A Comparison of Some Organizational Characteristics of the Mouse Central Retina and the Human Macula

Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch’s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.     

Orally active multi-functional antioxidants are neuroprotective in a rat model of light-induced retinal damage

Background

Progression of age-related macular degeneration has been linked to iron dysregulation and oxidative stress that induce apoptosis of neural retinal cells. Since both antioxidants and chelating agents have been reported to reduce the progression of retinal lesions associated with AMD in experimental animals, the present study evaluates the ability of multi-functional antioxidants containing functional groups that can independently chelate redox metals and quench free radicals to protect the retina against light-induced retinal degeneration, a rat model of dry atrophic AMD.

Methods/Results

Proof of concept studies were conducted to evaluate the ability of 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8) to reduce retinal damage in 2-week dark adapted Wistar rats exposed to 1000 lx of light for 3 hours. Assessment of the oxidative stress markers 4- hydroxynonenal and nitrotyrosine modified proteins and Thioredoxin by ELISA and Western blots indicated that these compounds reduced the oxidative insult caused by light exposure. The beneficial antioxidant effects of these compounds in providing significant functional and structural protection were confirmed by electroretinography and quantitative histology of the retina.

Conclusions/Significance

The present study suggests that multi-functional compounds may be effective candidates for preventive therapy of AMD.     

Plasma Proteome Analysis on Cynomolgus Monkey (Macaca Fascicularis) Pedigrees with Early Onset Drusen Formation

The central region of the primate retina is called macula. The fovea is located at the center of the macula, where the photoreceptors are concentrated to create neural network adapted for high visual acuity. Damage to the fovea by macular dystrophies and age-related macular degeneration (AMD) can reduce the central visual acuity. The molecular mechanisms leading to these diseases are most likely dependent on the proteins in macula differ from that in peripheral retina in expression level. Previously, we reported an early onset macular degeneration with drusen in cynomolgus monkey pedigrees. These monkeys show similar fundus findings of early stage of AMD at 2 years after birth. To elucidate mechanism of drusen formation and to find disease biomarkers for early stage of AMD, we performed plasma proteome analysis. Plasma samples were collected from four affected and control monkeys within the same pedigree. Successful fractionation of the plasma proteins by ProteoMiner and Gelfree8100 were confirmed by SDS-PAGE. Total of 245 proteins were identified from eight samples. From the results of spectral counting, we selected some proteins, Apolipoprotein E, Histidine-rich glycoprotein, and Retinol-binding protein 4 as candidate proteins that would be related with drusen formation. Candidate proteins would be potentially beneficial as biomarkers for human AMD. One of the identified proteins, Apolipoprotein E (ApoE), is structural component of drusen and also related with other neurodegenerative disease like Alzheimer disease. In this plasma proteome analysis, ApoE would be one of the possible factors of early drusen formation in these cynomolgus monkey pedigrees.     

Light distributions on the retina: relevance to macular pigment photoprotection

Light exposure has been implicated in age-related macular degeneration (AMD). This study was designed to measure cumulative light distribution on the retina to determine whether it peaked in the macula. An eye-tracker recorded the subject's field of view and pupil size, and superimposed the gaze position. Fifteen na?ve subjects formed a test group; 5 formed a control group. In phase 1, all subjects viewed a sequence of photographic images. In phase 2, the na?ve subjects observed a video; in phase 3, they performed computer tasks; in phase 4, the subjects walked around freely. In phase 1, control subjects were instructed to gaze at bright features in the field of view and, in a second test, at dark features. Test group subjects were allowed to gaze freely for all phases. Using the subject's gaze coordinates, we calculated the cumulative light distribution on the retina. As expected for control subjects, cumulative retinal light distributions peaked and dipped in the fovea when they gazed at bright or dark features respectively in the field of view. The light distribution maps obtained from the test group showed a consistent tendency to peak in the macula in phase 3, a variable tendency in phase 4, but little tendency in phases 1 and 2. We conclude that a tendency for light to peak in the macula is a characteristic of some individuals and of certain tasks. In these situations, risk of AMD could be increased but, at the same time, mitigated by the presence of macular carotenoids.     

