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Hearing and cognition are commonly involved in both normal and pathological aging. Current clinical interest lies in whether peripheral hearing loss promotes cognitive decline. In our previous publication, the authors have shown a causal relationship between hearing and cognitive impairments in C57BL/6 mice. Here we extended the follow-up to 12 months to determine the long-term effects of hearing loss on cognition and to observe hippocampal p-tau and lipofuscin. One month old male mice were randomly allocated into two groups, the control (n = 12) and noise-induced hearing loss (NIHL) (n = 12). After baseline hearing and cognitive measurements, the mice in the NIHL group were exposed to 110 dB SPL white noise for 1 h every day for 20 consecutive days. Cognitive function was assessed by radial arm maze and novel object recognition tests. p-Tau was observed by the western blot, immunofluorescence, and immunogold staining. The mice in the NIHL group showed elevated auditory brainstem response thresholds and poorer performances in spatial working and recognition memories than the controls. They exhibited more p-tau and lipofuscin in the hippocampus. The cognitive impact of hearing loss varied with the types of memory. Working memory impairment was reversible, whereas recognition memory impairment was permanent. Our results provide behavioral and histopathological evidence for hearing-related cognitive decline. Early hearing loss is suggested to be one of the important determinants between normal and pathological cognitive aging., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor's age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.

The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols., (Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.)

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contain water-solubilizing amine groups, and several were more potent inhibitors of A2E photooxidation than quercetin.

Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch's membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host's cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

Lipofuscin, an autofluorescent biomarker of physiological wear-and-tear, was concentrated in those areas of a fish's midbrain responsible for visual performance, suggesting a potentially strong link between physiological specialization, ecological adaptation and senescence., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation mechanisms to remove damaged structures completely results in the progressive accumulation of garbage, including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging. The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mitochondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery, which eventually is incompatible with survival.

Aims: To analyse the autofluoresence (AF) properties of pingueculae and compare the size of AF with the extent of the visible lesion., Methods: Forty eyes of 23 patients with pingueculae were included in the study. AF images were obtained using an HRA2 confocal scanning laser ophthalmoscope; anterior segment photographies were obtained using TRC-50IX, IMAGEnet 2000 Digital Imaging System. The AF characteristics of pingueculae were analysed. The extent of visible lesion in anterior segment photography and AF image was compared., Results: AF images revealed well-defined hyper-autofluorescence in the area of pinguecula, which was greater than the extent of visible pinguecula in the slit-lamp examination, in 40 of 56 lesions (71.4%). In none of the eyes was the hyperautofluorescent area smaller than the extent of visible lesion., Conclusion: Pingueculae display hyperautofluorescence in AF imaging. The real size of pingueculae may be estimated by its AF characteristics, which is mostly larger than the visible lesion.

Aim: To evaluate the distribution of fundus autofluorescence in patients with age-related macular degeneration and choroidal neovascularisation (CNV)., Methods: Colour fundus photographs, fundus fluorescein angiograms (FFA) and fundus autofluorescence images were obtained from a group of 40 patients (43 eyes) with age-related macular degeneration and purely classic or occult CNV. Only patients with newly diagnosed CNV and in whom autofluorescence images were obtained within 2 weeks from FFA were included. The distribution of autofluorescence was qualitatively evaluated, and the findings compared with those from colour fundus photographs and FFA., Results: 29 (67%) eyes had classic CNV and 14 (33%) had occult CNV. In 26 (90%) eyes with classic CNV, a low autofluorescence signal was detected at the site of the CNV; in 7 (50%) eyes with occult CNV, multiple foci of low autofluorescence signal were detected. Outside the area affected by the lesion, homogeneous autofluorescence was observed in most of the cases (n = 33, 77%). Similarly, homogeneous autofluorescence was commonly observed in fellow eyes (62%). A pattern of focal increased autofluorescence was rarely seen in eyes with CNV (n = 4, 9%) or in fellow eyes (n = 4, 15%). In 11 of 43 (25%) eyes, areas of increased autofluorescence, other than a pattern of focal increased autofluorescence, were detected. In four patients, autofluorescence images had been obtained before the development of CNV; in none was any increased autofluorescence detected before the formation of CNV., Conclusions: Distinct patterns of autofluorescence were observed in eyes with pure classic and occult CNV. Increased autofluorescence was rarely seen in eyes with CNV and in fellow eyes, suggesting that increased autofluorescence, and thus, retinal pigment epithelium lipofuscin, may not play an essential part in the formation of CNV.

