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Age-related macular degeneration (AMD) is a major leading cause of irreversible visual impairment in the world with limited therapeutic interventions. Histological, biochemical, genetic, and epidemiological studies strongly implicate dysregulated lipid metabolism in the retinal pigmented epithelium (RPE) in AMD pathobiology. However, effective therapies targeting lipid metabolism still need to be identified and developed for this blinding disease. To test lipid metabolism-targeting therapies, preclinical AMD mouse models are needed to establish therapeutic efficacy and the role of lipid metabolism in the development of AMD-like pathology. In this review, we provide a comprehensive overview of current AMD mouse models available to researchers that could be used to provide preclinical evidence supporting therapies targeting lipid metabolism for AMD. Based on previous studies of AMD mouse models, we discuss strategies to modulate lipid metabolism as well as examples of studies evaluating lipid-targeting therapeutics to restore lipid processing in the RPE. The use of AMD mouse models may lead to worthy lipid-targeting candidate therapies for clinical trials to prevent the blindness caused by AMD.

A pathologic feature of late-onset retinal degeneration caused by the S163R mutation in C1q-tumor necrosis factor-5 (C1QTNF5) is the presence of unusually thick deposits between the retinal pigmented epithelium (RPE) and the vascular choroid, considered a hallmark of this disease. We have previously demonstrated that following its specific expression in mouse RPE, the S163R mutant exhibits a reversed polarized distribution relative to the apically secreted wild-type C1QTNF5, and forms widespread, prominent deposits that gradually increase in size with aging. This study shows that S163R deposits expand to a considerable thickness through a progressive increase in the basolateral RPE membrane, substantially raising the total RPE height, and enabling their clear imaging as a distinct hyporeflective layer by noninvasive optical coherence tomography in advanced aged animals. This phenotype bears a striking similarity to ocular pathology previously documented in patients harboring the S163R mutation. Therefore, we used a similar viral vector-based gene delivery approach to investigate the behavior of two novel pathogenic C1QTNF5 mutants recently reported in patients, P188T and G216C, for which histopathologic data are lacking. We found that both mutants primarily impact the RPE/photoreceptor interface and do not generate basal laminar deposits. The results demonstrate distinct distribution patterns and phenotypic consequences of C1QTNF5 mutants in vivo, and suggest that multiple pathobiological mechanisms contribute to RPE dysfunction and vision loss in this disorder.

Item does not contain fulltext Age-related macular degeneration (AMD) is a disease that affects the macula - the central part of the retina. It is a leading cause of irreversible vision loss in the elderly. AMD onset is marked by the presence of lipid- and protein-rich extracellular deposits beneath the retinal pigment epithelium (RPE), a monolayer of polarized, pigmented epithelial cells located between the photoreceptors and the choroidal blood supply. Progression of AMD to the late nonexudative "dry" stage of AMD, also called geographic atrophy, is linked to progressive loss of areas of the RPE, photoreceptors, and underlying choriocapillaris leading to a severe decline in patients' vision. Differential susceptibility of macular RPE in AMD and the lack of an anatomical macula in most lab animal models has promoted the use of in vitro models of the RPE. In addition, the need for high throughput platforms to test potential therapies has driven the creation and characterization of in vitro model systems that recapitulate morphologic and functional abnormalities associated with human AMD. These models range from spontaneously formed cell line ARPE19, immortalized cell lines such as hTERT-RPE1, RPE-J, and D407, to primary human (fetal or adult) or animal (mouse and pig) RPE cells, and embryonic and induced pluripotent stem cell (iPSC) derived RPE. Hallmark RPE phenotypes, such as cobblestone morphology, pigmentation, and polarization, vary significantly betweendifferent models and culture conditions used in different labs, which would directly impact their usability for investigating different aspects of AMD biology. Here the AMD Disease Models task group of the Ryan Initiative for Macular Research (RIMR) provides a summary of several currently used in vitro RPE models, historical aspects of their development, RPE phenotypes that are attainable in these models, their ability to model different aspects of AMD pathophysiology, and pros/cons for their use in the RPE and AMD fields. In addition, due to the burgeoning use of iPSC derived RPE cells, the critical need for developing standards for differentiating and rigorously characterizing RPE cell appearance, morphology, and function are discussed.

Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.

