals showed a higher intensity and extent of Ang II staining compared with nondiabetic eyes, but lower intensity with a reduced distribution of Ang-(1-7) immunoreactivity. Treatment of the diabetic animals with the angiotensin-converting enzyme inhibitor (ACEI) captopril showed a reduced intensity of Ang II staining, whereas increased intensity and distribution were evident with Ang-(1-7) staining., Conclusions: These studies reveal that pharmacological inhibition with ACEIs may provide a specific intervention for the management of the diabetes-induced decline in retinal function, reversing the profile of the endogenous angiotensin peptides closer to the normal condition.

In the retina, oxidative stress can initiate a cascade of events that ultimately leads to a focal loss of RPE cells and photoreceptors, a major contributing factor in geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative metabolism under physiological and pathological conditions remains largely unknown. DJ-1 functions as an antioxidant, redox-sensitive molecular chaperone, and transcription regulator, which protected cells from oxidative stress. Here we discuss our progress toward characterization of the DJ-1 function in the protection of RPE to oxidative stress.

Noninvasive ocular imaging platforms are undeniably useful in identifying retinal abnormalities. The purpose of this study was to investigate a novel method for integrating information acquired from two independent imaging platforms, AF-SLO and SDOCT, in order to demonstrate retinal perturbations as a result of genetic or pharmacological manipulation. Two cohorts of mice were investigated, Nyx nob and C57BL/6 J. In Nyx nob mice, SLO revealed an atypical but variable amount of autofluorescent foci (AFF); SDOCT showed altered photoreceptor outer segment architecture. Naïve Nyx nob had significantly more AFF than C57BL/6 J, suggesting that Nyx nob have some predisposition for developing AFF. Interestingly, both findings were significantly ameliorated in diabetic Nyx nob mice as compared to the controls. These data were incorporated into a novel analysis plot comparing AF-SLO and SDOCT results. The integration of the qualitative changes and accompanying quantitative analysis approach described herein provide a sensitive means for detecting whether a mouse model is susceptible to degeneration before other hallmark indicators are observed.

Circadian clocks regulate various aspects of photoreceptor physiology, but their contribution to photoreceptor development and function is unclear. Cone photoreceptors are critical for color vision. Here, we define the molecular function of circadian activity within cone photoreceptors and reveal a role for the clock genes Bmal1 and Per2 in regulating cone spectral identity. ChIP analysis revealed that BMAL1 binds to the promoter region of the thyroid hormone (TH)-activating enzyme type 2 iodothyronine deiodinase (Dio2) and thus regulates the expression of Dio2. TH treatment resulted in a partial rescue of the phenotype caused by the loss of Bmal1, thus revealing a functional relationship between Bmal1 and Dio2 in establishing cone photoreceptor identity. Furthermore, Bmal1 and Dio2 are required to maintain cone photoreceptor functional integrity. Overall, our results suggest a mechanism by which circadian proteins can locally regulate the availability of TH and influence tissue development and function., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oxidative stress alters physiological function in most biological tissues and can lead to cell death. In the retina, oxidative stress initiates a cascade of events leading to focal loss of RPE and photoreceptors, which is thought to be a major contributing factor to geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative stress under normal and pathological conditions remains largely unknown. A better understanding of the mechanisms involved in regulating RPE and photoreceptors oxidative stress response is greatly needed. To this end we evaluated photoreceptor and RPE changes in mice deficient in DJ-1, a protein that is thought to be important in protecting cells from oxidative stress. Young (3 months) and aged (18 months) DJ-1 knockout (DJ-1 KO) and age-matched wild-type mice were examined. In both group of aged mice, scanning laser ophthalmoscopy (SLO) showed the presence of a few autofluorescent foci. The 18 month-old DJ-1 KO retinas were also characterized by a noticeable increase in RPE fluorescence to wild-type. Optical coherence tomography (OCT) imaging demonstrated that all retinal layers were present in the eyes of both DJ-1 KO groups. ERG comparisons showed that older DJ-1 KO mice had reduced sensitivity under dark- and light-adapted conditions compared to age-matched control. Histologically, the RPE contained prominent vacuoles in young DJ-1 KO group with the appearance of enlarged irregularly shaped RPE cells in the older group. These were also evident in OCT and in whole mount RPE/choroid preparations labeled with phalloidin. Photoreceptors in the older DJ-1 KO mice displayed decreased immunoreactivity to rhodopsin and localized reduction in cone markers compared to the wild-type control group. Lower levels of activated Nrf2 were evident in retina/RPE lysates in both young and old DJ-1 KO mouse groups compared to wild-type control levels. Conversely, higher levels of protein carbonyl derivatives and iNOS immunoreactivity were detected in retina/RPE lysates from both young and old DJ-1 KO mice. These results demonstrate that DJ-1 KO mice display progressive signs of retinal/RPE degeneration in association with higher levels of oxidative stress markers. Collectively this analysis indicates that DJ-1 plays an important role in protecting photoreceptors and RPE from oxidative damage during aging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The goal of this study was to define the histopathology of the retina in donor eyes from a patient with Stargardt disease (STGD1) due to compound mutations in the ABCA4 gene., Materials and Methods: Eyes were obtained from a 66-year-old female and fixed within 18 hours postmortem. The fundi of the posterior globes were evaluated with macroscopic, SLO and OCT imaging. The perifoveal and peripheral regions were processed for electron microscopy and immunocytochemistry using cell specific antibodies. Two age-similar normal eyes were used as controls. Prior ophthalmic examinations and genetic test results were also reviewed., Results: All imaging modalities showed scattered bone spicules in the peripheral retina. Atrophy of the RPE was present around the optic nerve as evidenced by the absence of SLO autofluorescence. Histology analysis showed a severely degenerated fovea with little evidence of any retinal layering or remaining RPE. The fovea was severely degenerated, with little evidence of any retinal cell layer, including the RPE. In contrast, retinal nuclear layers were present in the periphery. The perifoveal region contained few cones labeled with cone-specific antibodies; some rhodopsin-labeled cells, reactive glia labeled with GFAP; and decreased autofluorescence of the RPE. The fovea was free of cone-specific labeling, contained a few disorganized rhodopsin-labeled cells and showed substantial GFAP labeling and no autofluorescent material in the retina. The periphery displayed stubby cells labeled with cone-specific antibodies, decreased rhodopsin-labeled cells, increased GFAP staining, and autofluorescent granules in the RPE., Conclusions: The histopathology of the retina in this patient with Stargardt disease displayed a highly degenerated fovea. In all retinal locations studied, cones were more severely affected than rods.