Oxidative mechanisms are considered to contribute to the aging changes in retinal pigment epithelial (RPE) cells that underlie the pathogenesis of age-related macular degeneration. An important source of oxidative damage is likely to be the photoreactive pigments that progressively accumulate and constitute the lipofuscin of retinal pigment epithelial cells. Evidence for a link between RPE lipofuscin and cellular dysfunction is also provided by the understanding of disease progression in Stargardt disease. Using a culture model previously used to demonstrate photooxidative damage to retinal pigment epithelial cells that have accumulated the lipofuscin fluorophore A2E, it was shown that the propensity for cell death is increased under conditions that deplete cellular levels of glutathione. Additionally, sulforaphane, a phytochemical and inducer of phase 2 enzymes, protected RPE cells that accumulated A2E and were irradiated at 430 nm. The protection afforded by sulforaphane was paralleled by elevated levels of glutathione and increases in the activities of the phase 2 enzymes NAD(P)H:quinone reductase and glutathione-S-transferases. Moreover, transcriptional induction of NAD(P)H:quinone reductase was indicated by the increases in mRNA determined by real time RT-PCR. There has been considerable interest in the intake of carotenoids and antioxidant vitamins and the related incidence of age-related macular degeneration. The present results indicate that the indirect antioxidant activity of plant-derived phase 2 inducers also may be potentially important.

Lipid-deposits in internal organs, e.g. nephrons, are discussed as reliable marker to determine hypothermia as cause of death. While investigations concerning lipid vacuoles in the epithelium of the renal tubules are already published, there is no systematic information available about hypothermia and lipid deposits in cardiomyocytes. Therefore, this retrospective study presents the first results of lipid-stainings of myocardial samples taken by autopsies in hypothermia-cases in comparison to samples from a control group. It was the aim of the study to clarify the conceivable causal relationship between death due to hypothermia and lipid-deposits apart from lipofuscin and fatty degeneration, respectively, in cardiomyocytes.

Normal metabolism is associated with unavoidable mild oxidative stress resulting in biomolecular damage that cannot be totally repaired or removed by cellular degradative systems, including lysosomes, proteasomes, and cytosolic and mitochondrial proteases. Consequently, irreversibly damaged and functionally defective structures (biological 'garbage') accumulate within long-lived postmitotic cells, such as cardiac myocytes and neurons, leading to progressive loss of adaptability and increased probability of death and characterizing a process called aging, or senescence. Intralysosomal 'garbage' is represented by lipofuscin (age pigment), an undegradable autophagocytosed material, while extralysosomal 'garbage' involves oxidatively modified cytosolic proteins, altered biomembranes, defective mitochondria and other organelles. In aged postmitotic cells, heavily lipofuscin-loaded lysosomes perform poorly, resulting in the enhanced accumulation of defective mitochondria, which in turn produce more reactive oxygen species causing additional damage (the mitochondrial-lysosomal axis theory). Potential anti-aging strategies may involve not only overall reduction of oxidative stress, but also the use of intralysosomal iron chelators hampering Fenton-type chemistry as well as the stimulation of cellular degradative systems.

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photooxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.

Reflection densitometry has been widely used to measure the density difference of the bleachable cone photopigments in human eyes. Most such measurements make a series of assumptions concerning the amount of scattered light to derive an estimate of the true cone photopigment density from the density difference measurements. The current study made three types of measurements of the light returning from the eye before and after bleaching: the amount of light returning in the "directed" reflection, which is a double-pass estimate of the cone photopigment density; the amount of light in undirected or diffuse reflection; and the amount of fluorescence from lipofuscin in the RPE, which provides a single-pass measurement of optical density difference. For a 1 deg foveally fixated field, the density difference estimates for the three measurements were 0.68, 0.21, and 0.22 respectively. The lipofuscin fluorescence was found to be unguided. The background density difference was non-negligible and very close to the single pass estimate from fluorescence. These measurements each involve potentially different pathways of light through the retina, and therefore place different constraints on models of these pathways. A simple model comparing the directional and the fluorescence optical densities produced retinal coverage estimates around 70-75%. Estimates of the shape factor of the single pass optical Stiles-Crawford effect were evaluated from the dark-adapted and bleached fluorescence measurements. The values were closer to those obtained from psychophysical methods than to the double pass optical Stiles-Crawford shape factors obtained directly from retinal reflectometry.

