Lipofuscin In Retinal Pigment Epithelium Research Articles

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. A repeatability analysis revealed change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.

Removal of RPE lipofuscin results in rescue from retinal degeneration in a mouse model of advanced Stargardt disease: Role of reactive oxygen species

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging and is associated with retinal degeneration encountered in age-related macular degeneration (AMD) and Stargardt disease (SD). Currently, treatment for lipofuscin-induced retinal degeneration is unavailable. Here, we report that Remofuscin (INN: soraprazan, a tetrahydropyridoether small molecule) reverses lipofuscin accumulation in aged primary human RPE cells and is non-cytotoxic in aged SD mouse RPE cells in vitro. In addition, we show that the removal of lipofuscin after a single intravitreal injection of Remofuscin results in a rescue from retinal degeneration in a mouse model of advanced SD which is even accompanied by an amelioration of the retinal dysfunction. Finally, we demonstrate that the mechanism causing lipofuscinolysis may involve the reactive oxygen species generated via the presence of Remofuscin. These data suggest a possible therapeutic approach to untreatable lipofuscin-mediated diseases like AMD, SD and lipofuscinopathies in neurodegenerative diseases.

Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls.

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF–SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Best Vitelliform Dystrophy; Pathophysiology, Findings, Diagnosis, and Treatment

Best vitelliform dystrophy is the second most common hereditary macular dystrophy. It is an early-onset, progressive disease with autosomal dominant inheritance. The BEST1 gene is located on the long arm of the 11th chromosome and is responsible for the production of a transmembrane protein called bestrophin-1. The disease takes its name from the classic egg yolk-like macular lesion. Its histopathology shows increased lipofuscin in retinal pigment epithelium (RPE), photoreceptor loss, sub RPE deposits, and cell and material accumulation in the subretinal area. As long as complications such as choroidal neovascularization, macular scar, and geographical atrophy do not develop, the disease has a relatively good prognosis. The aim of this review is to summarize the possible etiopathogenesis, examination findings, imaging and electrophysiology characteristics, and treatment approaches of Best vitelliform dystrophy in the light of current literature.

Relative Contributions of All-Trans and 11-Cis Retinal to Formation of Lipofuscin and A2E Accumulating in Mouse Retinal Pigment Epithelium.

Purpose Bis-retinoids are a major component of lipofuscin that accumulates in the retinal pigment epithelium (RPE) in aging and age-related macular degeneration (AMD). Although bis-retinoids are known to originate from retinaldehydes required for the light response of photoreceptor cells, the relative contributions of the chromophore, 11-cis retinal, and photoisomerization product, all-trans retinal, are unknown. In photoreceptor outer segments, all-trans retinal, but not 11-cis retinal, is reduced by retinol dehydrogenase 8 (RDH8). Using Rdh8−/− mice, we evaluated the contribution of increased all-trans retinal to the formation and stability of RPE lipofuscin.Methods Rdh8−/− mice were reared in cyclic-light or darkness for up to 6 months, with selected light-reared cohorts switched to dark-rearing for the final 1 to 8 weeks. The bis-retinoid A2E was measured from chloroform-methanol extracts of RPE-choroid using HPLC-UV/VIS spectroscopy. Lipofuscin fluorescence was measured from whole flattened eyecups (excitation, 488 nm; emission, 565–725 nm).ResultsCyclic-light-reared Rdh8−/− mice accumulated A2E and RPE lipofuscin approximately 1.5 times and approximately 2 times faster, respectively, than dark-reared mice. Moving Rdh8−/− mice from cyclic-light to darkness resulted in A2E levels less than expected to have accumulated before the move.ConclusionsOur findings establish that elevated levels of all-trans retinal present in cyclic-light-reared Rdh8−/− mice, which remain low in wild-type mice, contribute only modestly to RPE lipofuscin formation and accumulation. Furthermore, decreases in A2E levels occurring after moving cyclic-light-reared Rdh8−/− mice to darkness are consistent with processing of A2E within the RPE and the existence of a mechanism that could be a therapeutic target for controlling A2E cytotoxicity.

