Ulf T. Brunk

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.

Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species

The objective of this study was to examine the possible involvement of oxygen free radicals in the aging process. Rates of mitochondrial O2.- and H2O2 production and oxygen consumption in the kidney and the heart were compared among seven different mammalian species namely, mouse, hamster, rat, guinea pig, rabbit, pig, and cow, whose maximum life span potential (MLSP) varies from 3.5 to 30 years. The rates of mitochondrial O2.- and H2O2 generation were inversely correlated to MLSP, and directly related to specific metabolic rate and state 4 mitochondrial respiration. Results of this study indicate that under identical conditions, mitochondria from shorter-lived species produce relatively higher amounts of reactive oxygen species than those from the longer-lived species, and, thus, support the free radical hypothesis of aging.

Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H2O2-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H2O2-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A2 activities, whereas H2O2-induced necrosis requires iron-dependent Fenton reactions.

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.

Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.

Acridine orange (AO) is a lysosomotropic weak base, a metachromatic fluorochrome, and a photosensitizer, as well. Living cells that are exposed for a short period of time to this compound at low concentration, and under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular fluoresence upon excitation with blue light. When AO-loaded cells are irradiated with intense blue light, AO soon starts to leak from late endosomes and lysosomes, partially shifting the fluorescence to a green, nuclear and diffuse cytosolic, one. This AO-relocalization is a consequence of photo-oxidation of the lysosomal membranes, which initially results in disruption of their proton-gradients and later, in leakage into the cytosol of a host of hydrolytic enzymes--as was here demonstrated by immunocytochemistry--which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of reparative autophagocytosis. Severe photo-oxidation, which resulted in severe lysosomal damage, caused cellular necrosis; whereas moderate stress, resulting in only partial lysosomal leakiness lead to apoptosis with TUNEL-positive nuclei and shrunken cytoplasm. The findings of the present study show that photo-oxidative damage to the membranes that surround the acidic vacuolar compartment, is an event that results in release of proteolytic and DNA-fragmenting enzymes into the cytosol, which may induce either necrosis, apoptosis, or reparable sublethal damage, depending on the magnitude of lysosomal rupture. Furthermore, the results strongly suggest that proteases and endonucleases of lysosomal origin may induce apoptosis if relocalized from the acidic vacuolar compartment into the cytosol.

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.

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture.

We have re-examined the lysosomal hypothesis of oxidative-stress-induced apoptosis using a new technique for exposing cells in culture to a low steady-state concentration of H(2)O(2). This steady-state technique mimics the situation in vivo better than the bolus-administration method. A key aspect of H(2)O(2)-induced apoptosis is that the apoptosis is evident only after several hours, although cells may become committed within a few minutes of exposure to this particular reactive oxygen species. In the present work, we were able to show, for the first time, several correlative links between the triggering effect of H(2)O(2) and the later onset of apoptosis: (i) a short (15 min) exposure to H(2)O(2) caused almost immediate, albeit limited, lysosomal rupture; (ii) early lysosomal damage, and later apoptosis, showed a similar dose-related response to H(2)O(2); (iii) both events were inhibited by pre-treatment with iron chelators, including desferrioxamine. This compound is known to be taken up by endocytosis only and thus to become localized in the lysosomal compartment. After exposure to oxidative stress, when cells were again in standard culture conditions, a time-dependent continuous increase in lysosomal rupture was observed, resulting in a considerably lowered number of intact lysosomes in apoptotic cells, whereas non-apoptotic cells from the same batch of oxidative-stress-exposed cells showed mainly intact lysosomes. Taken together, our results reinforce earlier findings and strongly suggest that lysosomal rupture is an early upstream initiating event, and a consequence of intralysosomal iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.

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

Induction of cell death by the lysosomotropic detergent MSDH

Controlled lysosomal rupture was initiated in lysosome‐rich, macrophage‐like cells by the synthetic lysosomotropic detergent, O‐methyl‐serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro‐caspase‐3‐like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro‐caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro‐apoptotic factors.