Cellular senescence may be accompanied by accumulation of large aggregates of oxidized proteins, also known as lipofuscin. The hypothesis that cellular accumulation of lipofuscin-like materials (LIP) results in cell death as a result of proteasome inhibition was examined. Rat neonatal cardiomyocytes were incubated with synthetic LIP for up to 48 h. This was accompanied by increases in cellular autofluorescence (207% by 48 h; p < 0.05) and electron microscopic evidence of internalization of LIP particles. LIP incubation resulted in loss of viability (-46% by 48 h; p < 0.05) through apoptotic cell death. Although 20S-proteasome activity was increased by 74% after 6 h, both 20S- and 26S-proteasome activities were decreased after 48 h of incubation (-54% (p < 0.05) and -50%, respectively), accompanied by large increases in ubiquitinated proteins. Several proteasome-regulated proapoptotic proteins, including c-Jun (2.9-fold; p < 0.05), Bax (1.8-fold; p < 0.05), and p27(kip1) (3.2-fold; p < 0.05), were observed to be increased by 48 h. Observation of ubiquitinated homologues of Bax and p27(kip1) suggested that part of the increase was due to decreased proteasomal degradation of these proteins. The results of this study are consistent with the conclusion that accumulation of LIP results in inhibition of the proteasome, which initiates an apoptotic cascade as a result of dysregulation of several proapoptotic proteins.

Lipofuscin is membrane-bound cellular waste that can be neither degraded nor ejected from the cell but can only be diluted through cell division and subsequent growth. The fate of postmitotic cells is to accumulate lipofuscin, which as an "aging pigment" has been considered a reliable biomarker for the age of cells such as neurons and, by extension, their hosts. In the aging human brain, deposits of lipofuscin are not uniformly distributed but are concentrated in specific regions of functional interest. The prevailing thought is that the major source of lipofuscin is incomplete lysosomal degradation of damaged mitochondria. Accumulating evidence suggests that lipofuscin is not benign but can impair the functioning of seemingly unrelated cellular systems, including the ubiquitin/proteasome pathway. A damaging feedback loop of lysosomal and proteasomal inhibition may occur as lipofuscin accumulates, leading to what has been appropriately named a "garbage catastrophe." Reversing this catastrophe presents a formidable challenge.

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.

Protein aggregation seems to be a common feature of several neurodegenerative diseases and to some extent of physiological aging. It is not always clear why protein aggregation takes place, but a disturbance in the homeostasis between protein synthesis and protein degradation seems to be important. The result is the accumulation of modified proteins, which tend to form high molecular weight aggregates. Such aggregates are also called inclusion bodies, plaques, lipofuscin, ceroid, or 'aggresomes' depending on their location and composition. Such aggregates are not inert metabolic end products, but actively influence the metabolism of cells, in particular proteasomal activity and protein turnover. In this review we focus on the influence of oxidative stress on protein turnover, protein aggregate formation and the various interactions of protein aggregates with the proteasome. Furthermore, the formation and effects of protein aggregates during aging and neurodegeneration will be highlighted.

Lipofuscin or "age pigment" is a lipid-protein complex which accumulates in a variety of postmitotic, metabolically active cells throughout the body. These complexes, which are thought to result from the incomplete degradation of oxidised substrate, have the potential for photoreactivity. This is particularly so in the retina in which the lipofuscin not only contains retinoid metabolites but is also exposed to high oxygen and fluxes of visible light all of which provide an ideal environment for the generation of reactive oxygen species (ROS). Lipofuscin is a potent photoinducible generator of ROS with the potential to damage proteins, lipids and DNA. Retinal cell dysfunction may be strongly associated with photoreactivity of lipofuscin and may contribute to age-related disease and vision loss.