Purpose: Subretinal delivery of stem cells and gene therapy vectors hold great promise in clinical care. Delivery techniques are evolving. We investigate the utility of 4-dimensional (volumes over time) microscope-integrated optical coherence tomography (4D-MIOCT) to guide such maneuvers. Methods: This investigational 4D-MIOCT system displayed heads-up cross-sectional and stereoscopic volumetric views through the microscope oculars at 3.3–10 volumes/second. In enucleated porcine eyes, we evaluated novel information available to the surgeon during subretinal: 1) injection of fluid and cell suspensions, 2) retinotomy creation, 3) insertion of membranes with a retinal pigment epithelium (RPE) cell monolayer and 4) subretinal instrument maneuvers, all in postmortem porcine eyes. Results: Interactions between tools, membranes, retina and RPE were often not detectable on the conventional microscope view but were obvious with 4D-MIOCT. 4D-MIOCT visualization was essential to: monitor the extent of induced retinal detachment, guide the cannula tip and instrument maneuvers in the subretinal space, and identify efflux of cells into the vitreous cavity. This allowed adjustment by the surgeon to avoid complications—e.g. scissors scraping RPE. Conclusions: 4D-MIOCT is useful for continuous monitoring of model subretinal surgery and may help avoid technique-related complications. 4D-MIOCT can contribute to the advancement of subretinal delivery of therapies under development.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly in the developed world. While treatment is effective for the neovascular or “wet” form of AMD, no therapy is successful for the non-neovascular or “dry” form. Here we discuss the current knowledge on dry AMD pathobiology and propose future research directions that would expedite the development of new treatments. In our view, these should emphasize system biology approaches that integrate omic, pharmacological, and clinical data into mathematical models that can predict disease onset and progression, identify biomarkers, establish disease causing mechanisms, and monitor response to therapy., No effective therapies exist for dry age-related macular degeneration. In this perspective, the authors propose that research should emphasize system biology approaches that integrate various ‘omics’ data into mathematical models to establish pathogenic mechanisms on which to design novel treatments, and identify biomarkers that predict disease progression and therapeutic response.

Significance The complement factor H (CFH) Y402H polymorphism (rs1061170) imparts the strongest risk for age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Popular thinking holds that the CFH H402 variant increases complement activation in the eye, predisposing susceptibility to disease. However, clinical trials of complement inhibitors in AMD patients have failed. Here we provide an explanation, showing CFH variant-specific differences in the presentation of AMD-like pathologies. We show that aged mice expressing the human H402, but not Y402 variant, (i) develop AMD-like symptoms and (ii) display differences in their systemic and ocular lipoprotein levels, but not in their complement activation, after diet. These findings support targeting lipoproteins for the treatment of AMD., One of the strongest susceptibility genes for age-related macular degeneration (AMD) is complement factor H (CFH); however, its impact on AMD pathobiology remains unresolved. Here, the effect of the principal AMD-risk–associated CFH variant (Y402H) on the development and progression of age-dependent AMD-like pathologies was determined in vivo. Transgenic mice expressing equal amounts of the full-length normal human CFH Y402 (CFH-Y/0) or the AMD-risk associated CFH H402 (CFH-H/H) variant on a Cfh−/− background were aged to 90 weeks and switched from normal diet (ND) to a high fat, cholesterol-enriched (HFC) diet for 8 weeks. The resulting phenotype was compared with age-matched controls maintained on ND. Remarkably, an AMD-like phenotype consisting of vision loss, increased retinal pigmented epithelium (RPE) stress, and increased basal laminar deposits was detected only in aged CFH-H/H mice following the HFC diet. These changes were not observed in aged CFH-Y/0 mice or in younger (36- to 40-week-old) CFH mice of both genotypes fed either diet. Biochemical analyses of aged CFH mice after HFC diet revealed genotype-dependent changes in plasma and eyecup lipoproteins, but not complement activation, which correlated with the AMD-like phenotype in old CFH-H/H mice. Specifically, apolipoproteins B48 and A1 are elevated in the RPE/choroid of the aged CFH-H/H mice compared with age-matched control CFH-Y/0 fed a HFC diet. Hence, we demonstrate a functional consequence of the Y402H polymorphism in vivo, which promotes AMD-like pathology development and affects lipoprotein levels in aged mice. These findings support targeting lipoproteins as a viable therapeutic strategy for treating AMD.

