Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (ß-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of ß-CD. Importantly, ß-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of ß-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of ß-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Retinal pigment epithelium lipofuscin proteomics.

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

Accumulation of ceroid-like pigments in macrophages cultured with phosphatidylcholine liposomes in vitro.

When mouse peritoneal macrophages as well as P388D1 cells, an established macrophage-like cell line, were cultured with liposomes composed of rat liver phosphatidylcholine and phosphatidylserine, storage of fluorescent products, ceroid-like pigments, within those cells was observed with light and fluorescence microscopy, and fluorescence spectrophotometry. The amounts of thiobarbituric acid-reactive substances and fluorescent products in macrophages were increased gradually to reach a maximal level to between 6 and 8 days of culture. The involvement of peroxidation of liposomal lipids in the formation of the pigments was further suggested by the 6 days that incorporation of alpha-tocopherol into liposomes decreased the storage of the pigments. No appreciable formation of the pigments was observed in macrophages cultured with liposomes containing dipalmitoylphosphatidylcholine instead of rat liver phosphatidylcholine. The fluorescent products formed in cultured cells were found in lipid-soluble and -insoluble fractions. Lipid-insoluble fluorescent products had an excitation maximum at 360 nm and a fluorescence maximum at 430 nm in SDS-aqueous solution (pH 7.4) and the intensity of the fluorescence was quenched at base pH, but it was not changed in acidic media. These findings indicate that the macrophages can store Schiff base fluorescent substances formed by the reaction between peroxidation products of exogenous lipids and amino compounds in the cells, under some pathological conditions.

Proteome analysis of lipofuscin in human retinal pigment epithelial cells.

Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age-related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.

Preparation of artificial ceroid/lipofuscin by UV-oxidation of subcellular organelles.

Recent studies have consistently shown that, during oxidative damage, glycation, and other oxygen stress-related reactions, various biomolecules are converted into ceroid- and lipofuscin-like fluorescent pigments. In this study, artificial ceroid/lipofuscin was produced by exposing rat liver fractions to UV-light overnight. Thiobarbituric acid reactive substances (TBARS) were formed in increasing amounts during the early stages of the process, but decreased as the material was later converted into a polymeric structure with few remaining peroxides. In the transmission electron microscope the artificial pigment showed lamellar structures and was osmiophilic. By energy-dispersive X-ray analysis the material was found to contain Ca and Fe in the same way as natural ceroid/lipofuscin. Moreover, it exhibited ceroid/lipofuscin-like, greenish-yellowish autofluorescence when assayed by microfluorometry, with a fluorescence maximum consistently found at 430 nm when excited at 350 nm. Identical fluorescence maxima were found for each fraction of rat liver that was used as the origin of the pigments, i.e. nuclei, mitochondria, lysosomes and microsomes. Extracts with either chloroform-methanol, or sodium dodecylsulphate, showed identical complex fluorescence. When the pigments were extracted by chloroform-methanol, five fluorescent bands were obtained after thin-layer chromatographic separation. Fibroblasts were found to endocytose the material, a process that converted them into lipofuscin-loaded cells of an aged phenotype as observed by light and electron microscopy. Similar fluorescence emission spectra were obtained from cells grown at 40% O2, in order to stimulate endogenous lipofuscin-formation, and from cells exposed to artificial ceroid/lipofuscin. The described technique for creating artificial ceroid/lipofuscin is relatively easy to perform and should provide a useful new tool to study the possible influences of ceroid/lipofuscin on lysosomal and cellular functions.

Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium.

PURPOSE: Lipofuscin (LF) accumulation in the retinal pigment epithelium (RPE) is associated with age and various retinal diseases. Toxic LF compounds may interfere with normal RPE function. Oxidative modification of proteins was determined in LF granules from human eyes. METHODS: LF was isolated from the RPE-choroid complex of 10 pairs of donor eyes by gradient ultracentrifugation. Protein compounds were separated by two-dimensional (2-D) gel electrophoresis and screened by Western blot analysis for lipid peroxidation- or glucoxidation-induced damage-in particular, by malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGEs). Identity of the immunostained proteins was revealed using 2-D software for comparison of the spot position with Coomassie-stained 2-D gels of the same samples. RESULTS: By comparing the results taken from the authors' previous proteome analysis of RPE LF with an immunoblot analysis of the same samples, this study shows that a variety of LF-associated proteins were damaged by aberrant covalent modifications of MDA, 4-HNE, and AGEs. Several proteins were altered by two or three different modification types. Modified mitochondrial proteins indicated that autophagy of altered proteins also contributed to lipofuscin formation. CONCLUSIONS: The identification of lipid peroxidation and glucoxidation products in proteinaceous LF components in human RPE supports the hypothesis that these compounds are involved in lipofuscinogenesis and may contribute to the cytotoxic effects of LF in retinal diseases such as age-related macular degeneration and Stargardt disease. Their identification may help to identify potential future treatment targets.