Over time, postmitotic cells accumulate a non-degradable intralysosomal substance, lipofuscin, which forms due to iron-catalyzed oxidation/polymerization of protein and lipid residues. Lipofuscin is often considered a hallmark of aging, showing an accumulation rate that inversely correlates with longevity. There is an emerging impression that lipofuscin, although still typically considered a harmless wear-and-tear product, may have multiple negative effects. By interfering with the important autophagic process, by which most worn out cellular components are degraded, it may prevent cellular renewal and advance the accumulation of damaged cellular constituents. Due to binding of transition metals, such as iron and copper, lipofuscin also seems to sensitize lysosomes and cells to oxidative stress. Of importance for the pathogenesis of age-related macular degeneration, lipofuscin deposition interferes with the phagocytic activity of retinal pigment epithelial cells and also sensitizes their lysosomes to blue light.

Melano-macrophage centres, also known as macrophage aggregates, are distinctive groupings of pigment-containing cells within the tissues of heterothermic vertebrates. In fish they are normally located in the stroma of the haemopoietic tissue of the spleen and the kidney, although in amphibians and reptiles, and some fish, they are also found in the liver. They may also develop in association with chronic inflammatory lesions elsewhere in the body and during ovarian atresia. In higher teleosts, they often exist as complex discrete centres, containing lymphocytes and macrophages, and may be primitive analogues of the germinal centres of lymph nodes. Melano-macrophage centres usually contain a variety of pigments, including melanins, and these increase in range and volume in older fish or in the presence of cachectic disease. Melano-macrophage centres act as focal depositories for resistant intracellular bacteria, from which chronic infections may develop. Iron capture and storage in haemolytic diseases appears to be a primary function, but antigen trapping and presentation to lymphocytes, sequestration of products of cellular degradation and potentially toxic tissue materials, such as melanins, free radicals and catabolic breakdown products are among other functions that have been ascribed. Recent work suggests that they are a site of primary melanogenesis rather than mere storage. Melano-macrophage centres increase in size or frequency in conditions of environmental stress and have been suggested as reliable biomarkers for water quality in terms of both deoxygenation and iatragenic chemical pollution.

Mutation in any of the four clock genes (clk-1, clk-2, clk-3, gro-1) causes an average slowing down of many temporal processes, and an increase of mean life span. The latter effect has been linked to the slow phenotype, and it has been reasoned that any reduction of the rate of living would reduce the load of oxidative damage, which is thought to drive the ageing process. To test this model we measured several parameters describing metabolic output in wild type worms and all four Clk mutants. We found no gross changes in metabolic output, as assessed from oxygen consumption and heat production rates, lucigenin-mediated light production capacity, ATP content, and lipofuscin autofluorescence. Catalase and superoxide dismutase (SOD) were variably altered, but not cooperatively, as would be expected to enhance reactive oxygen species (ROS) scavenging activity. Thus we conclude that the prolonged life span of Clk mutants cannot be attributed to reduced metabolic rate or an increased activity of the major antioxidant enzymes catalase and SOD.

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occurring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.

Increasing evidence suggests an important role of oxidant-induced damage in the progress of senescent changes, providing support for the free radical theory of aging proposed by Harman in 1956. However, considering that biological organisms continuously renew their structures, it is not clear why oxidative damage should accumulate with age. No strong evidence has been provided in favor of the concept of aging as an accumulation of synthetic errors (e.g. Orgel's 'error-catastrophe' theory and the somatic mutation theory). Rather, we believe that the process of aging may derive from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions. From this perspective, it might be predicted that: (i) suppression of oxidative damage would enhance longevity; (ii) accumulation of incompletely digested material (e.g. lipofuscin pigment) would interfere with cellular functions and increase probability of death; (iii) rejuvenation during reproduction is mainly provided by dilution of undigested material associated with intensive growth of the developing organism; and (iv) age-related damage starts to accumulate substantially when development is complete, and mainly affects postmitotic, cells and extracellular matrix, not proliferating cells. There is abundant support for all these predictions.