Lipofuscin in keratinocytes: Production, properties, and consequences of the photosensitization with visible light

A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by the maximization of lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (~450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ~1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (1O2) when irradiated with blue light (420 nm), but there was no 1O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.

Quantitative Fundus Autofluorescence in Rhesus Macaques in Aging and Age-Related Drusen.

PurposeTo employ quantitative fundus autofluorescence (qAF) imaging in rhesus macaques to noninvasively assess retinal pigment epithelial (RPE) lipofuscin in nonhuman primates (NHPs) as a model of aging and age-related macular degeneration (AMD).MethodsThe qAF imaging was performed on eyes of 26 rhesus macaques (mean age 18.8 ± 8.2 years, range 4–27 years) with normal-appearing fundus or with age-related soft drusen using a confocal scanning laser ophthalmoscope with 488 nm excitation and an internal fluorescence reference. Eyes with soft drusen also underwent spectral-domain optical coherence tomography imaging to measure drusen volume and height of individual drusen lesions. The qAF levels were measured from the perifoveal annular ring (quantitative autofluorescence 8 [qAF8]) using the Delori grid, as well as focally over individual drusen lesions in this region. The association between qAF levels and age, sex, and drusen presence and volume were determined using multivariable regression analysis.ResultsMean qAF levels increased with age (P < 0.001) and were higher in females (P = 0.047). Eyes with soft drusen exhibited reduced mean qAF compared with age-matched normal eyes (P = 0.003), with greater drusen volume showing a trend toward decreased qAF levels. However, qAF levels are focally increased over most individual drusen (P < 0.001), with larger drusen appearing more hyperautofluorescent (R2 = 0.391, P < 0.001).ConclusionsIn rhesus macaques, qAF levels are increased with age and female sex, but decreased in eyes with soft drusen, similar to human AMD. However, drusen lesions appear hyperautofluorescent unlike those in humans, suggesting similarities and differences in RPE lipofuscin between humans and NHPs that may provide insight into drusen biogenesis and AMD pathogenesis.

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Quantifying lipofuscin in retinal pigment epithelium in vivo by visible-light optical coherence tomography-based multimodal imaging

Lipofuscin in the retinal pigment epithelium (RPE) is the major source of fundus autofluorescence (FAF). A technical challenge to accurately quantify the FAF intensities, thus the lipofuscin concentration, is to compensate the light attenuation of RPE melanin. We developed the VIS-OCT-FAF technology to accomplish optical coherence tomography (OCT) and FAF simultaneously with a single broadband visible light source. We demonstrated that light attenuation by RPE melanin can be assessed and corrected using the depth-resolved OCT signals. FAF images from albino and pigmented rats showed that without compensation, FAF signals from pigmented rats are lower than that from albinos. After compensation, however, FAF signals from pigmented rats are higher. This finding is supported by measurements of lipofuscin fluorophore A2E in the RPE using liquid chromatography/mass spectrometry (LC/MS) showing that compensated FAF intensities correlate linearly with A2E contents. The present work represents an important step toward accurately assessing RPE lipofuscin concentrations by FAF.

Visible-light optical coherence tomography-based multimodal system for quantitative fundus autofluorescence imaging.

Quantitative fundus autofluorescence imaging with simultaneous visible-light optical coherence tomography-based multimodal technology has potential significant impact on the diagnosis and monitoring the progression of retinal diseases.