The title of the chapter is "Melatonin as the Possible Link Between Age-Related Retinal Degeneration and the Disrupted Circadian Rhythm in Elderly" but degeneration was incorrectly published as regeneration. Now this has been corrected to degeneration.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel that is a key player in oxidative stress under pathological conditions. The P2X7R is expressed in the retinal pigmented epithelium (RPE) and neural retina. Chronic oxidative stress contributes to the pathogenesis of age-related macular degeneration (AMD). Mice lacking Cu, Zn superoxide dismutase (Sod1) developed chronic oxidative stress as well as AMD-like features, but whether the P2X7R plays a causative role in oxidative stress-induced AMD is unknown. Thus, the main purpose of this study was to test if concurrent knockout (KO) of P2X7R could block AMD-like defects seen in Sod1 KO mice. Using multiple approaches, we demonstrate that Sod1 KO causes AMD-like defects, including positive staining for oxidative stress markers, 3-nitrotyrosine and carboxymethyl lysine, thinning of the RPE and retina, thickening of Bruch's membrane, presence of basal laminar and linear deposits, RPE barrier disruption and accumulation of microglia/macrophages. Moreover, we find that Sod1 KO mice accumulate more microparticles (MPs) within RPE/choroid tissues. Concurrent KO of the P2X7R protects against AMD-like defects and MP accumulation in Sod1 KO mice. Together, we show for the first time, that deficiency of P2X7R prevents in vivo oxidative stress-induced accumulation of MPs and AMD-like defects. This work could potentially lead to novel therapies for AMD and other oxidative stress-driven diseases.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier and provides nutrients and recycling of visual pigment to the photoreceptors, among many other functions. The RPE is also a key site of pathophysiological changes in age-related macular degeneration, making it an important focus of study in both visual health and disease. Exosomes are nanometer-sized vesicles that are released by cells in a controlled fashion and mediate a range of extra- and intercellular activities. Some key exosome actions include cell-cell communication, immune modulation, extracellular matrix turnover, stem cell division/differentiation, neovascularization, and cellular waste removal. While much is known about their role in cancer and cardiovascular disease, exosome function in the many specialized tissues of the eye is just beginning to undergo rigorous study. Here we review current knowledge of the functions and roles of exosomes and other small extracellular vesicles released from the RPE. In particular, we discuss the potential role and importance of polarized exosome release from the RPE.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier and provides nutrients and recycling of visual pigment to the photoreceptors, among many other functions. The RPE is also a key site of pathophysiological changes in age-related macular degeneration, making it an important focus of study in both visual health and disease. Exosomes are nanometer-sized vesicles that are released by cells in a controlled fashion and mediate a range of extra- and intercellular activities. Some key exosome actions include cell-cell communication, immune modulation, extracellular matrix turnover, stem cell division/differentiation, neovascularization, and cellular waste removal. While much is known about their role in cancer and cardiovascular disease, exosome function in the many specialized tissues of the eye is just beginning to undergo rigorous study. Here we review current knowledge of the functions and roles of exosomes and other small extracellular vesicles released from the RPE. In particular, we discuss the potential role and importance of polarized exosome release from the RPE.

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Age-Related Macular Degeneration (AMD) is a complex multifactorial disease characterized in its early stages by lipoprotein accumulations in Bruch's Membrane (BrM), seen on fundoscopic exam as drusen, and in its late forms by neovascularization (“wet”) or geographic atrophy of the Retinal Pigmented Epithelial (RPE) cell layer (“dry”). Genetic studies have strongly supported a relationship between the alternative complement cascade, in particular the common H402 variant in Complement Factor H (CFH) and development of AMD. However, the functional significance of the CFH Y402H polymorphism remains elusive. In this article, we critically review the literature surrounding the functional significance of this polymorphism. Furthermore, based on our group's studies we propose a model in which CFH H402 affects CFH binding to heparan sulfate proteoglycans leading to accelerated lipoprotein accumulation in BrM and drusen progression. We also review the literature on the role of other complement components in AMD pathobiologies, including C3a, C5a and the membrane attack complex (MAC), and on transgenic mouse models developed to interrogate in vivo the effects of the CFH Y402H polymorphism.