Purpose: A fluorescent component of lipofuscin, A2-E (N-retinylidene-N-retinylethanol-amine) has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. In addition to its lysosomotropic properties A2-E is known to be photoreactive. The purpose of this study was to determine the phototoxic potential of A2-E on RPE cells., Methods: A2-E (synthesized by coupling all-trans-retinaldehyde to ethanolamine) was complexed to low-density lipoprotein (LDL) to allow for specific loading of the lysosomal compartment. Human RPE cell cultures were loaded with the A2-E-LDL complex four times within 2 weeks. A2-E accumulation was confirmed by fluorescence microscopy and flow cytometry analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. The phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short wavelength visible light (400-500 nm) and assessing cell viability and lysosomal integrity., Results: Fluorescence microscopy and flow cytometry analysis demonstrated that the intralysosomal accumulation of A2-E in cultured RPE cells increased with the number of feedings. Acridine orange staining confirmed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E-fed cells to light resulted in a significant loss of cell viability by 72 hours, which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity., Conclusions: A2-E is detrimental to RPE cell function by a variety of mechanisms: inhibition of lysosomal degradative capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging as well as retinal diseases associated with excessive lipofuscin accumulation-for example, age-related macular degeneration and Stargardt's disease.

This article provides current information on the potential role of oxidation in relation to age-related macular degeneration (AMD). The emphasis is placed on the generation of oxidants and free radicals and the protective effects of antioxidants in the outer retina, with specific emphasis on the photoreceptor cells, the retinal pigment epithelium and the choriocapillaris. The starting points include a discussion and a definition of what radicals are, their endogenous sources, how they react, and what damage they may cause. The photoreceptor/pigment epithelium complex is exposed to sunlight, is bathed in a near-arterial level of oxygen, and membranes in this complex contain high concentrations of polyunsaturated fatty acids, all considered to be potential factors leading to oxidative damage. Actions of antioxidants such as glutathione, vitamin C, superoxide dismutase, catalase, vitamin E and the carotenoids are discussed in terms of their mechanisms of preventing oxidative damage. The phototoxicity of lipofuscin, a group of complex autofluorescent lipid/protein aggregates that accumulate in the retinal pigment epithelium, is described and evidence is presented suggesting that intracellular lipofuscin is toxic to these cells, thus supporting a role for lipofuscin in aging and AMD. The theory that AMD is primarily due to a photosensitizing injury to the choriocapillaris is evaluated. Results are presented showing that when protoporphyric mice are exposed to blue light there is an induction in the synthesis of Type IV collagen synthesis by the choriocapillary endothelium, which leads to a thickened Bruch's membrane and to the appearance of sub-retinal pigment epithelial fibrillogranular deposits, which are similar to basal laminar deposits. The hypothesis that AMD may result from oxidative injury to the retinal pigment epithelium is further evaluated in experiments designed to test the protective effects of glutathione in preventing damage to cultured human pigment epithelial cells exposed to an oxidant. Experiments designed to increase the concentration of glutathione in pigment epithelial cells using dimethylfumarate, a monofunctional inducer, are described in relation to the ability of these cells to survive an oxidative challenge. While all these models provide undisputed evidence of oxidative damage to the retinal pigment epithelium and the choriocapillaris that is both light- and oxygen-dependent, it nevertheless is still unclear at this time what the precise linkage is between oxidation-induced events and the onset and progression of AMD.

The influence of lipofuscin granules and melanosomes from human retinal pigment epithelium on the light-induced photooxidation of cardiolipin liposomes and the generation of superoxide radicals was studied. Lipofuscin granules were able to stimulate, while melanosomes inhibited, the cardiolipin photooxidation. The visible light irradiation of both melanosomes and lipofuscin granules generated superoxide radicals with mean rates of 1.5 nmole/min/10(7) and 38 nmole/min/10(7) granules, accordingly. However, melanosomes but not lipofuscin granules reacted readily with superoxide radicals. Moreover, the rate constant of degradation of superoxide radicals in the presence of melanosomes was about five orders of magnitude higher than the rate constant of its photogeneration. Therefore, we propose that melanosomes in retinal pigment epithelium cells have a photoprotective role whereas lipofuscin granules may stimulate photodestructive reactions.

Retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that is strategically placed between the rod and cone photoreceptors and the vascular bed of the choriocapillaris. It has many important functions, such as phagocytic uptake and breakdown of the shedded photoreceptor membranes, uptake, processing, transport and release of vitamin A (retinol), setting up the ion gradients within the interphotoreceptor matrix, building up the blood-retina barrier, and providing all transport from blood to the retina and back. This short review focuses on the role of the pigment granules in RPE. Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of zinc and drugs, and, therefore, serve as a versatile partner of the RPE cell. Melanin plays a role in the development of the fovea and routing of optic nerves. New findings show that the melanin granules are connected to the lysosomal degradation pathway. Most of these functions are not yet understood. Deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness.