Purpose: To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations., Methods: Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells., Results: DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea., Conclusions: Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina.

DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) imaging. Age-similar normal eyes and an eye donated by donor 1's asymptomatic mother (donor 4, family 1) were used as controls. The perifovea and peripheral retina were processed for microscopy and immunocytochemistry with markers for cone and rod photoreceptor cells. DNA analysis revealed EYS mutations c.2259 + 1G > A and c.2620C > T (p.Q874X) in family 1, and c.4350&#95;4356del (p.I1451Pfs*3) and c.2739-?&#95;3244 + ?del in family 2. Imaging studies revealed the presence of bone spicule pigment in arRP donor retinas. Histology of all three affected donor eyes showed very thin retinas with little evidence of stratified nuclear layers in the periphery. In contrast, the perifovea displayed a prominent inner nuclear layer. Immunocytochemistry analysis demonstrated advanced retinal degenerative changes in all eyes, with near-total absence of rod photoreceptors. In addition, we found that the perifoveal cones were more preserved in retinas from the donor with the midsize genomic rearrangement (c.4350&#95;4356del (p.I1451Pfs*3) and c.2739-?&#95;3244 + ?del) than in retinas from the donors with the truncating (c.2259 + 1G > A and c.2620C > T (p.Q874X) mutations. Advanced retinal degenerative changes with near-total absence of rods and preservation of some perifoveal cones are observed in arRP donor retinas with EYS mutations.

Purpose: To define the retinal pathology in a 3 year-old eye donor who died from complications of an undiagnosed genetic syndrome., Methods: Eyes were fixed and analyzed using macroscopic fundus photography (MF), confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT). Small areas from the perifovea and periphery were processed for histology and indirect immunofluorescence, using antibodies specific to retinal proteins such as rhodopsin, cone arrestin, RPE65 and others. Available medical records were also reviewed., Results: With all three imaging modalities, the affected donor's eyes lacked the distinct morphological detail typically observed with these techniques in postmortem control eyes. MF images showed a "photonegative effect" due to a hypopigmented macula relative to a hyperpigmented retinal background. cSLO imaging demonstrated a weak autofuorescence signal that was largely devoid of the usual retinal structures compared to the control. SD-OCT suggested disorganization of the affected retina, absence of a photoreceptor layer, and degeneration of the choroid in the macular area. Histologic findings indicated a highly disorganized photoreceptor layer in the macula and periphery. The RPE layer displayed thinning in some regions of the periphery and decreased pigmentation in most areas. Rods and cones were significantly reduced in the affected retina but a few cones were detected in the perifovea. Centrin-2 labeling was mostly absent from the connecting cilium of the photoreceptor cells. Medical record review pointed to a possible clinical diagnosis of Joubert syndrome., Conclusions: The retinal degenerative findings, and absence of centrin-2 labeling are compatible with the expected retinal phenotype in patients with Joubert syndrome.

