Autophagy and UPS pathway contribute to nicotine-induced protection effect in Parkinson's disease.

The gradual nature of age-related neurodegeneration causes Parkinson's disease (PD) and impairs movement, memory, intellectual ability, and social interaction. One of the most prevalent neurodegenerative conditions affecting the central nervous system (CNS) among the elderly is PD. PD affects both motor and cognitive functions. Degeneration of dopaminergic (DA) neurons and buildup of the protein α-synuclein (α-Syn) in the substantia nigra pars compacta (SNpc) are two major causes of this disorder. Both UPS and ALS systems serve to eliminate α-Syn. Autophagy and UPS deficits, shortened life duration, and lipofuscin buildup accelerate PD. This sickness has no cure. Innovative therapies are halting PD progression. Bioactive phytochemicals may provide older individuals with a natural substitute to help delay the onset of neurodegenerative illnesses. This study examines whether nicotine helps transgenic C. elegans PD models. According to numerous studies, nicotine enhances synaptic plasticity and dopaminergic neuronal survival. Upgrades UPS pathways, increases autophagy, and decreases oxidative stress and mitochondrial dysfunction. At 100, 150, and 200 µM nicotine levels, worms showed reduced α-Syn aggregation, repaired DA neurotoxicity after 6-OHDA intoxication, increased lifetime, and reduced lipofuscin accumulation. Furthermore, nicotine triggered autophagy and UPS. We revealed nicotine's potential as a UPS and autophagy activator to prevent PD and other neurodegenerative diseases.

Sertraline Promotes Health and Longevity in Caenorhabditis elegans.

INTRODUCTION: While several antidepressants have been identified as potential geroprotectors, the effect and mechanism of sertraline on healthspan remain to be elucidated. Here, we explored the role of sertraline in the lifespan and healthspan of Caenorhabditis elegans. METHODS: The optimal effect concentration of sertraline was first screened in wild-type N2 worms under heat stress conditions. Then, we examined the effects of sertraline on lifespan, reproduction, lipofuscin accumulation, mobility, and stress resistance. Finally, the expression of serotonin signaling and aging-related genes was investigated to explore the underlying mechanism, and the lifespan assays were performed in ser-7 RNAi strain, daf-2, daf-16, and aak-2 mutants. RESULTS: Sertraline extended the lifespan in C. elegans with concomitant extension of healthspan as indicated by increasing mobility and reducing fertility and lipofuscin accumulation, as well as enhanced resistance to different abiotic stresses. Mechanistically, ser-7 orchestrated sertraline-induced longevity via the regulation of insulin and AMPK pathways, and sertraline-induced lifespan extension in nematodes was abolished in ser-7 RNAi strain, daf-2, daf-16, and aak-2 mutants. CONCLUSION: Sertraline promotes health and longevity in C. elegans through ser-7-insulin/AMPK pathways.

Developmental exposure to methylmercury alters GAD67 immunoreactivity and morphology of endothelial cells and capillaries of midbrain and hindbrain regions of adult rat offspring.