Lipofuscin (age pigment) is a brown-yellow, electron-dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen-derived free radicals generated in reactions catalyzed by redox-active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age-independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age-related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.

Lipofuscin is the autofluorescent material, which accumulates with aging in the cells of various tissues. However, its autofluorescence characteristics are different among tissues. In the present study, the autofluorescence features of lipofuscin in the brain and adrenal were compared. In 18-21-month-old rats, the brain lipofuscin was granular and its autofluorescence was bright whitish-yellow to bright orange. On the contrary, the adrenal lipofuscin was not demarkated as granules, and its autofluorescence was subdued orange. The emission maximum of the bright whitish-yellow brain lipofuscin was 540 nm to 570 nm and that of the adrenal lipofuscin was 640 nm to 660 nm, when excited at 330 nm to 380 nm. When the spectra were drawn after correcting the wavelength-dependent bias of microspectrofluorometer, the autofluorescence spectra were consistent with microscopically observable tint. To conclude, the present results showed that the autofluorescence features of the bright whitish-yellow brain lipofuscin and the adrenal lipofuscin were quite different.

Published attempts to extract lipofuscin from crustaceans and fish to assess age for fisheries research purposes have used essentially the same extraction methodology applied to insects, but have neither shown a conclusive age-dependence of spectrally similar fluorescence nor proved its association with lipofuscin. The reported lipofuscin solvent extraction method for fleshflies, Sarcophaga bullata, was manipulated by varying wash volume. This revealed that almost all age-dependent blue fluorescent material persisting in lipid fractions was actually pteridine-like. This finding was consistent with some previous independent results for Musca domestica. Examination of reported lipofuscin extraction protocols for other insects suggested that this problem was probably widespread. The pteridines are known to occur in unusually high amounts in insects, accumulating with age specifically in some members of this group by storage excretion, probably as a terrestrial water conservation strategy. In addition, there is growing evidence in the gerontological literature for other groups that solvent extracted blue fluorescence is not a true measure of lipofuscin content in tissues. These findings provide considerable insight into anomalies in putative lipofuscin fluorescence data between the insects and various aquatic species and suggest that there may be little basis for expectations of age-dependent fluorescence from aquatic species when the same gross extraction and crude purification methods are used.

Accumulation of lipofuscin is an important phenomenon of the cellular aging process. The first appearance of cardiac lipofuscin showed a good correlation with sexual maturation, which was correlated with maximum life-span of mammals. Large metabolic changes occurred at sexual maturation. From these results, it is suggested that sexual maturation of mammals is the initiation period of the aging process. Correlation between sexual maturation and longevity was re-evaluated using many mammals. Domestic and laboratory animals showed an earlier sexual maturation than other mammals, including rodents.

It is well known that the amplitude of accommodation deteriorates with aging. However, it is not clear what kinds of morphological changes of age-dependent deterioration of amplitude of accommodation accompany in the crystalline lens and the ciliary muscle, which are the most important involved tissues in the accommodation mechanism. In the present study morphological changes of these tissues were investigated in relation to the deterioration of the accommodative amplitude. Age-dependent deterioration of the subjective amplitude of accommodation obtained by an accommodometer (VDT Accommodometer NP-200, Toyo-Medical, Nagoya) and the objective amplitude by an autorefractometer with a built-in infrared optometer (NIDEK AA 2000 Accommodometer, NIDEK, Gamagori, Japan) were compared. The objective amplitude was 2-3 dptr less than the subjective amplitude for all ages, but change of both curves with aging were parallel. The crystalline lens measured by an ultrasonic A-scan instrument, Alpha 20/20 (Storz, U.S.A.), showed continuous increase in thickness with aging and by accommodative contraction. The crystalline lens in the fifties was as same in thickness as the accommodatively contracted lens in the twenties but the increasing ratio of lens thickness in accommodative contraction decreased with aging and it reached a minimum in the sixties. Scanning electron microscopy (JSM F-15, JOEL Co, Tokyo) revealed three types of characteristic substructures on the cell surface of the crystalline lens fiber, ie, interlocking protrusion, ball-and-socket junction and microplica or the tongue-and-groove junction. In the lenticular nucleus the microplica was prominent on the cell surface throughout all ages but the ball-and-socket junction was sparse in number. On the other hand, the ball-and-socket junction was well developed on the cell surface in the superficial cortex of the lens. The microplica on the cell surface in the superficial cortex was recognizable but indistinct. The microplica and ball-and-socket junction seemed to shift relative to each other in the deeper cortex of the lens. The interlocking protrusion was recognized throughout the whole lens, but was prominent in the lenticular nucleus. The crystalline lens fibers of advanced age were irregural in shape and the spherical substructure, large and small in size, and frequently formed suggesting the cataractous change. Delicate shift of the ciliary processes after conjunctival application of 4% pilocarpine was confirmed by an angioendoscope (Angio Fiber Imaging System FCA-8000. Fukuda Electronics K.K., Tokyo) which was inserted into the vitreous space of cynomorgus monkey eyes.(ABSTRACT TRUNCATED AT 400 WORDS)

