Lysosomal Lipofuscin Accumulation Research Articles

Women’s health update: A literature review impacting primary care

The authors review studies on key issues in women's health with potential impact on internal medicine practice. The reviewed articles discuss cardiovascular disease risks, bone health, breast cancer genetics, cervical cancer prevention, depression in the peripartum period, pelvic pain, and emergency contraception.

Crosstalk of proteasomes and autophagy in RPE cells

AbstractThe pathogenesis of AMD involves impaired protein degradation in RPE cells. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress‐induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 has been shown to be a key player linking the proteasomal and lysosomal clearance systems.The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates drusens. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that p62, together with autophagy, seems to have a role in the pathology of different degenerative diseases. In ARPE‐19 cells proteasomal inhibition caused accumulation of p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro‐survival and promoted cleansing by autophagy of the former complex. Human AMD donor samples showed strong p62 accumulation in the drusen rich macula revealing impaired autophagy flux.

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

Why are retinal pigment epithelial cells so resistant to oxidative stress?

Abstract Purpose The functional integrity of postmitotic retinal pigment epithelial (RPE) cells is crucial for photoreceptor survival, and RPE malfunction is considered an important factor behind age‐related macular degeneration (AMD). Although RPE cells daily phagocytose large amounts of fatty acid‐rich distal photoreceptor material, their lysosomal accumulation of lipofuscin (LF) is nevertheless slow. Only late in life do humans have LF‐loaded RPE cells, which then not only have a reduced capacity to endocytose photoreceptor material but also are less capable of keeping up with normal autophagy. The latter activity is important for normal turnover of the cell’s own components. Methods ARPE‐19 cells in culture were evaluated with respect to lysosomal stability at oxidative stress. Redox‐active iron, autophagic activity (evaluation of LC3‐I and LC3‐II protein), and anti‐oxidative proteins were assayed using cytochemistry (Sulphide‐Silver Method), electron spin resonance (ESR) and western blotting for a variety of stress proteins. Results The lysosomal compartment of the ARPE‐19 cells was found remarkably resistant to oxidative stress. The SSM and ESR methods suggested low lysosomal amounts of redox‐active iron and no enhanced autophagy following oxidative stress. The normal autophagy rate was higher than in other professional scavengers. Conclusion The found low amount of lysosomal redox‐active iron explains the slow formation of lipofuscin over time and the remarkable stability that ARPE‐19 cells and their lysosomes show even under severe oxidative stress. The molecular mechanisms behind this phenomenon may be an unusual capacity to quickly export low mass iron from lysosomes to the cytosol and/or a high normal degree of autophagy of iron‐binding stress proteins.

Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis

In bivalve molluscs, responses to bacterial infection have been largely characterized in terms of both functional responses and gene expression in the immune cells, the hemocytes. The effects of bacterial challenge at the tissue level, where bacterial infection may cause stressful conditions, have not been so far specifically investigated. Biomarkers are widely utilised to evaluate the health status of bivalves, from the molecular to the organism level, in response to both natural and anthropogenic stressors. In this work, the effects of in vivo challenge with heat-killed vibrio species, Vibrio splendidus LGP32 and Vibrio anguillarum (ATCC19264), on different biomarkers in the digestive gland of the marine bivalve Mytilus galloprovincialis were investigated. Mussels were injected with either vibrio and tissues sampled at 3, 6 and 24 h post injection (p.i.). Lysosomal biomarkers, such as lysosomal membrane stability (LMS) and lipofuscin accumulation, as well as specific activities of antioxidant enzymes (catalase and glutathione transferase-GST) were evaluated. Moreover, the expression of antioxidant molecules (catalase, GST-π and metallothioneins MT10 and MT20) was determined by quantitative RT-PCR. Both V. splendidus and V. anguillarum significantly affected all parameters measured, to a different extent and at different times p.i. Interestingly, whereas both vibrios induced lysosomal membrane destabilisation and increases in the activities of antioxidant enzymes, distinct responses were observed in terms of lysosomal lipofuscin accumulation and expression of antioxidant molecules. In particular, V. splendidus induced a general increase in the transcription of antioxidant genes, indicating that Mytilus digestive gland can mount an efficient antioxidant response towards this vibrio species. On the other hand, a general down-regulation or no effect was observed with V. anguillarum. The lack of this response was reflected in stronger oxidative stress conditions in the digestive gland of mussels challenged with V. anguillarum, as indicated by higher levels of lysosomal lipofuscin observed at longer times p.i. Overall, these data indicate that lysosomal and oxidative stress biomarkers could be usefully applied in order to monitor early changes in the health status of bivalves induced by bacteria. Moreover, the results support the hypothesis that host responses to bacteria may be taken into account when interpreting biomarker data in ecotoxicological studies.