Exosomes are nanometer-sized vesicles that are released by cells in a controlled fashion and mediate a plethora of extra- and intercellular activities. Some key functions of exosomes include cell-cell communication, immune modulation, extracellular matrix turnover, stem cell division/differentiation, neovascularization and cellular waste removal. While much is known about their role in cancer, exosome function in the many specialized tissues of the eye is just beginning to undergo rigorous study. Here we review current knowledge of exosome function in the visual system in the context of larger bodies of data from other fields, in both health and disease. Additionally, we discuss recent advances in the exosome field including use of exosomes as a therapeutic vehicle, exosomes as a source of biomarkers for disease, plus current standards for isolation and validation of exosome populations. Finally, we use this foundational information about exosomes in the eye as a platform to identify areas of opportunity for future research studies.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel that is a key player in oxidative stress under pathological conditions. The P2X7R is expressed in the retinal pigmented epithelium (RPE) and neural retina. Chronic oxidative stress contributes to the pathogenesis of age-related macular degeneration (AMD). Mice lacking Cu, Zn superoxide dismutase (Sod1) developed chronic oxidative stress as well as AMD-like features, but whether the P2X7R plays a causative role in oxidative stress-induced AMD is unknown. Thus, the main purpose of this study was to test if concurrent knockout (KO) of P2X7R could block AMD-like defects seen in Sod1 KO mice. Using multiple approaches, we demonstrate that Sod1 KO causes AMD-like defects, including positive staining for oxidative stress markers, 3-nitrotyrosine and carboxymethyl lysine, thinning of the RPE and retina, thickening of Bruch’s membrane, presence of basal laminar and linear deposits, RPE barrier disruption and accumulation of microglia/macrophages. Moreover, we find that Sod1 KO mice accumulate more microparticles (MPs) within RPE/choroid tissues. Concurrent KO of the P2X7R protects against AMD-like defects and MP accumulation in Sod1 KO mice. Together, we show for the first time, that deficiency of P2X7R prevents in vivo oxidative stress-induced accumulation of MPs and AMD-like defects. This work could potentially lead to novel therapies for AMD and other oxidative stress-driven diseases.

Age-related macular degeneration (AMD) is an ocular neurodegenerative disorder and is the leading cause of legal blindness in Western societies, with a prevalence of up to 8 % over the age of 60, which continues to increase with age. AMD is characterized by the progressive breakdown of the macula (the central region of the retina), resulting in the loss of central vision including visual acuity. While its molecular etiology remains unclear, advances in genetics and genomics have illuminated the genetic architecture of the disease and have generated attractive pathomechanistic hypotheses. Here, we review the genetic architecture of AMD, considering the contribution of both common and rare alleles to susceptibility, and we explore the possible mechanistic links between photoreceptor degeneration and the alternative complement pathway, a cascade that has emerged as the most potent genetic driver of this disorder.

β‐Secretase (BACE1) is a major drug target for combating Alzheimer9s disease (AD). Here we show that BACE1 −/− mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial‐derived factor, a known regulator of γ‐secretase, inhibits vascular endothelial growth factor (VEGF)‐induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ‐secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age‐related macular degeneration.

This paper presents a generalized framework for segmenting closed-contour anatomical and pathological features using graph theory and dynamic programming (GTDP). More specifically, the GTDP method previously developed for quantifying retinal and corneal layer thicknesses is extended to segment objects such as cells and cysts. The presented technique relies on a transform that maps closed-contour features in the Cartesian domain into lines in the quasi-polar domain. The features of interest are then segmented as layers via GTDP. Application of this method to segment closed-contour features in several ophthalmic image types is shown. Quantitative validation experiments for retinal pigmented epithelium cell segmentation in confocal fluorescence microscopy images attests to the accuracy of the presented technique.

It has long been established that β-Secretase (BACE) plays a critical role in the formation of amyloid plaques in Alzheimer's Disease patients, but it is only recently that the importance of β-secretases in retinal pathophysiology has been recognized. BACE expression is elevated in response to stress, and downregulation results in lysosomal abnormalities and mitochondrial changes. Inhibition of BACE can lead to reduced retinal function, retinal thinning, lipofuscin accumulation and vascular dysfunction in mice. Furthermore, BACE inhibition accelerates choroidal neovascularization (CNV) in mice. We propose that BACE plays an important role in retinal homeostasis and that BACE upregulation in response to stress is a protective measure.

It has long been established that β-Secretase (BACE) plays a critical role in the formation of amyloid plaques in Alzheimer’s Disease patients, but it is only recently that the importance of β-secretases in retinal pathophysiology has been recognized. BACE expression is elevated in response to stress, and downregulation results in lysosomal abnormalities and mitochondrial changes. Inhibition of BACE can lead to reduced retinal function, retinal thinning, lipofuscin accumulation and vascular dysfunction in mice. Furthermore, BACE inhibition accelerates choroidal neovascularization (CNV) in mice. We propose that BACE plays an important role in retinal homeostasis and that BACE upregulation in response to stress is a protective measure.

An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase–based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch’s membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch’s membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch’s membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch’s membrane/choroid.