Sexually mature Rana temporaria L. frogs were captured in their natural habitat in seven stages of the the annual cycle. The volume of the nuclei of neurocytes of the pars dorsalis of the tuber cinereum containing lipofuscin pigment underwent significant changes in the course of the year. In both sexes the nuclear volume of the neurocytes was greatest in the terminal stage of winter sleep (1st decade of March), and smallest at the beginning of active life (3rd decade of May). The accumulation of lipofiscin pigment varied parallel to the changes in mean volume of the neurocyte nuclei of the pars dorsalis of the tuber cinereum.

The neurons of the supraoptic nucleus of male C57BL/Icrfat mice at 6 or 28 months of age were examined from normally hydrated, osmotically loaded and osmotically loaded-rehydrated animals. Using quantitative morphological techniques, a reduction in the concentration of lipofuscin in the neurons was observed in osmotically loaded mice at both ages, and these levels were restored to control values during rehydration. In addition, there was a significant difference in the pattern of response of lipofuscin levels between the two age groups during the experiment. The concentration of hormone containing neurosecretory granules in the neurons of the supraoptic nucleus did not differ significantly between the two age groups during the experiment. However, the surface area of rough endoplasmic reticulum per unit volume of the supraoptic nucleus cell did differ significantly between the two age groups over the course of the experiment. It is concluded that increasing concentrations of lipofuscin do not affect the ability of the cell to control the concentration of neurosecretory granules or rough endoplasmic reticulum. The simplistic view that lipofuscin accumulates with age to the detriment of cell function must be revised.

Extra-cutaneous melanocytes derive from either the neural crest, the outer wall of the optic cup, or the cranial neural tube. Those of neural crest origin reach most bodily regions, and may give rise to primary melanoma in various tissues. The Kupffer cell produces a form of melanin, but is hardly a melanocyte. Melanocytes of the internal ear may be concerned with secretion of endolymph, trans-epithelial ion transport, and with protection against ototoxic drugs and high-intensity noise damage. There is evidence from albino animals that retinal pigment epithelium determines co-ordinates of the neural retina, and its decussation pattern during development. Neuromelanin derives from Dopamine, and is found in dopaminergic neurons widely distributed throughout the brain-stem and hypothalamus, and which project to the striatum and limbic system. Parkinsonism is due to degeneration of melanin-containing dopaminergic neurons of locus coeruleus and substantia nigra, and MPTP provides an investigative probe for studying the causes of Parkinsonism. Neuromelanin should not be regarded as a waste-product, but as something which can affect the firing properties of neurons with specific functional effect.

Lipofuscin is defined as being a yellowish brown, lipid-rich, heterogeneous, cytoplasmic granular pigment emitting an intense yellow autofluorescence when excited with ultraviolet light, which accumulates in various tissues of animals during their aging. It is believed that the pigments are derived from the reaction of some of reactive secondary products including malonaldehyde, formed during membranous lipid peroxidation, with amino groups of phospholipids and proteins, etc., and that these formations are accompanied by alteration of the membrane structure and inactivation of the enzymes. The fluorescence measurement of the pigments is widely used as a parameter of lipid peroxidation in vivo as well as in vitro. However, their origin, chemical structure, biological significance or fate has not as yet been fully elucidated. This article introduces and discusses the recent studies on these problems.