Biomarkers in Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon black, C60 fullerene, Nano-TiO 2, Nano-SiO 2)

The potential for ecological toxicity associated with nanomaterials is a growing area of investigation, in particular in the aquatic environment. In suspension feeding invertebrates, the cellular immune system and digestive gland are likely to be targeted, due to their highly developed processes for the cellular internalisation of nano- and micro-scale particles that are integral to key physiological functions such as cellular immunity and intracellular digestion. We have recently demonstrated that suspensions of selected commercial nanomaterials, namely Nano carbon black (NCB), C60 fullerene (C60), Nano-titanium dioxide (n-TiO 2) and Nanosilica (n-SiO 2) induce oxyradical production and lysosomal enzyme release in the hemocytes of the marine mussel Mytilus in vitro. In this work the possible effects of in vivo exposure to these NPs were investigated. Mussels were exposed to different concentrations (0.05–0.2–1–5 mg/l) of NP suspensions for 24 h and different biomarkers were evaluated in hemocytes, digestive gland and gills. Characterisation of NP suspensions in artificial sea water (ASW) was performed, indicating the formation of agglomerates of different sizes in the nano-micromolar range for different types of NPs. Formation of larger agglomerates was observed at the end of exposure. The results show that all NP suspensions induced significant lysosomal membrane destabilisation in both the hemocytes and the digestive gland, with NCB ≫ C60 > n-TiO 2, >n-SiO 2. In the digestive gland, all NPs induced lysosomal lipofuscin accumulation only at the highest concentrations tested to a different extent depending on the NP type. NCB, TiO 2 and SiO 2 also induced lysosomal neutral lipid accumulation. Moreover, all NPs increased the activity of the antioxidant enzyme catalase, with n-SiO 2 > NCB ≅ TiO 2 > C60; NCB and n-TiO 2 also stimulated glutathione transferase (GST). Changes in catalase and GST activities were also observed in gills, with both increases and decreases depending on NP type and concentration. The reported results demonstrate that in mussels responses to exposure to NP suspensions involve changes in lysosomal and oxidative stress biomarkers in the digestive gland, suggesting uptake of NP aggregates/agglomerates mainly through the digestive system. Overall, these data further support the hypothesis that suspension feeding invertebrates represent a significant target for NPs in the aquatic environment.

Role of HSP70 and p62 in regulation of autophagy clearance in ARPE‐19 cells

Abstract Purpose A hallmark of RPE cell aging is lysosomal lipofuscin accumulation reflecting a weakened capacity for protein degradation in lysosomes. The presence of lipofuscin increases the misfolding of intracellular proteins, which evokes additional stress in the RPE cells. If the capacity of molecular chaperones to repair protein damages is overwhelmed, then the proteins are mainly cleared in proteasomes or in lysosomes including autophagy. We demonstrate that autophagy is a master clearance mechanism in proteasome inhibitor ‐induced aggregation in ARPE‐19 cells. Methods The HSP70, p62 and ubiquitin expression levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate morphological changes. HSP70 and p62 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results The proteasome inhibitor MG132 evoked the formation of juxtanuclear protein aggregates in ARPE‐19 cells. It also caused a robust accumulation of HSP70 and p62 proteins and ubiquitin‐protein conjugates that all colocalized with the formed protein aggregates. We found that protein aggregation is a temporary process, a cessation of proteasome inhibition led to autophagosome‐mediated removal of cytoplasmic protein aggregates. Interestingly, the p62 rather than the HSP70 regulate autophagy activity. However, both of them have essential function in regulation of cellular viability. Conclusion In addition to classical lysosomal proteolysis, there is the increasing evidence that heat shock proteins, proteasomes and autophagy regulate protein turnover in the RPE cells and thus have important roles in AMD disease.