The retinal pigment epithelium (RPE) constitutes a monolayer of cuboidal cells that interact apically with the interphotoreceptor matrix (IPM) and outer segments of the photoreceptor cells and basally with the subjacent Bruch's membrane. This highly polarized structure is maintained by the cytoskeleton of individual cells and their interactions at the basolateral junctional complexes that stabilize this epithelial structure. This RPE complex network of filaments, tubules and associated proteins is modeled by the cellular environment, the RPE intercellular interactions, and by its interactions with the extracellular matrix. This is a review of the key features of the RPE cytoskeleton in vivo and in vitro., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

DJ-1 is a protein expressed in many tissues including the brain where it has been extensively studied due to its association with Parkinson's Disease (PD). DJ-1 was reported to function as an antioxidant, redox-sensitive molecular chaperone, and transcription regulator, which protected cells from oxidative stress by modifying signaling pathways that regulate cell survival. Here we discuss our progress toward characterization of the DJ-1 function in the protection of RPE to oxidative stress.

Background: DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress., Methodology: Retinal pigment epithelial (RPE) cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM)/choroid from AMD eyes., Principal Findings: DJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes., Conclusions/significance: DJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toxoplasma gondii infects both hematopoietic and nonhematopoietic cells and can cause cerebral and ocular toxoplasmosis, as a result of either congenital or postnatally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is a key factor for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti-T. gondii activity at the level of nonhematopoietic cells. Engagement of CD40 on various endothelial cells including human microvascular brain endothelial cells, human umbilical vein endothelial cells, and a mouse endothelial cell line as well as human and mouse retinal pigment epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux, and led to recruitment of LC3 around the parasite. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40-stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various nonhematopoietic cells dependent on proteins of the autophagy machinery.

Background: Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood., Methodology: Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis., Principal Findings: A total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described., Conclusions/significance: The data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.

Purpose: Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51., Methods: Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors., Results: Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons., Conclusions: The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.

Purpose: To define the retinal pathology in an 88-year-old male affected with Goldmann-Favre syndrome with a 2 bp 5' A>C splice site mutation in the NR2E3 gene., Methods: Retinal tissue from the macula and periphery was processed for immunohistochemistry. Perimacular retina was processed for transmission electron microscopy. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to rhodopsin, cone cytoplasm, and cone opsins. The affected donor eye was compared to a postmortem matched normal eye., Results: The retina was highly disorganized without laminar organization. The RPE was discontinuous in some perimacular regions. Large (>1 mm) spherical electrondense melanosomes were observed in the RPE and choroid by TEM. Rods were virtually absent in the affected retina. Cones were present in the macula, but were mostly absent from the retinal periphery. In addition, cone rosettes were observed in the perimacular area. Both red/green and blue cone opsins were distributed along the entire cellular expanse of the cone photoreceptors in the affected eye, but were restricted to the cone outer segments in the control retina., Conclusions: The histological data obtained from the retina of an elderly male patient with Goldmann-Favre syndrome showed an absence of rods and abnormal distribution of red/green and blue cone opsins.

Purpose: To compare mechanical and ethanol epithelial removal with respect to myofibroblast development and haze formation after photorefractive keratectomy (PRK)., Methods: Seventeen rabbits underwent mechanical or ethanol debridement, and the opposite eye of each rabbit served as an unwounded control. In both groups, the epithelium was removed with a spatula and discarded. A -9.00-diopter PRK was performed in each eye. The level of haze in each cornea at 4 weeks was graded at the slit-lamp microscope according to the Fantes scale. Myofibroblast generation was detected with immunocytochemistry for alpha-smooth muscle actin (alpha-SMA) and cells were quantitatively analyzed., Results: No difference was noted between the two groups in alpha-SMA + myofibroblasts 4 weeks after surgery (43.6+/-2.0/400x field and 45.7+/-4.8/400x field in ethanol and mechanical groups, respectively) (P=.10). A slight difference was noted but did not reach statistical significance with regard to stromal haze between ethanol and mechanical groups (2.0+/-0.5 and 2.3+/-0.4, respectively, P=.063). The ethanol and mechanical groups were statistically different when compared to controls regarding stromal haze and alpha-SMA+ cells (P<.0001for all comparisons)., Conclusions: No difference was noted in clinical haze or myofibroblast generation between corneas that had PRK with mechanical or ethanol epithelial debridement.

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.

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

As the retinal pigment epithelium (RPE) ages, a number of structural changes occur, including loss of melanin granules, increase in the density of residual bodies, accumulation of lipofuscin, accumulation of basal deposits on or within Bruch's membrane, formation of drusen (between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane), thickening of Bruch's membrane, microvilli atrophy and disorganization of the basal infoldings. Although these changes are well known, the basic mechanisms involved in them are frequently poorly understood. These age-related changes progress slowly and vary in severity in different individuals. These changes are also found in age-related macular degeneration (AMD), a late onset disease that severely impacts the RPE, but they are much more pronounced than during normal aging. However, the changes in AMD lead to severe loss of vision. Given the many supporting functions which the RPE serves for the retina, it is important to decipher the age-related changes in this epithelium in order to understand age-related changes in vision.