The nematode Caenorhabditis elegans contains autofluorescent lipofuscin granules, located exclusively in the 32-34 intestinal cells. Using epifluorescence microscopy on live adult animals, we have shown that fluorescent-labeled exogenous probes are taken up by endocytosis and accumulate within the granules. Macromolecular solutes such as proteins and dextran appear to be taken up by fluid-phase pinocytosis. There is no phagocytosis of latex particles with diameter greater than or equal to 0.25 micron. The granules concentrate the lysosomotropic weak base acridine orange, indicating that they have an acidic internal milieu. These observations imply that the lipofuscin granules in the intestinal cells are secondary lysosomes which remain active recipients of endocytosed materials.

The progressive loss of visual faculties with advancing age is attributed, sometimes, mainly to allegedly inevitable optical deterioration, such as clouding of the media, or again to neurosenescence, as typified by cell-death. The relevant factors are assessed, and it is not thought likely that changes in normal ocular optics account for changes in contrast sensitivity and visual acuity with age. The role of senescent changes in the retina is analyzed. The progressive temporal and spatial patterns of the accumulation of lipofuscin in the retinal pigment epithelium (RPE) and the putative control of its concentration by the crystalline lens are examined following an earlier suggestion that lenticular UVA transmissivity may control the rate of UVA entry into the eye and so perhaps the amount of lipofuscin formed in the RPE. It is suggested that morphological changes attributed to senescence may partly be due to cumyulative effects of irradiation.

Relatively high proportions of long-chain, polyunsaturated fatty acids seem to be required in rod photoreceptor membranes in order to provide the precise microenvironment for the proper function of the visual pigment rhodopsin. At the same time, such high levels of lipid unsaturation put the photoreceptor membranes at a high risk for autoxidation. The antioxidant vitamin E which can minimize autoxidation of polyunsaturated fatty acids is found in rather high concentrations in the outer segment membranes. Dietary deficiency in vitamin E induces disintegration of rod outer segment membranes, probably by increasing autoxidation. Also, it greatly accelerates the accumulation of aging pigments in the retinal pigment epithelium, probably because these lipofuscin granules do indeed represent the end products of lipid peroxidation. Vitamin E supplements, up to threefold normal levels, appear to provide no significant protection of the retina from light damage produced either by short but acute or by long-term, low level exposures to light. This is not consistent with current theories which implicate lipid peroxidation in the destruction of rod outer segments in light damaged retinas; more work is needed before any relation between retinal light damage and vitamin E levels can be assessed. Surprisingly, the amount of lipofuscin granule accumulation in the retinal pigment epithelium is influenced dramatically by dietary levels of vitamin A. Even retinas lacking a source of polyunsaturated fatty acids from rod outer segments still may accumulate massive lipofuscin if dietary vitamin A is provided. Perhaps vitamin A, which has such a dynamic relationship with the retinal pigment epithelium, becomes oxidized, and then contributes to the formation of a lipofuscin-like pigment. Centrophenoxine, a drug claimed to be effective in reversing the accumulation of age-related lipofuscin in the central nervous system, has no obvious effect in the eye or uterus in removing the lipofuscin granules induced by vitamin E deficiency. Microperoxisomes are abundant in the retinal pigment epithelium, and may be associated with rapid lipid turnover and/or utilization of lipid soluble vitamins. Their potential roles, however, need further documentation and clarification. Recently developed techniques and new discoveries in lipid research open the way for many fruitful studies on the interactions and precise roles of lipids and lipid-soluble vitamins in vision.

The study of the ultrastructure of the sural nerve and peripheral blood lymphocytes of a boy with late-infantile neuronal ceroid-lipofuscinosis revealed the presence of 'curvilinear bodies' and 'fingerprint profiles'. The elder sister of the patient died at the age of 7 years after progressive mental and motor deterioration. The same kind of cytoplasmic inclusions was found in the lymphocytes of the father of these children, who had had epilepsy since the age of 32. Clinical data and the results of the ultrastructural study suggest that in the same family two different forms of ceroid-lipofuscinosis appear and that the disease is inherited as an autosomal dominant trait. This family seems to suggest the nosological unity of clinically different forms of ceroid-lipofuscinosis.