Heat shock proteins as gatekeepers of proteolytic pathways—Implications for age-related macular degeneration (AMD)

Age-related macular degeneration (AMD) is the major diagnosis for severe and irreversible central loss of vision in elderly people in the developed countries. The loss of vision involves primarily a progressive degeneration and cell death of postmitotic retinal pigment epithelial cells (RPE), which secondarily evokes adverse effects on photoreceptor cells. The RPE cells are exposed to chronic oxidative stress from three sources: their high levels of oxygen consumption, their exposure to the high levels of lipid peroxidation derived from the photoreceptor outer segments and their exposure to constant light stimuli. Cells increase the expression of heat shock proteins (HSPs) in order to normalize their growth conditions in response to various environmental stress factors, e.g. oxidative stress. The HSPs function as molecular chaperones by preventing the accumulation of cellular cytotoxic protein aggregates and assisting in correct folding of both nascent and misfolded proteins. Increased HSPs levels are observed in the retina of AMD patients, evidence of stressed tissue. A hallmark of RPE cell aging is lysosomal lipofuscin accumulation reflecting a weakened capacity to degrade proteins in lysosomes. The presence of lipofuscin increases the misfolding of intracellular proteins, which evokes additional stress in the RPE cells. If the capacity of HSPs to repair protein damages is overwhelmed, then the proteins are mainly cleared in proteasomes or in lysosomes. In this review, we discuss the role of heat shock proteins, proteasomes, and lysosomes and autophagic processes in RPE cell proteolysis and how these might be involved in development of AMD. In addition to classical lysosomal proteolysis, we focus on the increasing evidence that, HSPs, proteasomes and autophagy regulate protein turnover in the RPE cells and thus have important roles in AMD disease.

Application of a biomarker battery for the evaluation of the sublethal effects of pollutants in the earthworm Eisenia andrei

We applied a battery of biomarkers in the earthworm Eisenia andrei to assess the pollutant-induced stress syndrome. Earthworms were exposed to sublethal concentrations of copper (1–10 μM) and benzo[ a]pyrene (0.01–10 μM) as models of inorganic and organic pollutants for 1, 3 and 7 days. Four potential biomarkers of stress were measured: lysosomal membrane stability of coelomocytes, lysosomal accumulation of lipofuscin in chloragogenous tissue and of neutral lipids in coelomatic cells, plasma membrane Ca 2+-ATPase activity in the intestinal epithelium; metallothionein content was also evaluated as a biomarker of exposure. Significant changes were observed in the parameters measured in earthworms exposed to both contaminants. Certain biomarkers, such as lysosomal membrane stability and Ca 2+-ATPase activity, that showed a decreasing trend with increasing pollutant concentration and time of exposure, proved to be particularly suitable to describe the evolution of a stress syndrome from its early phase to the development of pathological conditions. On the other hand, contaminant-induced changes in lysosomal lipofuscin and neutral lipid accumulation showed a bell-shaped trend, indicating that these biomarkers are able to follow the development of pollutant-induced stress syndrome as far as an equilibrium in the cell functions is maintained; therefore, they are particularly useful to describe mild stress conditions.

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu 2+, Cd 2+ or Zn 2+ (40 μg/l/animal) were investigated. 2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed. 3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules. 4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects. 5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.