Alexei Terman

Lipofuscin: mechanisms of age-related accumulation and influence on cell function.

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occuring 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.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.

Autophagy and Aging The Importance of Maintaining "Clean" Cells

A decrease in the rate of protein turnover and the intracellular accumulation of altered proteins in cytosol and membranes are features common to all aged cells. Diminished autophagic activity plays a major role in these age-related manifestations. In this work we review the molecular defects responsible for the malfunctioning of two forms of autophagy - macroautophagy and chaperone-mediated autophagy - in old mammals, and highlight general and cell-type specific consequences of dysfunction of the autophagic system with age. Dietary caloric restriction and antilipolytic agents have been proven to efficiently stimulate autophagy in old rodents. These and other possible restorative efforts are discussed.

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.

Lipofuscin: mechanisms of formation and increase with age.

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.

Autophagy, organelles and ageing

As a result of insufficient digestion of oxidatively damaged macromolecules and organelles by autophagy and other degradative systems, long‐lived postmitotic cells, such as cardiac myocytes, neurons and retinal pigment epithelial cells, progressively accumulate biological ‘garbage’ (‘waste’ materials). The latter include lipofuscin (a non‐degradable intralysosomal polymeric substance), defective mitochondria and other organelles, and aberrant proteins, often forming aggregates (aggresomes). An interaction between senescent lipofuscin‐loaded lysosomes and mitochondria seems to play a pivotal role in the progress of cellular ageing. Lipofuscin deposition hampers autophagic mitochondrial turnover, promoting the accumulation of senescent mitochondria, which are deficient in ATP production but produce increased amounts of reactive oxygen species. Increased oxidative stress, in turn, further enhances damage to both mitochondria and lysosomes, thus diminishing adaptability, triggering mitochondrial and lysosomal pro‐apoptotic pathways, and culminating in cell death. Copyright

Lysosomes and oxidative stress in aging and apoptosis

The lysosomal compartment consists of numerous acidic vesicles (pH approximately 4-5) that constantly fuse and divide. It receives a large number of hydrolases from the trans-Golgi network, while their substrates arrive from both the cells outside (heterophagy) and inside (autophagy). Many macromolecules under degradation inside lysosomes contain iron that, when released in labile form, makes lysosomes sensitive to oxidative stress. The magnitude of generated lysosomal destabilization determines if reparative autophagy, apoptosis, or necrosis will follow. Apart from being an essential turnover process, autophagy is also a mechanism for cells to repair inflicted damage, and to survive temporary starvation. The inevitable diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow oxidative formation of lipofuscin in long-lived postmitotic cells, where it finally occupies a substantial part of the volume of the lysosomal compartment. This seems to result in a misdirection of lysosomal enzymes away from autophagosomes, resulting in depressed autophagy and the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. This scenario might put aging into the category of autophagy disorders.

Lysosomes in iron metabolism, ageing and apoptosis

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ∼4 to 5) that constantly fuse and divide. It receives a large number of hydrolases (∼50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.

Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis.

The mechanisms involved in the accumulation of ceroid/lipofuscin within non-dividing cells are not totally understood. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce ceroid/lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in ceroidogenesis/lipofuscinogenesis, and to study the fate of already formed ceroid/lipofuscin, confluent cultures of AG-1518 human fibroblasts were exposed to oxidative stress (40% ambient oxygen) and/or treated with the thiol protease inhibitor leupeptin for 2 weeks. Both oxidative stress and protease inhibition caused accumulation of ceroid/lipofuscin per se (estimated by fluorescent, confocal and electron microscopy). The combined effect of these factors was, however, almost three times as large as the sum of their isolated effects. The pigment accumulated progressively as long as the oxidative stress and/or protease inhibition acted; was not eliminated after re-establishment of normal conditions; and decreased in amount after subsequent passage. The results suggest that (i) ceroid/lipofuscin forms within secondary lysosomes due to peroxidative damage of autophagocytosed material, and (ii) it is not substantially eliminated from non-dividing cells by degradation or exocytosis.

Aging of Cardiac Myocytes in Culture: Oxidative Stress, Lipofuscin Accumulation, and Mitochondrial Turnover

Abstract: Oxidative stress is believed to be an important contributor to aging, mainly affecting long‐lived postmitotic cells such as cardiac myocytes and neurons. Aging cells accumulate functionally effete, often mutant and enlarged mitochondria, as well as an intralysosomal undegradable pigment, lipofuscin. To provide better insight into the role of oxidative stress, mitochondrial damage, and lipofuscinogenesis in postmitotic aging, we studied the relationship between these parameters in cultured neonatal rat cardiac myocytes. It was found that the content of lipofuscin, which varied drastically between cells, positively correlated with mitochondrial damage (evaluated by decreased innermembrane potential), as well as with the production of reactive oxygen species. These results suggest that both lipofuscin accumulation and mitochondrial damage have common underlying mechanisms, likely including imperfect autophagy and ensuing lysosomal degradation of oxidatively damaged mitochondria and other organelles. Increased size of mitochondria (possibly resulting from impaired fission due to oxidative damage to mitochondrial DNA, membranes, and proteins) also may interfere with mitochondrial turnover, leading to the appearance of so‐called “giant” mitochondria. This assumption is based on our observation that pharmacological inhibition of autophagy with 3‐methyladenine induced only moderate accumulation of large (senescent‐like) mitochondria but drastically increased numbers of small, apparently normal mitochondria, reflecting their rapid turnover and suggesting that enlarged mitochondria are poorly autophagocytosed. Overall, our findings emphasize the importance of mitochondrial turnover in postmitotic aging and provide further support for the mitochondrial‐lysosomal axis theory of aging.

Ceroid/lipofuscin-loaded human fibroblasts show decreased survival time and diminished autophagocytosis during amino acid starvation.

To test whether heavy accumulation of ceroid/lipofuscin can disturb important functions of the lysosomal system, AG-1518 human fibroblasts, ceroid/lipofuscin-loaded (following prolonged culture at normobaric hyperoxia) or not, were exposed to amino acid starvation. Ceroid/lipofuscin-loading resulted in decreased cellular survival. Also, there was an inverse relationship between amounts of ceroid/lipofuscin and the survival time of individual cells within the same cultures. Ceroid/lipofuscin-loaded fibroblasts displayed diminished autophagocytotic capacity, as demonstrated by electron microscopy and by treatment of cell cultures with NH4Cl (which inhibits autophagocytotic degradation by increasing intralysosomal pH) for 1 week before ensuing starvation. The latter treatment increased survival of control cells (due to deposition of nondegraded autophagocytosed material before start of starvation), but not that of ceroid/lipofuscin-loaded cells. Moreover, when NH4Cl treatment was combined with starvation, both groups of cells showed approximately the same shortened survival times, testifying to the causal relationship between diminished autophagocytosis and decreased survival of starving ceroid/lipofuscin-loaded cells. We hypothesize that large amounts of undegradable ceroid/lipofuscin within the acidic vacuolar compartment may interfere with lysosomal function, resulting in poor renewal of long-lived proteins and worn-out/damaged organelles, decreased adaptability, and cell death.