INTRODUCTION: Methylmercury (MeHg) is an environmental contaminant that is of particular concern in Northern Arctic Canadian populations. Specifically, organic mercury compounds such as MeHg are potent toxicants that affect multiple bodily systems including the nervous system. Developmental exposure to MeHg is a major concern, as the developing fetus and neonate are thought to be especially vulnerable to the toxic effects of MeHg. The objective of this study was to examine developmental exposure to low doses of MeHg and effects upon the adult central nervous system (CNS). The doses of MeHg chosen were scaled to be proportional to the concentrations of MeHg that have been reported in human maternal blood samples in Northern Arctic Canadian populations. METHOD: Offspring were exposed to MeHg maternally where pregnant Sprague Dawley rats were fed cookies that contained MeHg or vehicle (vehicle corn oil; MeHg 0.02 mg/kg/body weight or 2.0 mg/kg/body weight) daily, throughout gestation (21 days) and lactation (21 days). Offspring were not exposed to MeHg after the lactation period and were euthanized on postnatal day 450. Brains were extracted, fixed, frozen, and sectioned for immunohistochemical analysis. A battery of markers of brain structure and function were selected including neuronal GABAergic enzymatic marker glutamic acid decarboxylase-67 (GAD67), apoptotic/necrotic marker cleaved caspase-3 (CC3), catecholamine marker tyrosine hydroxylase (TH), immune inflammatory marker microglia (Cd11b), endothelial cell marker rat endothelial cell antigen-1 (RECA-1), doublecortin (DCX), Bergmann glia (glial fibrillary acidic protein (GFAP)), and general nucleic acid and cellular stains Hoechst, and cresyl violet, respectively. Oxidative stress marker lipofuscin (autofluorescence) was also assessed. Both male and female offspring were included in analysis. Two-way analysis of variance (ANOVA) was utilized where sex and treatment were considered as between-subject factors (p* <0.05). ImageJ was used to assess immunohistochemical results. RESULTS: In comparison with controls, adult rat offspring exposed to both doses of MeHg were observed to have (1) increased GAD67 in the cerebellum; (2) decreased lipofuscin in the locus coeruleus; and (3) decreased GAD67 in the anterior CA1 region. Furthermore, in the substantia nigra and periaqueductal gray, adult male offspring consistently had a larger endothelial cell and capillary perimeter in comparison to females. The maternal high dose of MeHg influenced RECA-1 immunoreactivity in both the substantia nigra and periaqueductal gray of adult rat offspring, where the latter neuronal region also showed statistically significant decreases in RECA-1 immunoreactivity at the maternal low dose exposure level. Lastly, males exposed to high doses of MeHg during development exhibited a statistically significant increase in the perimeter of endothelial cells and capillaries (RECA-1) in the cerebellum, in comparison to male controls. CONCLUSION: Findings suggest that in utero and early postnatal exposure to MeHg at environmentally relevant doses leads to long-lasting and selective changes in the CNS. Exposure to MeHg at low doses may affect GABAergic homeostasis and vascular integrity of the CNS. Such changes may contribute to neurological disturbances in learning, cognition, and memory that have been reported in epidemiological studies.

Lipofuscin Granule Accumulation Requires Autophagy Activation.

Lipofuscins are oxidized lipid and protein complexes that accumulate during cellular senescence and tissue aging, regarded as markers for cellular oxidative damage, tissue aging, and certain aging-associated diseases. Therefore, understanding their cellular biological properties is crucial for effective treatment development. Through traditional microscopy, lipofuscins are readily observed as fluorescent granules thought to accumulate in lysosomes. However, lipofuscin granule formation and accumulation in senescent cells are poorly understood. Thus, this study examined lipofuscin accumulation in human fibroblasts exposed to various stressors. Our results substantiate that in glucose-starved or replicative senescence cells, where elevated oxidative stress levels activate autophagy, lipofuscins predominately appear as granules that co-localize with autolysosomes due to lysosomal acidity or impairment. Meanwhile, autophagosome formation is attenuated in cells experiencing oxidative stress induced by a doxorubicin pulse and chase, and lipofuscin fluorescence granules seldom manifest in the cytoplasm. As Torin-1 treatment activates autophagy, granular lipofuscins intensify and dominate, indicating that autophagy activation triggers their accumulation. Our results suggest that high oxidative stress activates autophagy but fails in lipofuscin removal, leaving an abundance of lipofuscin-filled impaired autolysosomes, referred to as residual bodies. Therefore, future endeavors in treating lipofuscin pathology-associated diseases and dysfunctions through autophagy activation demand meticulous consideration.

Antioxidant and anti-aging activities of Laminaria japonica polysaccharide in Caenorhabditis elegans based on metabonomic analysis.

In this study, Laminaria japonica polysaccharide (LJP) was measured in vitro against three antioxidant indicators: DPPH, ABTS, and hydroxyl. In vivo, LJP investigated thermal tolerance, H2O2-induced oxidative stress tolerance, and lipofuscin in Caenorhabditis elegans (C. elegans). Following that, after LJP treatment, the effects and underlying mechanisms were investigated at the mRNA and metabolite levels. We discovered the free radical scavenging activity of LJP. The thermal tolerance of C. elegans improved significantly, lowering levels of malondialdehyde, lipofuscin, and reactive oxygen species. Upregulation of Glp-1, Daf-16, Skn-1, and Sod-3 expression and downregulation of Age-1 and Daf-2 expression increased the ability to resist oxidative stress. Metabolomic analysis revealed that LJP promoted alanine, aspartate, and glutamate metabolism, the TCA cycle, butanoate metabolism, and the FOXO signaling pathway expression, resulting in significant changes in (R)-3-hydroxybutyric acid, palmitic acid, L-glutamic acid, L-malic acid, and oleic acid. The present study shows that LJP, as a functional food, has the potential to boost antioxidant capacity and delay aging.

NMDA Receptor Antagonists Degrade Lipofuscin via Autophagy in Human Retinal Pigment Epithelial Cells.

Background and Objectives: Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Results: Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. Conclusions: This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.

