Cognitive decline and increased hippocampal p-tau expression in mice with hearing loss.

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.

Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors' age and antioxidants.

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.

Studying melanin and lipofuscin in RPE cell culture models.

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.

Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium.

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.

Synthesis of antioxidants for prevention of age-related macular degeneration.

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.

Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells.

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.

[New insights into intracellular inclusions and intracellular proteolysis]. / Les inclusions intracellulaires sont-elles toujours des témoins d'un trouble de la protéolyse intracellulaire?

Intracellular inclusions seen by the pathologist may have variable significance. Although they are excellent markers of proteolytic disorders, they can also be due to several other mechanisms. This article examines recent data on the morphology, significance and consequences of aging lipofuscins in the brain and retina, neurofibrillary tangles and Lewy bodies, and Birbeck granules associated with Langerhans histiocytosis. Some of these disorders involve increased protein production, misfolding and aggregation, and altered intracellular proteolysis, but other cell constituents may also play a role. Proteolytic mechanisms do not appear to be involved in the formation of Birbeck granules, which helped to reveal the Langerhans origin of histiocytosis X. Analyses of intracellular inclusions, together with genetic and epigenetic studies, are highly informative in various degenerative diseases.

Use it and lose it: lipofuscin accumulation in the midbrain of a coral reef fish.

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.

Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging.

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.

Autofluorescence imaging of pingueculae.

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.

Fundus autofluorescence in exudative age-related macular degeneration.

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.

Indirect antioxidant protection against photooxidative processes initiated in retinal pigment epithelial cells by a lipofuscin pigment.

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.

Oxidative stress, accumulation of biological 'garbage', and aging.

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.

Fatty degeneration of myocardial cells as a sign of death due to hypothermia versus degenerative deposition of lipofuscin.

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.

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin.

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.

Investigating the light absorption in a single pass through the photoreceptor layer by means of the lipofuscin fluorescence.

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.

Aggregates of oxidized proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins.

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 and aging: a matter of toxic waste.

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.

Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain.

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.