Purpose A large body of evidence supports a central role for complement activation in the pathobiology of age-related macular degeneration (AMD), including plasma complement component 5a (C5a). Interestingly, C5a is a chemotactic agent for monocytes, a cell type also shown to contribute to AMD. However, the role monocytes play in the pathogenesis of "dry" AMD and the pharmacologic potential of targeting C5a to regulate these cells are unclear. We addressed these questions via C5a blockade in a unique model of early/intermediate dry AMD and large panel flow cytometry to immunophenotype monocytic involvement. Methods Heterozygous complement factor H (Cfh+/-) mice aged to 90 weeks were fed a high-fat, cholesterol-enriched diet (Cfh+/-∼HFC) for 8 weeks and were given weekly intraperitoneal injections of 30 mg/kg anti-C5a (4C9, Pfizer). Flow cytometry, retinal pigmented epithelium (RPE) flat mounts, and electroretinograms were used to characterize anti-C5a treatment. Results Aged Cfh+/- mice developed RPE damage, sub-RPE basal laminar deposits, and attenuation of visual function and immune cell recruitment to the choroid that was accompanied by expression of inflammatory and extracellular matrix remodeling genes following 8 weeks of HFC diet. Concomitant systemic administration of an anti-C5a antibody successfully inhibited local recruitment of mononuclear phagocytes to the choroid-RPE interface but did not ameliorate these AMD-like pathologies in this mouse model. Conclusions These results show that immunotherapy targeting C5a is not sufficient to block the development of the AMD-like pathologies observed in Cfh+/-∼HFC mice and suggest that other complement components or molecules/mechanisms may be driving "early" and "intermediate" AMD pathologies.

Aging-associated neurodegenerative diseases significantly influence the quality of life of affected individuals. Genetic approaches, combined with genomic technology, have provided powerful insights into common late-onset diseases, such as age-related macular degeneration (AMD). Here, we discuss current findings on the genetics of AMD to highlight areas of rapid progress and new challenges. We also attempt to integrate available genetic and biochemical data with cellular pathways involved in aging to formulate an integrated model of AMD pathogenesis.

To compare spectral domain optical coherence tomography (SDOCT) cross-sectional images of human central retina obtained from donor eyes with and without age-related macular degeneration (AMD) to corresponding histopathology from light micrographs. To establish the utility of SDOCT for localizing pathology in the posterior eyecup, for identifying ocular disease in donor eyes, or for directing subsequent sectioning of retinal lesions for research. Seven consecutive human donor eyes were selected based on age. The eyes, with the anterior segment removed, were imaged by SDOCT with a focusing aspheric lens. Four eyes were from donors with a clinical history of AMD, and three were from age-matched donors with no history of AMD. Histopathological correlation of morphological changes detected in three eyes by SDOCT was obtained for comparison to step serial-sectioned light microscopy images of the formalin-fixed, paraffin-embedded retina. A simplified imaging setup was tested on an enucleated porcine eye for comparison. AMD pathology was detected and localized in four eyes by SDOCT. The SDOCT images correlated with the histopathology observed by light microscopy in each sectioned eye. Pathologies included a subfoveal neovascular lesion with subretinal fluid, peripapillary neovascularization, epiretinal membrane, foveal cyst, choroidal folds, and drusen. Similar imaging was possible with the simplified setup. SDOCT imaging identified retinal disease of the posterior eyecup in human donor eyes. Pathology detected with SDOCT was verified by light microscopy in three eyes, supporting the utility of SDOCT as a screening tool for research.

Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient's blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes.A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes.The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402.A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.

Age-related macular degeneration (AMD) is a late-onset, neurodegenerative retinal disease that shares several clinical and pathological features with Alzheimer's disease (AD) including extracellular deposits containing amyloid-beta (Abeta) peptides. Immunotherapy targeting the Abeta protein has been investigated as a potential treatment for AD. Here, we present the rationale for extending this approach to treat AMD. We tested an anti-Abeta antibody administered systemically in a mouse model of AMD. Histological and functional measurements in treated animals compared to controls showed that following immunotherapy, the amounts of Abeta in the retina and brain were decreased and the ERG deficits in the retina were attenuated. These data support the hypothesis that Abeta is a therapeutic target for AMD.

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh(+/-) and Cfh(-/-) mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch's membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh(+/-) and Cfh(-/-) mice, RPE damage accompanied by loss of vision occurred only in old Cfh(+/-) mice. We demonstrate that such pathology is a function of excess complement activation in Cfh(+/-) mice versus complement deficiency in Cfh(-/-) animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch's membrane lipoprotein binding and show, using human Bruch's membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.

