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The mitochondrial-lysosomal axis theory of aging
Accumulation of damaged mitochondria as a result of imperfect autophagocytosis
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Abstract
Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological ‘garbage’). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.
Abbreviations
-
- mtDNA
-
- mitochondrial DNA
-
- ROS
-
- reactive oxygen species.
Introduction
Different types of cells within multicellular organisms are known to age at different rates. Age-related changes are most prominent in postmitotic cells, such as neurons and cardiac myocytes, whereas in bone marrow, intestinal epithelium and other proliferating cell populations, only minor or undetectable changes occur with age [1,2]. Aging affects not only cells, but also the extracellular matrix [3], possibly causing disorders of diffusion and intercellular communication. However, being responsible for age-related disturbances of vitally important brain and heart functions, senescent changes in postmitotic cells play an essential role in the overall aging process.
Although aging affects many cellular components, perhaps the most remarkable changes occur in mitochondria and lysosomes of postmitotic cells. Mitochondria undergo gradual structural alterations associated with decreased capacity to produce energy [4–6], while lysosomes progressively accumulate lipofuscin (age pigment), a brown-yellow, autofluorescent, undegradable, polymeric material [7,8].
Accumulating evidence suggests that age-related damage is an ineluctable consequence of normal oxygen metabolism, which is associated with a relentless formation of reactive oxygen species (ROS) [9–11]. Superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and other antioxidant defense systems reduce, but do not prevent, macromolecular damage such as single- and double DNA-strand breaks, DNA–protein and protein–protein cross-linking, protein fragmentation, and oxidation and decomposition of lipids, resulting in the formation of dangerous products such as hydroperoxides, alkyl radicals, cyclic endoperoxides, and aldehydes [11–13]. Cellular aging is also characterized by disturbances in protein synthesis, decreased enzyme activity and progressive impairment of the functions of mitochondria and other organelles [11]. Damaged and effete cellular structures may well be considered as biological ‘garbage’.
In proliferating cells, oxidant-induced damage does not accumulate substantially with age, apparently because the process of cell division efficiently dilutes damaged structures [14,15]. Indeed, Hydra, a primitive multicellular organism composed only of cells that are continuously renewed by proliferation, do not seem to show any signs of aging [16]. Similarly, age-related changes are not seen in cultured neoplastic cells or in early passages of normal, actively proliferating cells. However, when cell proliferation is inhibited in confluent cultures of normal diploid cells, or at late passages of such cells (phase III according to Hayflick), senescent alterations start to accumulate [17,18].
Postmitotic cells accumulate biological ‘garbage’ because they are not renewed by cell division. Both proliferating and postmitotic cells can, however, renew themselves by degrading defective macromolecules and organelles into low-molecular-mass compounds that are then either cleared or re-utilized for the construction of new biological structures. Cells possess several mechanisms to degrade their components. Some proteins, mainly those that are short-lived, are decomposed by cytosolic cysteine proteases, known as calpains, while others are degraded by special mini-organelles, called proteasomes [19,20]. In contrast, most long-lived proteins, other macromolecules, and all organelles are degraded within the cellular acidic vacuolar compartment, the lysosomes, which contain various lytic enzymes with pH optima in the acidic range. Large portions of cytoplasm (often containing whole organelles) enter lysosomes by macroautophagy, a process that initially involves formation of double-membrane-bound autophagosomes and their subsequent fusion with lysosomes [21,22]. Alternatively, the material enters lysosomes by invagination of the membrane (microautophagy) [23] or by chaperone-mediated selective autophagy [24].
Despite such continuous recycling of cellular components, postmitotic cells age, suggesting that the recycling mechanisms are inherently imperfect [15,25], and this may provide an attractive explanation for many of the features of aging. A number of early explanations of aging, such as Orgel's error catastrophe theory and the somatic mutation theory, were based on the idea that aging results from the accumulation of synthetic errors [26,27]. Adequate support for these theories, however, could not be found, suggesting that organisms age because they cannot completely remove ‘biological garbage’, rather than because they incorrectly synthesize new constituents [15]. Despite functioning proteasomes and lysosomes, postmitotic cells progressively accumulate oxidatively modified proteins, other macromolecules and defective organelles. Even when these defective structures are autophagocytosed, they are not totally degraded, and lipofuscin forms.
Here we discuss the involvement of mitochondria and lysosomes in the aging process, based on our opinion that structural alterations and misfunction of these two organelles account for the majority of senescent changes in postmitotic cells.
Intralysosomal accumulation of lipofuscin
Deposition of lipofuscin pigment within postmitotic cells, one of the most prominent signs of aging, has been known for more than 150 years [28]. However, the relation of lipofuscin to lysosomes and the basic mechanisms of lipofuscin formation have been determined only recently [29,30]. Lipofuscin accumulates within lysosomes, mainly due to the fact that it is undegradable and cannot be removed from postmitotic cells via exocytosis [31,32].
Lipofuscin is a mixture of substances, mainly of protein and lipid origin (30–70% and 20–50%, respectively). It also contains small amounts of carbohydrates (up to 7%) and traces of metals, especially iron [7,33]. There is strong evidence that macromolecules become indigestible as a result of oxidative modification (associated with aggregation due to aldehyde-mediated cross-linking), and that this modification occurs primarily during autophagocytotic degradation inside lysosomes [30,32]. An undegradable lysosomal pigment (occasionally called ‘ceroid’) also forms independently of age due to enhanced reparative autophagocytosis or delayed lysosomal degradation in a number of pathological conditions such as malnutrition, lysosomal storage diseases, ionizing radiation, tumors, etc. [7,8]. However, despite different etiology, the mechanisms of lysosomal pigment formation and its composition are basically the same in aging and disease.