Lutein, zeaxanthin, and the macular pigment

The predominant carotenoids of the macular pigment are lutein, zeaxanthin, and meso-zeaxanthin. The regular distribution pattern of these carotenoids within the human macula indicates that their deposition is actively controlled in this tissue. The chemical, structural, and optical characteristics of these carotenoids are described. Evidence for the presence of minor carotenoids in the retina is cited. Studies of the dietary intake and serum levels of the xanthophylls are discussed. Increased macular carotenoid levels result from supplementation of humans with lutein and zeaxanthin. A functional role for the macular pigment in protection against light-induced retinal damage and age-related macular degeneration is discussed. Prospects for future research in the study of macular pigment require new initiatives that will probe more accurately into the localization of these carotenoids in the retina, identify possible transport proteins and mechanisms, and prove the veracity of the photoprotection hypothesis for the macular pigments.     

Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.     

Complement dysregulation in AMD: RPE-Bruch's membrane-choroid

The question as to why the macula of the retina is prone to an aging disease (age-related macular degeneration) remains unanswered. This unmet challenge has implications since AMD accounts for approximately 54% of blindness in the USA (Swaroop, Chew, Bowes Rickman and Abecasis, 2009). While AMD has onset in the elder years, it likely develops over time. Genetic discovery to date has accounted for approximately 50% of the inheritable component of AMD. The polymorphism that has been most widely studied is the Y402H allele in the complement factor H gene. The implication of this genetic association is that in a subset of AMD cases, unregulated complement activation is permissive for AMD. Given that this gene variant results in an amino acid substitution, it is assumed that this change will have functional consequences although the precise mechanisms are still unknown. Genetic predisposition is not the only factor however, since in this complex disease there is substantial evidence that lifestyle factors such as diet and smoking contribute to risk. Here we provide an overview of current knowledge with respect to factors involved in AMD pathogenesis. Interwoven with these issues is a discussion of the significant role played by aging processes, some of which are unique to the retina and retinal pigment epithelium. One recurring theme is the potential for disease promotion by diverse types of oxidation products.     

Identification of StARD3 as a lutein-binding protein in the macula of the primate retina

Lutein, zeaxanthin, and their metabolites are the xanthophyll carotenoids that form the macular pigment of the human retina. Epidemiological evidence suggests that high levels of these carotenoids in the diet, serum, and macula are associated with a decreased risk of age-related macular degeneration (AMD), and the AREDS2 study is prospectively testing this hypothesis. Understanding the biochemical mechanisms underlying the selective uptakes of lutein and zeaxanthin into the human macula may provide important insights into the physiology of the human macula in health and disease. GSTP1 is the macular zeaxanthin-binding protein, but the identity of the human macular lutein-binding protein has remained elusive. Prior identification of the silkworm lutein-binding protein (CBP) as a member of the steroidogenic acute regulatory domain (StARD) protein family and selective labeling of monkey photoreceptor inner segments with an anti-CBP antibody provided an important clue for identifying the primate retina lutein-binding protein. The homology of CBP with all 15 human StARD proteins was analyzed using database searches, Western blotting, and immunohistochemistry, and we here provide evidence to identify StARD3 (also known as MLN64) as a human retinal lutein-binding protein. Antibody to StARD3, N-62 StAR, localizes to all neurons of monkey macular retina and especially cone inner segments and axons, but does not colocalize with the Mu?ller cell marker, glutamine synthetase. Further, recombinant StARD3 selectively binds lutein with high affinity (K(D) = 0.45 μM) when assessed by surface plasmon resonance (SPR) binding assays. Our results demonstrate previously unrecognized, specific interactions of StARD3 with lutein and provide novel avenues for exploring its roles in human macular physiology and disease.     

Transformation of the proteasome with age-related macular degeneration

The proteasome mediates pathways associated with oxidative stress and inflammation, two pathogenic events correlated with age-related macular degeneration (AMD). In human donor eyes corresponding to four stages of AMD, we found the proteasomal chymotrypsin-like activity increased in neurosensory retina with disease progression. Increased activity correlated with a dramatic increase in the inducible subunits of the immunoproteasome, which was not due to an increase in CD45 positive immune cells in the retina. The novel observation of proteasome transformation may reflect retinal response to local inflammation or oxidative stress with AMD.     