To examine the effects and potential implications for the expression of the two basic fibroblast growth factor (bFGF) receptors, FGFR-1 and FGFR-2, in retinal pigment epithelial (RPE) cells, bFGF-dependent changes in gene expression and RPE cell function were studied. bFGF increased L-type Ca2+ channel activity of RPE cells, which in turn resulted in an increase of vascular endothelial growth factor A (VEGF-A) secretion from RPE cells. Also, both bFGF and direct stimulation of L-type Ca2+ channels by BayK8644 increased the expression of c-fos in RPE cells, to the same extent. bFGF-induced-c-fos expression was reduced by inhibition of FGFR-1, but not by L-type Ca2+ channel inhibition, demonstrating that stimulation of FGFR-1 results in a Ca2+ channel-independent change of gene expression. In contrast, stimulation of FGFR-2 results in a Ca2+ channel-dependent stimulation of VEGF secretion. Furthermore, immunohistological investigation of neovascular tissues obtained from patients with age-related macular degeneration (AMD) revealed FGFR-1 and FGFR-2 expression in the RPE of the diseased tissue. Our findings support the hypothesis that there are two different FGFR-1- and FGFR-2-dependent pathways that modulate the role of bFGF in induction of neovascularisation in AMD.

Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. IGF-1 is produced in neurons and retinal pigment epithelium (RPE) but its targets and impact in CNV are not understood. IGF-1 immunoreactivity was abundant throughout surgically isolated human CNV tissues and RPE cells were immunopositive for IGF-1R. Cultured RPE cells obtained from CNV tissues expressed IGF-1R. IGF-1 stimulation of cultured cells from CNV tissues induced monophasic sustained rises in intracellular free Ca(2+). VEGF concentration in the medium of unstimulated RPE cell cultures from CNV tissues increased with time to a steady-state (8h) which was increased twofold by IGF-1 stimulation. Thus, in RPE cells IGF-1 stimulates the second messenger Ca(2+) and increases VEGF secretion which, in turn, induces neovascularization.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Variations in several complement genes are now known to be significant risk factors for the development of age-related macular degeneration (AMD). Despite dramatic effects on disease susceptibility, the underlying mechanisms by which common polymorphisms in complement proteins alter disease risk have remained unclear. Genetically modified mice in which the activity of the complement has been altered are available and can be used to investigate the role of complement in the pathogenesis of AMD. In this mini review, we will discuss some existing complement models of AMD and our efforts to develop and characterize the ocular phenotype in a variety of mice in which complement is either chronically activated or inhibited. A spectrum of complement dysregulation was modeled on the APOE4 AMD mouse model by crossing these mice to complement factor H knockout (cfh-/-) mice to test the impact of excess complement activation, and by crossing them to soluble-complement-receptor-1-related protein y (sCrry) mice, in which sCrry acts as a potent inhibitor of mouse complement acting in a manner similar to CFH. In addition, we have also generated humanized CFH mice expressing normal and risk variants of CFH.

Variations in several complement genes are now known to be significant risk factors for the development of age-related macular degeneration (AMD). Despite dramatic effects on disease susceptibility, the underlying mechanisms by which common polymorphisms in complement proteins alter disease risk have remained unclear. Genetically modified mice in which the activity of the complement has been altered are available and can be used to investigate the role of complement in the pathogenesis of AMD. In this mini review, we will discuss some existing complement models of AMD and our efforts to develop and characterize the ocular phenotype in a variety of mice in which complement is either chronically activated or inhibited. A spectrum of complement dysregulation was modeled on the APOE4 AMD mouse model by crossing these mice to complement factor H knockout (cfh−/−) mice to test the impact of excess complement activation, and by crossing them to soluble-complement-receptor-1-related protein y (sCrry) mice, in which sCrry acts as a potent inhibitor of mouse complement acting in a manner similar to CFH. In addition, we have also generated humanized CFH mice expressing normal and risk variants of CFH.

The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells.ApoER2knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development.VLDLRKO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in bothApoER2andVLDLRKOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid β (Aβ) peptides, Aβ1–40 (Aβ40) and Aβ1–42 (Aβ42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aβ, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aβ-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aβ40 and Aβ42. Concomitant reduction in the levels of Aβ and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aβ40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aβ antibodies. They also implicate Aβ in the pathogenesis of AMD and identify Aβ as a viable therapeutic target for its treatment.