Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

Retinal long-chain PUFAs (LC-PUFAs, C₁₂-C₂₂) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, C_n>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology.     

Comparisons between the inorganic content of healthy and hypertensive rat tissues by inductively coupled plasma-mass spectrometry

The inorganic contents of bone, brain, erythrocyte, heart, kidney cortex, kidney medulla, liver, lung, muscle and plasma from spontaneously hypertensive rats were compared with those of the same tissues from healthy Sprague-Dawley rats. A general inductively coupled plasma-mass spectrometry method developed for multi-element determinations of most of the elements present in biological tissues was used. Variations were found not only for major elements, as expected, but also for many trace elements in several tissues.     

Evaluation of the role of the retinal G protein-coupled receptor (RGR) in the vertebrate retina in vivo

The retinal G protein-coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein-coupled receptors. RGR may play a role as a photoisomerase in the production of 11-cis-retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11-cis-retinol dehydrogenase (RDH5), is to regenerate 11-cis-retinal under light conditions and RDH5 is expected to function in the light-independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr-/- single and rdh5-/-rgr-/- double knockout mice under a number of light conditions. The most striking phenotype of rgr-/- mice after a single flash of light includes light-dependent formation of 9-cis- and 13-cis-retinoid isomers. These isomers are not formed in wild-type mice because either all-trans-retinal is bound to RGR and protected from isomerization to 9-cis- or 13-cis-retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all-trans-retinyl esters and an attenuated recovery of 11-cis-retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic-like photoisomerization-driven production of 11-cis-retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod-dominated mice.     

Fractionation analysis and bioavailability of manganese in spinach (Spinacia oleracea L.) leaves

Abstract

This paper introduces a fractionation scheme using water, acetone, chloroform, diethyl ether, ethanol, n-hexane, and methanol as extractants for the determination of manganese in spinach samples by inductively coupled plasma-mass spectrometry (ICP-MS). Simulated gastric and intestinal digestions as well as n-octanol extraction and activated carbon adsorption were performed for the bioavailability assessments. Comparative studies of the various extraction treatments were evaluated for confirmation analysis. The total elemental concentrations were determined after digesting the samples in a microwave digestion system. The method validation parameters were defined in terms of the detection limits, accuracy, and precision. Additional validation was performed by comparing the ICP-MS method with atomic absorption spectrometry. The limits of detection and quantification were 0.046 and 0.154 mg kg^-1, respectively. Additionally, the repeatability and reproducibility, calculated from the relative standard deviation (%RSD), were 2.4% and 3.7%, respectively.     

Studies on some trace and minor elements in blood

Blood is one of the widely used specimens for biological trace element research because of its biological significance and ease of sampling. We have conducted a study of the blood of the Kalpakkam township population for trace and minor elements. For this purpose, analytical methods have been developed and standardized in our laboratory for the elemental analysis of blood plasma and red cells. Inductively coupled plasma-mass spectrometry (ICP-MS), a relatively new technique, has been applied for the analysis of trace elements. Details regarding spectral interference and matrix interference encountered in the analysis of blood and the methods of correcting them have been discussed. Flame atomic absorption spectrometry (AAS)/atomic emission spectrometry (AES) has been applied for the determination of minor elements. Precision and accuracy of these methods have also been discussed.     

Rapid and direct determination of selenium, copper, and zinc in blood plasma by flow injection-inductively coupled plasma-mass spectrometry

A flow injection-inductively coupled plasma-mass spectrometry (FI-ICP-MS) with a simple sample preparation procedure was developed for the determination of selenium, copper, and zinc in blood serum/plasma. A serum/plasma sample was filtered through a 0.45-μm membrane filter and diluted with a mixture of trace elements in a standard solution (9∶1, v/v). Measurement of the reference serum sample confirmed the accuracy of our method for selenium, copper, and zinc concentration. In the case of blood plasma samples obtained from six healthy adult males, the selenium, copper, and zinc concentrations were similar to those of a typical healthy male in Japan. These results suggest that the sample prepartive procedure coupled with FI-ICP-MS can be used for the routine determination of selenium, copper, and zinc in human blood serum/plasma.     

Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1^flox/flox). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.     

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch''s membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch''s membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4^−/− and Rdh8^−/−/Abca4^−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4^−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4^−/− mice revealed that carbonyl adduct deposition in Bruch''s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4^−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch''s membrane that can confer risk of age-related macular degeneration.     

Regenerating Eye Tissues to Preserve and Restore Vision

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Molybdenum and Chromium Concentrations in Breast Milk from Japanese Women

Molybdenum (Mo) and chromium (Cr) in 79 Japanese breast milk samples were measured by inductively coupled plasma-mass spectrometry. For Mo, 51 samples (64.6%) showed less than 5 ng/ml and only 12 samples (15.2%) showed more than 10 ng/ml. The range and median were <0.1 to 25.91 and 3.18 ng/ml respectively. For Cr, 38 samples (48.1%) showed less than 1 ng/ml, 20 samples (25.3%) showed 1 to 2 ng/ml, and only six samples (7.6%) showed more than 5 ng/ml. The range and median were <0.1 to 18.67 and 1.00 ng/ml respectively.     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.     