Mutation analysis of oxisterol-binding-protein gene in patients with age-related macular degeneration

Allelic variants of several genes are increasingly recognized as susceptibility factors in age-related macular degeneration (AMD). Because of its metabolic characteristics the macula is sensitive to oxidative damage, and supplementation with antioxidants has been shown to be effective in slowing the progression of disease in AMD patients. The oxisterol-binding-protein (OSBP2) gene is expressed mainly in the retinal pigmented epithelium underlying the macular region. Its product specifically binds and transports oxisterols, the cytotoxic effects of which may be involved in macular damage. The aim of this study was to search for allelic variants of OSBP2 gene, as well as to evaluate several risk factors in 24 patients with AMD; 17 with nonexudative (NE) and 7 with neovascular (NV) form. Total cholesterol was elevated in 66% of the patients, high-density lipoprotein (HDL) cholesterol was reduced in 12%; vitamin A or vitamin E deficiency was not observed. OSBP2 gene analysis was performed in AMD patients and in 110 control subjects by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Six allelic variants were detected: 2 nonpolymorphic unique exonic variants in 2 AMD subjects and 4 polymorphic variants (2 exonic and 2 intronic). These data indicate a possible role of OSBP2 gene in the pathogenesis of oxidative damage to the macula induced by oxysterols in AMD patients.     

Genomic aspects of age-related macular degeneration

Age-related macular degeneration (AMD) is a major late-onset posterior eye disease that causes central vision to deteriorate among elderly populations. The predominant lesion of AMD is the macula, at the interface between the outer retina and the inner choroid. Recent advances in genetics have revealed that inflammatory and angiogenic pathways play critical roles in the pathophysiology of AMD. Genome-wide association studies have identified ARMS2/HTRA1 and CFH as major AMD susceptibility genes. Genetic studies for AMD will contribute to the prevention of central vision loss, the development of new treatment, and the maintenance of quality of vision for productive aging.     

年龄相关性黄斑变性患者黄斑区脉络膜新生血管特征分析

目的:探讨年龄相关性黄斑变性黄斑区脉络膜新生血管(CNV)的特征。方法:对41例(41只眼)年龄相关性黄斑变性患者CNV进行荧光素眼底血管造影(FFA)和相干光断层扫描(OCT)检查,并对两种不同的图像检查结果进行对比分析。结果:FFA发现典型CNV29眼,FFA早期黄斑区可见边界清晰的高荧光区,FFA晚期荧光扩大并增强;隐匿型CNV10眼,FFA早期可见边界欠清的强荧光,FFA晚期持续为荧光素渗漏或染色,边界多不清晰。OCT发现典型CNV33眼,显示CNV多位于视网膜神经上皮层下,呈边界清晰的纺锤形或圆形强反射,隐匿型CNV8眼,表现为视网膜色素上皮层平面不规则增强紊乱的反射信号。两种检查对黄斑区CNV的检出率对比差异无统计学意义(P0.05)。结论:结合FFA和OCT的图像特征对年龄相关性黄斑变性黄斑区脉络膜新生血管形态学改变进行对比分析,有助于明确病变的性质,为临床诊疗提供帮助。     

Autophagy,exosomes and drusen formation in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch’s membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch’s membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.     

Changes in Retinal Glial Cells with Age and during Development of Age-Related Macular Degeneration

Age is the major risk factor in the age-related macular degeneration (AMD) which is a complex multifactor neurodegenerative disease of the retina and the main cause of irreversible vision loss in people over 60 years old. The major role in AMD pathogenesis belongs to structure-functional changes in the retinal pigment epithelium cells, while the onset and progression of AMD are commonly believed to be caused by the immune system dysfunctions. The role of retinal glial cells (Muller cells, astrocytes, and microglia) in AMD pathogenesis is studied much less. These cells maintain neurons and retinal vessels through the synthesis of neurotrophic and angiogenic factors, as well as perform supporting, separating, trophic, secretory, and immune functions. It is known that retinal glia experiences morphological and functional changes with age. Age-related impairments in the functional activity of glial cells are closely related to the changes in the expression of trophic factors that affect the status of all cell types in the retina. In this review, we summarized available literature data on the role of retinal macro- and microglia and on the contribution of these cells to AMD pathogenesis.     