Oligosaccharides from agar extends lifespan through activation of unfolded protein response via SIR-2.1 in Caenorhabditis elegans.

PURPOSE: Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans. METHODS: Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity. RESULTS: AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes. CONCLUSION: AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway.

Impacts of engineered iron nanoparticles on oxidative stress, fatty acid composition, and histo-architecture of the smooth scallop Flexopecten glaber.

Engineered iron nanoparticles are widely used in environmental remediation, yet their potential toxic effects on marine biota remain poorly elucidated. This study aimed to gain insight into the nanoscale zero-valent iron (NZVI) toxicity mechanisms for marine invertebrates. Aside from the effect on oxidative status and histopathology, the effect of NZVI on lipid metabolism in bivalves was studied for the first time. To this end, specimens of Flexopecten glaber were exposed to ascending concentrations (0.5, 1, and 1.5 mg/L) of NZVI for 96 h. Results illustrate differential patterns of iron accumulation in the gills and the digestive gland. By increasing NZVI concentrations, the total iron level tended to markedly increase in the gills and decrease in the digestive gland, reaching 132 and 37.6 µg/g DW, respectively, in the specimens exposed to 1.5 mg/L. Biochemical and cellular biomarkers highlighted that NZVI caused oxidative stress (measured as hydrogen peroxide, malondialdehyde, and advanced oxidation protein product levels) and alterations of antioxidant defense systems, including reduced glutathione, non-protein thiol, glutathione peroxidase, superoxide dismutase, and catalase. Modulation of lipid metabolism with changed fatty acid compositions (mainly an increase in the saturation and a decrease in unsaturation levels) was also observed in both gills and digestive gland. Moreover, several histological damages, including lipofuscin accumulation, infiltrative inflammations, and digestive tubule alterations, were observed in the two studied organs, providing supplementary evidence regarding the toxic effect of NZVI. This study adds to the growing body of evidence pointing to the hazardous impacts of iron NPs on aquatic ecosystems.

Antiamyloid ß toxicity effect of genistein via activation of DAF-16 and HSP-16.2 signal pathways in Caenorhabditis elegans.

ß-Amyloid toxicity (Aß) is an important pathological factor of Alzheimer's disease (AD). Studies have shown that genistein can reduce the toxicity of Aß to a certain extent; however, the specific mechanism is still uncertain. In the study, we applied Caenorhabditis elegans strains expressing Aß peptides to evaluate the role of genistein inhibiting Aß toxicity and the undying mechanism. Genistein influencing the sterol metabolism pathway, the HSP-16.2 pathway, and lipofuscin in different strains of C. elegans were studied using reverse transcription-polymerase chain reaction, fluorescence labeling, RNA interference (RNAi), and so forth. Our results showed that genistein alleviated the paralysis of transgenic C. elegans strains. Furthermore, in AD C. elegans, genistein reduced the fluorescence of lipofuscin, downregulated the messenger RNA (mRNA) level of vit-3 and vit-6 which were related to the sterol metabolism pathway, significantly increased the mRNA level and protein level of HSP-16.2, increased the nuclear translocation of the DAF-16 transcription factor and increased the survival rate after heat stress, which was closely associated with HSP-16.2 levels. However, the paralysis-alleviating effect of genistein was greatly reduced because of RNAi-mediated inhibition of hsp-16.2, indicating that the anti-Aß toxicity effect of genistein was greatly dependent on HSP-16.2. The above results suggest that genistein inhibiting the toxicity of Aß in C. elegans, is involved in the modulation of the sterol metabolism pathway by promoting transcription factor DAF-16 translocation into the nucleus, increasing the expression level of HSP-16.2, and reducing the levels of lipofuscin through its antioxidant activity.

Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

Evaluation of senescent cells in intervertebral discs by lipofuscin staining.

Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-ß-gal staining or p16Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-ß-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.

Lipofuscin-dependent stimulation of microglial cells.