Conversion of all-trans-retinal into all-trans-retinal dimer reflects an alternative metabolic/antidotal pathway of all-trans-retinal in the retina

Free all-trans-retinal (atRAL) and retinal pigment epithelium (RPE) lipofuscin are both considered to play etiological roles in Stargardt disease and age-related macular degeneration. A2E and all-trans-retinal dimer (atRAL-dimer) are two well characterized bisretinoid constituents of RPE lipofuscin. In this study, we found that, after treatment of primary porcine RPE (pRPE) cells with atRAL, atRAL-dimer readily formed and accumulated in a concentration- and time-dependent manner, but A2E was barely detected. Cell-based assays revealed that atRAL, the precursor of atRAL-dimer, significantly altered the morphology of primary pRPE cells and decreased cell viability at a concentration of 80 μm regardless of light exposure. By contrast, atRAL-dimer was not cytotoxic and phototoxic to primary pRPE cells. Compared with atRAL and A2E, atRAL-dimer was more vulnerable to light, followed by the generation of its photocleaved products. Moreover, we observed the presence of atRAL-dimer in reaction mixtures of atRAL with porcine rod outer segments (ROS), RPE/choroid, or neural retina. Taken together, we here proposed an alternative metabolic/antidotal pathway of atRAL in the retina: atRAL that evades participation of the visual (retinoid) cycle undergoes a condensation reaction to yield atRAL-dimer in both ROS and RPE. Translocation of atRAL, all-trans N-retinylidene-phosphatidylethanolamine (NR-PE), atRAL-dimer, and photocleavage products of atRAL-dimer from ROS into RPE is accomplished by phagocytosing shed ROS on a daily basis. Without causing damage to RPE cells, light breaks up total atRAL-dimer within RPE cells to release low-molecular-weight photocleavage fragments. The latter, together with ROS-atRAL-dimer photocleavage products, may easily move across membranes and thereby be metabolically eliminated.

A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease.

To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5. Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins. EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 μm) than in the wild-type (61.40 ± 1.84 μm) or untreated ABCA4-/- (56.50 ± 3.24 μm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response. Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.

Omega‐3 fatty acids supplementation: therapeutic potential in a mouse model of Stargardt's disease

PurposeTo evaluate the therapeutic effects of omega‐3 (ω‐3) fatty acids in the ABCA4‐/‐ model of Stargardt's disease. Monitoring the blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA), served to adjust the treatment dosage (AA/EPA = 1‐1.5).MethodsEight‐month‐old mice were allocated to different groups: A) wild type (129S1), B) ABCA4‐/‐ untreated, C) ABCA4‐/‐ treated with ω‐3. Treatment was daily administered by gavage for 3 months. Eye cups from each group were histologically examined using confocal and transmission electron microscopy. Proteomic analysis and N‐retinylidene‐N‐retinylethanolamine (A2E) quantification was carried our using liquid chromatography mass spectrometry (LC‐MS/MS). Blood and retinal fatty acids analysis was performed using gas chromatography (GC).ResultsA significant decrease in melanolipofuscin granules in the retinal pigment epithelium (RPE) was observed in the treatment group, in parallel with a reduction in A2E level, a major component of RPE lipofuscin. Proteomic analysis indicated decreased levels of complement 3 (C3) in the treated group. The outer nuclear layer (ONL) thickness was significantly greater in group C (75.66 ± 4.8 μm), compared to group A (61.40 ± 1.84 μm) and B (56.50 ± 3.24 μm). Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment group.ConclusionsSupplementation with ω‐3 fatty acids (when AA/EPA = 1‐1.5) suggests a protective mechanism in the ABCA4‐/‐ animal model of Stargadt's disease.

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell.

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.

All-trans retinal levels and formation of lipofuscin precursors after bleaching in rod photoreceptors from wild type and Abca4-/- mice

The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.