The mitochondria-targeted antioxidant SkQ1 restores αB-crystallin expression and protects against AMD-like retinopathy in OXYS rats

Age-related macular degeneration (AMD), a neurodegenerative and vascular retinal disease, is the leading cause of blindness in the developed world. Accumulating evidence suggests that alterations in the expression of a small heat shock protein (αB-crystallin) are involved in the pathogeneses of AMD. Here we demonstrate that senescence-accelerated OXYS rats—an animal model of the dry form of AMD—develop spontaneous retinopathy against the background of reduced expression of αB-crystallin in the retina at the early preclinical stages of retinopathy (age 20 days) as well as at 4 and 24 months of age, during the progressive stage of the disease. The level of αA-crystallin expression in the retina of OXYS rats at all the ages examined was no different from that in disease-free Wistar rats. Treatment with the mitochondria-targeted antioxidant SkQ1 (plastoquinonyl-decyltriphenylphosphonium) from 1.5 to 4 months of age, 250 nmol/kg, increased the level of αB-crystallin expression in the retina of OXYS rats. SkQ1 slowed the development of retinopathy and reduced histological aberrations in retinal pigment epithelium cells. SkQ1 also attenuated neurodegenerative changes in the photoreceptors and facilitated circulation in choroid blood vessels in the retina of OXYS rats; this improvement was probably linked with the restoration of αB-crystallin expression.     

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch’s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch’s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch’s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch’s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch’s membrane biochemistry.     

Oxidative damage-induced inflammation initiates age-related macular degeneration

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.     

IFN-β signaling dampens microglia reactivity but does not prevent from light-induced retinal degeneration

Chronic activation of microglia is associated with retinal degeneration, which makes them a potential therapeutic target for retinal degenerative diseases including age-related macular degeneration (AMD). Interferon-beta (IFN-β) is a potent immune regulator, commonly used for the treatment of multiple sclerosis patients. We have previously shown that IFN-β prevents microgliosis and choroidal neovascularization in a laser model of wet AMD. Here, we hypothesized that microglia modulation via IFN-β may also dampen mononuclear phagocyte reactivity and thereby protect from retinal degeneration in a light-damage paradigm mimicking some features of dry AMD. BALB/cJ mice received intraperitoneal injections of 10,000 U IFN-β or vehicle every other day; starting at the day of exposure to 15,000 lux white light for 1 h. Systemic treatment with IFN-β partially enhanced IFN-α/β receptor (IFNAR) signaling in the retina and reduced the number of reactivated microglia in the subretinal space. However, four days after light damage neither decreased expression of complement factors nor rescue of retinal thickness was found. We conclude that IFNAR signaling modulate retinal microglia but cannot prevent strong retinal degeneration as elicited by acute white light damage.     

Treatment with 670 nm Light Up Regulates Cytochrome C Oxidase Expression and Reduces Inflammation in an Age-Related Macular Degeneration Model

Inflammation is an umbrella feature of ageing. It is present in the aged retina and many retinal diseases including age-related macular degeneration (AMD). In ageing and in AMD mitochondrial function declines. In normal ageing this can be manipulated by brief exposure to 670 nm light on the retina, which increases mitochondrial membrane potential and reduces inflammation. Here we ask if 670 nm exposure has the same ability in an aged mouse model of AMD, the complement factor H knockout (CFH^−/−) where inflammation is a key feature. Further, we ask whether this occurs when 670 nm is delivered briefly in environmental lighting rather than directly focussed on the retina. Mice were exposed to 670 nm for 6 minutes twice a day for 14 days in the form of supplemented environmental light. Exposed animals had significant increase in cytochrome c oxidase (COX), which is a mitochondrial enzyme regulating oxidative phosphorylation.There was a significant reduction in complement component C3, an inflammatory marker in the outer retina. Vimetin and glial fibrillary acidic protein (GFAP) expression, which reflect retinal stress in Muller glia, were also significantly down regulated. There were also significant changes in outer retinal macrophage morphology. However, amyloid beta (Aβ) load, which also increases with age in the outer retina and is pro-inflammatory, did not change. Hence, 670 nm is effective in reducing inflammation probably via COX activation in mice with a genotype similar to that in 50% of AMD patients even when brief exposures are delivered via environmental lighting. Further, inflammation can be reduced independent of Aβ. The efficacy revealed here supports current early stage clinical trials of 670 nm in AMD patients.