PURPOSE: To examine the reaction of microglial cells (MG) when incubated with lipofuscin (LP) in vitro with emphasis on the immunological reaction of the MG toward LP and the suppression of this reaction by immunomodulatory agents. MG are involved in the pathogenesis of degenerative eye disorders such as age-related macular degeneration (AMD). LP is a heterogeneous waste material that accumulates in the retinal pigment epithelium (RPE) cells with advancing age. LP is known to have toxic effects on RPE cells and therefore an elevated LP-derived fundus autofluorescence is a risk factor for AMD development. MG in the subretinal space have been reported in eyes affected by AMD. Moreover, in senescent mice, subretinal MG were found, which display an autofluorescence that may be derived from LP uptake. METHODS: In this study, we incubated MG (BV-2 cell line and primary cells from murine brain) in vitro with LP isolated from the human RPE. We observed phagocytosis, studied cell morphologies, and analyzed the cell culture supernatants. We also investigated the effect of the immunomodulatory agents hydrocortisone (HC), minocycline, and the tripeptide TKP. RESULTS: The MG phagocytosed the LP quickly and completely. We detected highly elevated levels of pro-inflammatory cytokines (especially of IL-6, IL-23p19, TNF-α, KC, RANTES, and IL-1α) in the cell culture supernatants. Furthermore, levels of vascular endothelial growth factor (VEGF) were raised in BV-2 cells. Anti-inflammatory agents added to the cell cultures inhibited the inflammatory reaction, in particular hydrocortisone (HC). Minocycline and TKP had less impact on the cytokine release. CONCLUSION: The interaction of MG and LP could play a role in the development of retinal degeneration by triggering an inflammatory reaction and angiogenesis.

Lipofuscin-mediated photodynamic stress induces adverse changes in nanomechanical properties of retinal pigment epithelium cells.

Retinal pigment epithelium (RPE) is an important part of the blood-retina barrier (BRB) that separates the retina from the choroid. Although melanin granules contribute to the mechanical stability of the BRB complex, it is unknown if the age pigment lipofuscin affects mechanical properties of the tissue. To address this issue the effect of sub-lethal photic stress mediated by phagocytized lipofuscin granules, isolated from RPE of human donors, on morphology and mechanical properties of ARPE-19 cells was investigated. Nanomechanical analysis using atomic force spectroscopy revealed that irradiation of cells containing lipofuscin granules with blue light induced significant softening of the cells, which was accompanied by substantial reorganization of the cell cytoskeleton due to peroxidation of cellular proteins. Our results indicate that lipofuscin-mediated photic stress can cause significant modification of the RPE cells with the potential to disturb biological function of the BRB complex.

Diet-derived advanced glycation end products or lipofuscin disrupts proteostasis and reduces life span in Drosophila melanogaster.

Advanced glycation end product (AGE)-modified proteins are formed by the nonenzymatic glycation of free amino groups of proteins and, along with lipofuscin (a highly oxidized aggregate of covalently cross-linked proteins, sugars, and lipids), have been found to accumulate during aging and in several age-related diseases. As the in vivo effects of diet-derived AGEs or lipofuscin remain elusive, we sought to study the impact of oral administration of glucose-, fructose-, or ribose-modified albumin or of artificial lipofuscin in a genetically tractable model organism. We report herein that continuous feeding of young Drosophila flies with culture medium enriched in AGEs or in lipofuscin resulted in reduced locomotor performance and in accelerated rates of AGE-modified proteins and carbonylated proteins accumulation in the somatic tissues and hemolymph of flies, as well as in a significant reduction of flies health span and life span. These phenotypic effects were accompanied by reduced proteasome peptidase activities in both the hemolymph and the somatic tissues of flies and higher levels of oxidative stress; furthermore, oral administration of AGEs or lipofuscin in flies triggered an upregulation of the lysosomal cathepsin B, L activities. Finally, RNAi-mediated cathepsin D knockdown reduced flies longevity and significantly augmented the deleterious effects of AGEs and lipofuscin, indicating that lysosomal cathepsins reduce the toxicity of diet-derived AGEs or lipofuscin. Our in vivo studies demonstrate that chronic ingestion of AGEs or lipofuscin disrupts proteostasis and accelerates the functional decline that occurs with normal aging.

Lipofuscin inhibits the proteasome by binding to surface motifs.

Lipofuscin, a highly oxidized aggregate, consists of covalently cross-linked proteins, lipids, and sugar residues and is one of the major life-span-limiting factors in postmitotic aging cells. An artificial model of this material, showing characteristics and effects comparable to those of the natural form, has turned out to be very useful for in vitro studies. Artificial lipofuscin was used to investigate its effects on the viability of human fibroblasts, its rate of uptake, and its ability to inhibit the proteasomal system. The inhibition of the proteasomal system is one of the major aspects of the cytotoxic effects of lipofuscin. We present here that this proteasomal inhibition is due to proteasomal binding to the lipofuscin surface motifs, degradable by protease K. Furthermore, removal of the surface peptide structures by protease K strongly reduces the cytotoxic effects of lipofuscin and binding of cellular proteins and proteasomes to intracellular protein aggregates.

The age lipid A2E and mitochondrial dysfunction synergistically impair phagocytosis by retinal pigment epithelial cells.