Concentrations of selected trace elements in human milk and in infant formulas determined by magnetic sector field inductively coupled plasma-mass spectrometry

Magnetic sector field inductively coupled plasma-mass spectrometry (ICP-MS) was applied to the reliable determination of the 8 essential trace elements cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), selenium (Se), and vanadium (V) as well as the 7 nonessential and toxic elements silver (Ag), aluminum (Al), arsenic (As), gold (Au), platinum (Pt), scandium (Sc), and titanum (Ti) in 27 transitory and mature human milk samples and in 4 selected infant formulas. This advanced instrumentation can separate spectral overlaps from the analyte signal hampering significantly the determination of many trace elements by conventional ICP-MS. Moreover, superior detection limits in the picogram per liter range can be obtained with such magnetic sector field instruments. Therefore, this is the first study to report the concentrations of the elements Ag, Au, Pt, Sc, Ti, and V in human milk and in infant formulas. Concentrations of Ag (median: 0.41 μg/L; range: <0.13–42 μg/L) and Au (median: 0.29 μg/L; range 0.10–2.06 μg/L) showed large variations in human milk that might be associated with dental fillings and jewelry. Pt concentrations were very low with most of the samples below the method detection limit of 0.01 μg/L. Human milk concentrations of Co (median: 0.19 μg/L), Fe (380 μg/L), Mn (6.3 μg/L), Ni (0.79 μg/L), and Se (17 μg/L) were at the low end of the corresponding reference ranges. Concentrations of Cr (24.3 μg/L) in human milk were five times higher than the high end of the reference range. For Al (67 μg/L), As (6.7 μg/L), and V (0.18 μg/L), most of the samples had concentrations well within the reference ranges. All elemental concentrations in infant formulas (except for Cr) were approximately one order of magnitude higher than in human milk.     

Pigmented anatomy in Carboniferous cyclostomes and the evolution of the vertebrate eye

The success of vertebrates is linked to the evolution of a camera-style eye and sophisticated visual system. In the absence of useful data from fossils, scenarios for evolutionary assembly of the vertebrate eye have been based necessarily on evidence from development, molecular genetics and comparative anatomy in living vertebrates. Unfortunately, steps in the transition from a light-sensitive ‘eye spot’ in invertebrate chordates to an image-forming camera-style eye in jawed vertebrates are constrained only by hagfish and lampreys (cyclostomes), which are interpreted to reflect either an intermediate or degenerate condition. Here, we report—based on evidence of size, shape, preservation mode and localized occurrence—the presence of melanosomes (pigment-bearing organelles) in fossil cyclostome eyes. Time of flight secondary ion mass spectrometry analyses reveal secondary ions with a relative intensity characteristic of melanin as revealed through principal components analyses. Our data support the hypotheses that extant hagfish eyes are degenerate, not rudimentary, that cyclostomes are monophyletic, and that the ancestral vertebrate had a functional visual system. We also demonstrate integument pigmentation in fossil lampreys, opening up the exciting possibility of investigating colour patterning in Palaeozoic vertebrates. The examples we report add to the record of melanosome preservation in Carboniferous fossils and attest to surprising durability of melanosomes and biomolecular melanin.     

Determination of total silver and silver species in coastal seawater by inductively-coupled plasma mass spectrometry after batch sorption experiments with Chelex-100 resin

Abstract

Sorption experiments in batch mode with Chelex-100 resin have been carried out for the determination of total Ag, labile and inert fractions in seawater from Ria de Vigo (NE Atlantic ocean) by inductively coupled plasma-mass spectrometry (ICP-MS). The method allows an accurate determination of the total Ag thus avoiding the effect of the saline matrix when direct determination by ICP-MS is performed. Similarly, the distribution patterns of Ag among species with different lability can be established in seawater as a function of pH. Species distribution and total Ag contents have been obtained in CRM CASS-4 (near shore seawater) and four coastal seawater samples. While the free silver ion concentration accounts for a significant fraction at low pH, the labile inorganic fraction is most important at natural pH (≈8) in non-polluted seawater. The inert and labile organic fractions were the most relevant in polluted coastal seawater. Complexation mechanisms of silver were also investigated from the sorption curves of silver on Chelex-100 according to the Gibbs-Donnan model. Two complexes were formed, AgL and AgL₂ with the following intrinsic complexation constants log β_(1np) = ?2.11 and ?10.02, respectively. The formation of the complex AgL predominates up to pH 4 but is negligible due to chloride ions effect, while the complex AgL₂ predominates from pH 4.     

Integration of eQTL and a Single-Cell Atlas in the Human Eye Identifies Causal Genes for Age-Related Macular Degeneration

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Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

In a previous study, we used differential proteomics to identify retinal proteins whose steady‐state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady‐state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina—retinal recycling and generation of retinoic acid—we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11‐cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 623–635, 2010     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

Changes in the Redox State in the Retina and Brain During the Onset of Diabetes in Rats

Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio.Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD⁺/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state     

Electron microscopic comparative studies of phagocytic processes between outer segments and latex microspheres in cultured human retinal pigment epithelial cells

Summary The process of phagocytosis in cultured human retinal pigment epithelium (RPE) cells was observed after more than 2 h of incubation with human outer segments and latex microspheres. Fingerlike microvilli were attached to outer segments and entwined around both its ends. The microvilli enveloped the outer segment and cut into the membranous structure, and the same process as that seen in in vivo shedding was observed. The looplike disk membranes and the whole of the outer segment were ingested into the cytoplasm and degraded by the lysosome. Latex microspheres were also ingested into the cytoplasm so as to be enveloped in many fingerlike microvilli. Microfilaments were concentrated in the vicinity of latex microspheres and outer segments, and latex microspheres were placed between two microtubules. Furthermore, when latex microspheres and outer segments were ingested into the cytoplasm, the dilated rough endoplasmic reticulum (rER) contained materials of high electron density and attached ribosomes increased. The rER of the cultured human RPE cells seemed to show a high level of protein synthesis during the phagocytic process. It was observed that cytoskeletons, such as microfilaments and microtubules, and lysosomes had important functions in the phagocytic process, and that there were basically no differences among the objects phagocytized by the cultured RPE cells. Supported by the Ministry of Education, Japan, through research grant 60771423.