Photophysical studies on human retinal lipofuscin.

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime ca 7 microseconds) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.

The photochemistry of human retinal lipofuscin as studied by EPR.

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light-induced contribution of these fluorophores to various diseases including age-related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen-free methanol led to the formation of a 5,5-dimethylpyrroline-N-oxide (DMPO) spin-trapped carbon-centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform-soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age-related decline of RPE function and blue light damage in the retina.

A2E: a component of ocular lipofuscin.

The presence of lipofuscin in postmitotic cells is considered a hallmark of the aging process. In the retinal pigment epithelium (RPE), lipofuscin is found as micrometer-sized spherical particles and characterized by its yellow autofluorescence when exposed to blue light. This exposure to light is also known to produce reactive oxygen intermediates (ROI), but the particular molecular constituent(s) responsible for this phototoxicity have yet to be completely identified. Resulting mostly from the autophagocytosis of intracellular organelles, the composition of lipofuscin is poorly defined but known to contain protein, lipids and several fluorophores. The subsequent identification of one of the fluorophores in lipofuscin, A2E, generated much interest and resulted in a variety of studies to understand its potential role in the phototoxicity of lipofuscin. Several modes of toxicity have been suggested through which A2E can affect the health of RPE cells. These modes include photoinduced production of ROI, which places additional oxidative stress on RPE cells, the disruption of membrane integrity through its natural role as an amphiphilic detergent and inhibition of key cellular functions. This article presents the current understanding of the photochemistry of A2E and its involvement as a phototoxic agent in RPE cells.

Studies on chemical nature of lipofusion (age pigment) isolated from normal human brain.

Human brain lipofusion isolate was studied for its purity and physical and chemical properties. Purification of the inpure material was achieved by gel permeation chromatography using Sephadex LH-20 and BioBeads S-X1 gels. The purified lipofusion polymer represented ca. 12% of the starting material with the rest of the material being various mixed lipids. The mol wt of the purified lipofuscin was determined to be between 6000-7000 daltons. IR, UV-visible, NMR, and fluorometric spectra were obtained, all indicating the fundamentally lipid nature of lipofuscin. The NMR spectrum strongly resembled that of a typical long chain fatty acid. Numbers of fatty acids and several amino acids were present as a portion of the lipofuscin structure. The results obtained suggested that the brain lipofuscin employed in the present study consisted mainly of polymeric lipid and phospholipid structures along with amino acids either bound to the lipids or as included proteins.

Organic solvent soluble lipofuscin pigment in brain tissues of mice fed large amounts of polyunsaturated fats in presence and absence of various antioxidants.

The amount of lipid soluble fluorescent pigment is compared in brain tissues of mice fed a diet containing 10% safflower oil with and without a supplement of antioxidants as Vitamin E, Ascorbyl-Palmitate and Sodium-Selenite. Addition of 0.2 g/kg Vitamin E to the diet gives a high raise in the mortality. The addition of 0.2 g/kg Vitamin E and 0.5 g/kg Ascorbyl-Palmitate leads to higher values of extractable lipid soluble fluorescent pigment in mice brain tissues.

Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.

Several lines of investigation suggest that the nondegradable fluorophores that accumulate as lipofuscin in retinal pigment epithelium (RPE) cells contribute to the etiology of macular degeneration. Despite evidence that much of this fluorescent material may originate as inadvertent products of the retinoid cycle, the enzymatic pathway by which the 11-cis-retinal chromophore of rhodopsin is generated, the only fluorophores of the RPE to be characterized as yet have been A2E and its isomers. Here, we report the isolation and structural characterization of an additional RPE lipofuscin fluorophore that originates as a condensation product of two molecules of all-trans-retinal (ATR) dimer and forms a protonated Schiff base conjugate with phosphatidylethanolamine (PE), the latter conjugate (ATR dimer-PE) having UV-visible absorbance maxima at 285 and 506 nm. ATR dimer was found to form natively in bleached rod outer segments in vitro and when rod outer segments were incubated with ATR. HPLC analysis of eye-cups that included RPE and isolated neural retina from Abcr-/- mice and RPE isolated from human donor eyes revealed the presence of a pigment with the same UV-visible absorbance and retention time as synthetic ATR dimer-PE conjugate. Evidence that ATR dimer undergoes a photooxidation process involving the addition of oxygens at double bonds as well as an aromatic demethylation also may indicate a role for this molecule, or its derivatives, in the photoreactivity of RPE lipofuscin.