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATP-dependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like age-related macular degeneration.

OT-674 suppresses photooxidative processes initiated by an RPE lipofuscin fluorophore.

The pathological processes involved in age-related macular degeneration (AMD) include retinal pigment epithelial (RPE) cell degeneration; oxidative mechanisms likely contribute to the demise of these cells. Indeed, RPE cells may be particularly susceptible to photooxidative mechanisms since they accumulate retinoid-derived photoreactive compounds that constitute the lipofuscin of the cell. Thus we undertook to test the capacity of OT-674, the reduction product (Tempol-H) of the nitroxide Tempol, to suppress photooxidative processes initiated by the RPE lipofuscin fluorophore A2E. Accordingly, when ARPE-19 cells that had accumulated A2E were irradiated at 430 nm, pretreatment with OT-674 (0.01-10 mM) was found to confer a resistance to cell death. Monitoring by quantitative HPLC also showed that OT-674 reduced A2E photooxidation in a cell-free system. Moreover, when presented with a singlet oxygen generator, OT-674 served as a quencher of singlet oxygen that was more effective than Trolox and alpha-tocopherol. We conclude that OT-674 is a potent antioxidant that suppresses photooxidative processes generated in cultured RPE cells by the lipofuscin fluorophore A2E. As oxidative damage to RPE cells is considered to be a risk factor for AMD, antioxidant therapy with OT-674 may serve a protective role.

The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.

Proteins involved in cholesterol trafficking are known to contribute to the pathogenesis of atherosclerosis and Alzheimer's disease. Allelic variants in the cholesterol transporters apolipoprotein E and ATP-binding cassette protein A1 (ABCA1) have recently been associated with susceptibility to age-related macular degeneration (AMD). Histopathological analyses of eyes with AMD demonstrate the presence of cholesterol and cholesteryl ester deposits beneath the retinal pigment epithelium (RPE), implicating abnormal cholesterol trafficking in disease progression. Here, we show that A2E, a quaternary amine and retinoid by-product of the visual cycle, causes the accumulation of free and esterified cholesterol in RPE cells. The mechanism involves neither generalized alterations in late endosomal/lysosomal pH nor a direct inhibition of acid lipase activity. Rather, A2E prevents cholesterol efflux from these organelles, which in turn indirectly inhibits acid lipase, leading to a subsequent accumulation of cholesteryl esters. Transcriptional activation of the ABCA1 cholesterol transporter by agonists of the liver X receptor/peroxisome proliferator-activated receptor pathway relieves the A2E-induced block on cholesterol efflux and restores cholesterol homeostasis in RPE cells. Our data also demonstrate that A2E, which is a cone-shaped lipid, increases the chemical activity and displacement of cholesterol from model membranes, providing a biophysical mechanism for cholesterol sequestration in A2E-loaded cells. Although endogenously produced A2E in the RPE has been associated with macular degeneration, the precise mechanisms are unclear. Our results provide direct evidence that A2E causes aberrant cholesterol metabolism in RPE cells which could likely contribute to AMD progression.

Inhibition of RPE lysosomal and antioxidant activity by the age pigment lipofuscin.

PURPOSE: To determine whether lipofuscin is detrimental to lysosomal and antioxidant function in cultured human retinal pigment epithelial (RPE) cells. METHODS: Isolated lipofuscin granules were fed to confluent RPE cultures and the cells maintained in basal medium for 7 days. Parallel cultures were established that did not receive lipofuscin. Cultures were either exposed to visible light (390-550 nm) at an irradiance of 2.8 mW/cm(2) or maintained in the dark at 37 degrees C for up to 24 hours. Cells were subsequently assessed for cell viability, lysosomal enzyme activity, and antioxidant capacity. RESULTS: There was no loss of cell viability during the first 3 hours of light exposure, whereas a 10% loss of viability was observed in lipofuscin-fed cultures after 6 hours' exposure to light. Activities of acid phosphatase, N-acetyl-beta-glucuronidase, and cathepsin D were decreased by up to 50% in lipofuscin-fed cells exposed to light compared with either unfed cells or cells maintained in the dark. There was also a decrease in the antioxidant potential of RPE cells. Catalase and superoxide dismutase activities decreased by up to 60% and glutathione levels by 28% in light-exposed lipofuscin-fed cells compared with unfed cells or cells maintained in the dark. CONCLUSIONS: Lipofuscin has the capacity to reduce the efficacy of the lysosomal and antioxidant systems in RPE cells that may play an important role in retinal ageing and the development of age-related macular degeneration.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.