178 - Oxidative, Morphological and Mechanical Changes in ARPE-19 Cells Induced by Lipofuscin-Mediated Photic Stress

Retinal pigment epithelium (RPE) provides important metabolic support for the entire retina and is involved in biological renewal of photoreceptor outer segments. It is believed that key changes involved in the pathology of age-related macular degeneration, the major cause of blindness in people over 60, occur in the RPE. Being exposed to high oxygen tension and intense visible light from focused irradiation, human RPR is at risk of oxidative stress, which is aggravated by the accumulating age pigment lipofuscin. In this work, we examined the effect of photic stress, induced by blue light irradiation of ARPE-19 cells containing phagocytized RPE lipofuscin granules isolated from human donors of different age, on cell survival, oxidation of cellular proteins, morphology of the cells and their mechanical properties, employing standard cell culture techniques, coumarin boronic acid (CBA) assay, immunofluorescence analysis with laser scanning confocal microscopy (LSCM), and atomic force microscopy (AFM) and spectroscopy. Photic stress mediated by lipofuscin from older donors reduced survival of ARPE-19 cells more efficiently than lipofuscin from younger donors. Sub-lethal stress induced oxidation of cellular proteins in a dose-dependent manner with the effect being stronger for lipofuscin from older donors. These biochemical changes correlated with modifications of the cell morphology, which indicated substantial decrease in the formation of actin stress fibers in lipofuscin-containing cells subjected to sub-lethal photic stress. In addition, nanomechanical analysis revealed that such cells were significantly softer than control non-irradiated cells, or irradiated cells without lipofuscin. Our study demonstrates that AFM analysis can be used for sensitive detection of early changes of cultured RPE cells subjected to sub-lethal oxidative stress. Supported by Poland National Science Centre (grant Maestro 2013/08/A/NZ1/00194).

Quantitative Fundus Autofluorescence in Early and Intermediate Age-Related Macular Degeneration

Increased lipofuscin accumulation is assumed to be an important factor in the pathogenesis of age-related macular degeneration (AMD), although direct evidence for this hypothesis is missing. To quantitatively investigate lipofuscin-associated fundus autofluorescence (AF) in patients with early and intermediate AMD. A prospective, single-center, case-control study was conducted from August 1, 2014, to October 31, 2015, at a university referral center. Participants included 40 patients aged 65 years or younger and 108 individuals without eye disease serving as controls. All participants underwent quantitative fundus AF (qAF) imaging with a modified scanning laser ophthalmoscope equipped with an internal fluorescent reference. Mean qAF values of an 8-segment circular ring centered on the fovea (qAF8) were measured and compared between patients and controls. For subgroup analysis, drusen were categorized as soft drusen, cuticular drusen, and/or reticular pseudodrusen (RPD). The qAF8 levels. In the 40 patients with AMD, mean (SD) age was 54.8 (5.6) years, and 32 (80%) were women. None of the investigated patients had qAF8 values above the 95% prediction interval (PI) of the 108 controls. In the soft drusen (28 [70%]) and cuticular drusen (8 [20%]) groups, qAF8 levels within the 95% PI were noted in 22 patients (79%; 95% CI, 60% to 90%) and 7 patients (88%; 95% CI, 51% to 99%) respectively. The qAF8 values in the RPD group (4 [10%]) were below the 95% PI in 3 patients (75%; 95% CI, 29% to 97%). Compared with the controls, statistical analysis revealed lower qAF8 values in the overall AMD cohort after adjusting for age (difference, -19.9% [95% CI, -25.6% to -12.7%], P < .001) as well as in all subgroups (soft drusen, -17.1% [95% CI, -24.1% to -9.5%], P < .001; cuticular drusen, -19.6% [95% CI, -30.3% to -7.2%], P = .003; and RPD, -34.5% [95% CI, -47.1% to -21.3%]; P < .001). The qAF8 measurements in this sample showed no increased lipofuscin-related fundus AF in patients with early and intermediate AMD. Lower qAF levels in certain subgroups may point to subnormal lipofuscin levels in the retinal pigment epithelium or, alternatively, limitations to detection of true retinal pigment epithelial lipofuscin content. The results of this study might expand the understanding of the pathogenesis of AMD and may have an effect on upcoming treatment trials that aim to modify lipofuscin accumulation.

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.