Extraction and purification of yellow-fluorescent lipofuscin in rat kidney.

Yellow fluorescent lipofuscin deposited in rat tissues was extracted in an aqueous solution. Yellow fluorescence with fluorescence maxima at 400/615-620 nm was detected in the aqueous extracts of brain and kidney of older rats. Purification and characterization of the yellow fluorescent components extracted from rat kidney were attempted. Centrifugal fractionation of the extract revealed that the flourescence was detected in the 105,000 g supernatant, and not in nuclei, mitochondria, lysosomes, microsomes and cell debris. Gel filtration of the supernatant through Sephadex columns gave at least 5 yellow fluorescent components with different molecular weights. The higher-molecular-weight components were converted into the smaller ones on treatment with alkali or ethanol, but may not be on protease treatment. These components were adherent to polymers composed of sugars. They were soluble in water and not in an organic solvent. While the yellow fluorophores were stable on borohydride treatment, they were destroyed on heavy metal ion treatment. The characteristics of the yellow fluorophores were different from those of bluish lipofuscin-like fluorophores generated by lipid peroxidation.

Isolation of melanin from human plasma lipofuscin.

Human plasma lipofuscin and its melanin component were isolated and quantified. Electron paramagnetic resonance, infrared, ultraviolet and visible spectra of this melanin exhibited absorption characteristics very similar to those of known melanins. The human plasma lipofuscin contained approximately 85% protein, 3% melanin, 0.4% lipid and 0.25% mucoprotein constituents and emitted yellow-green fluorescence in 366-nm light. The ethanol-ether lipid extract obtained after acid hydrolysis from the lipid-melanin fraction of this lipofuscin was also found to fluoresce in yellow-green color in 366-nm light and produced similar fluorescence excitation and emission spectra as those of the human plasma lipofuscin in water solution. The isolated melanin component was not fluorescent.

Structural basis of cation exchange, complex formation and redox properties in chloragosomes.

Components of chloragosomes isolated from Octolasium transpadanum ROSA were separated by fractional extraction. The chemical composition of the fractions was determined and their effect on the anion- and cation-binding capability and on the lipophilic property of chloragosomes was studied. The acid-alcoholic extract of chloragosomes contained, among others, metalloporphyrins with 2 or 4 free -COOH groups; the residuum consisted of basic proteins. The metalloporphyrins and phosphatides gained by extraction with chloroform-methanol neutralize the basic groups of the chloragosome proteins, thus, an excess of free acidic groups develops, which is responsible for the polyanionic nature and cation exchange of chloragosomes. The apolar groups of phosphatides and carotenoids are responsible for the lipophilic nature of chloragosomes. Under experimental circumstances both in vivo and in vitro, organic cations were bound to chloragosomes, partially by a cation-exchange mechanism and partially by formation of more stable complexes. A small amount of anions may be absorbed by the bound cations. The diluted acidic extract of chloragosomes predominantly contain organic cations, in the present study riboflavin, flavin nucle0tide and thiamine were determined. Chloragosomes, owing to their structure, form complexes of varying stability with anions, cations and hydrophilic compounds equally. This feature explains their capability of cumulating trophic and toxic substances, while their redox activity is explained by their metalloporphyrin, flavin, thiamine and carotene content.

Fluorophores of the human retinal pigment epithelium: separation and spectral characterization.

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

Lipofuscin pigment in cerebellar Purkinje neurones and choroid plexus epithelial cells of macaque monkeys with Plasmodium knowlesi cerebral malaria: an electron microscopical observation.

Experimental infection of rhesus monkeys (Macaca mulatta) with a virulent (W1) strain of Plasmodium knowlesi resulted in cerebral malaria. Electron microscopical examination of the brain revealed large numbers of intracytoplasmic lipofuscin pigment deposits in cerebellar Purkinje neurones and choroid plexus epithelium of the lateral ventricle. This lesion may be part of the nervous system response to ischaemic hypoxia.

Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T.

Amyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer-sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protein.