In a lysosome, where different organelles and a variety of proteins and other macromolecules are degraded, the milieu is acidic and reducing, partly due to the presence of the reducing amino acid cysteine [34]. Degradation of iron-containing metalloproteins, such as cytochromes and ferritin, results in the release of redox-active low-molecular-mass iron. The latter, in ferrous form, reacts with hydrogen peroxide (which easily diffuses throughout the cell), forming the extremely reactive hydroxyl radical (Fenton reaction):
Fe+H2O2→Fe3+OH−+HO
Hydroxyl radicals, which have a half-life on the order of 10−9 s, do not diffuse, making their reaction with surrounding biomolecules site-specific. In unsaturated fatty acids they initiate a chain-reaction:
As most organic peroxides are quite unstable, especially in the presence of iron (Fe2+↔Fe3+), these chain reactions result in the formation of aldehydes, such as malondialdehyde (CHO–CH2–CHO).
Aldehydes react with one or two free amino groups within proteins, forming Schiff bases (apparently responsible for lipofuscin autofluorescence), which undergo intramolecular Maillard-type rearrangements [13,35]. Formation of aldehyde bridges, an important mechanism of protein–protein cross-linking, is involved in lipofuscinogenesis. Lipofuscin seems to undergo maturation reactions and form aggregates that finally may take over whole lysosomes (which remain acidic and capable of taking up newly synthesized lysosomal hydrolases). Lipofuscin-loaded lysosomes may occupy considerable portions of postmitotic cells (up to 75% in some brain neurons) [36]. The basic principles of lipofuscin formation are summarized in Fig. 1.
Scheme illustrating the mechanisms of lipofuscin formation within postmitotic cells. A large variety of biomolecules is degraded by hydrolytic enzymes within the lysosomal compartment of the cell. Some macromolecules, e.g. mitochondrial cytochromes and ferritin, are iron-containing metalloproteins which would therefore release low molecular mass iron when degraded. Lysosomes have a low pH and are rich in reducing substances, such as cysteine. Some ferric iron would thus be reduced to redox-active ferrous form. Hydrogen peroxide, which freely diffuses into the lysosomes, may consequently undergo homolytic cleavage with formation of hydroxyl radicals which, in turn, induce peroxidative reactions and formation of undegradable material (lipofuscin) from macromolecules undergoing degradation.
Age-related mitochondrial damage
Mitochondrial alterations in aging postmitotic cells have been characterized extensively. Usually, mitochondrial size varies more in old cells, as compared to corresponding young cells, with a high proportion of large, sometimes extremely large (‘giant’), mitochondria [5,37]. Ultrastructural changes range from swelling and loss of cristae to complete deterioration and homogenization of matrix and mitochondrial membranes [4,5]. Mutations of mitochondrial DNA (mtDNA) accumulate progressively, often involving the sites coding for respiratory chain proteins [6,38]. Aging is associated with decreased activity of the citric acid cycle, beta-oxidation, and oxidative phosphorylation enzymes [39,40]. As a result, mitochondria of aged postmitotic cells have decreased membrane potential and produce less ATP than the mitochondria of young cells [6,38].
Mitochondria are the primary sites of ROS generation, which may be one reason why they are more affected by age than other organelles [38,41–43]. Certain properties specific to mtDNA also contribute to a high susceptibility of mitochondria to age-related damage. mtDNA is more vulnerable than nuclear DNA, because it is not protected by histones, contains a much larger proportion of expressed genes, and shows less efficient repair, at least for some types of lesions [6,38]. Consistent with this, aging primarily affects complexes I and IV of the electron-transport chain, encoded by mtDNA, but not complexes II and III, which are mainly coded by nuclear genes [44]. Obviously, mitochondrial proteins also suffer direct oxidative damage, although due to their disparate nature they are not affected equally. For example, as shown by Yan et al. [45] in aging houseflies, oxidative modification of aconitase is much more pronounced than that of other mitochondrial proteins. The authors suggest that this may be related to the iron–sulfur cluster in the active site of aconitase, promoting in situ iron-catalyzed oxidation reactions (see above) and resulting damage to the enzyme.
Why do defective mitochondria displace normal ones?
If damaged mitochondria were perfectly autophagocytosed and replaced by normal ones, dysfunctional mitochondria would not accumulate within aging postmitotic cells. But, in fact, such an accumulation does occur, implying that either cells autophagocytose defective mitochondria at a lower rate than normal mitochondria, or that these damaged mitochondria have a replicative advantage.
Decreased autophagocytosis of defective mitochondria
There are several reasons which favour the former hypothesis. One factor that can interfere with autophagocytosis of mitochondria is their enlargement. We recently observed that in cultured neonatal rat cardiac myocytes, inhibition of autophagocytosis by 3-methyladenine results in a dramatic accumulation of small mitochondria while the number of large ones increases only slightly (A. Terman, H. Dalen, J. Neuzil, J. W. Eaton & U. T. Brunk, unpublished results). This observation suggests that the majority of normally autophagocytosed mitochondria are small, while larger mitochondria are less well autophagocytosed. But what would make mitochondria large and, perhaps, exclude them from normal autophagocytotic degradation?
Regulation of mitochondrial division (fission) is poorly understood [46]. However, it is likely to be impaired by oxidative damage to mitochondrial DNA and proteins. Although mitochondrial fission is not absolutely dependent on mtDNA replication (as is the case for cell division which requires nuclear DNA replication); the amount of normal DNA per mitochondrion apparently must not be too low. Consistent with this, mtDNA-depleted cells usually contain substantially enlarged mitochondria. Therefore, age-dependent accumulation of mutations in mtDNA, perhaps especially in the control region for replication [47], may also diminish mitochondrial fission which latter could govern the appearance of abnormal, large mitochondria.
Once a mitochondrion starts to enlarge (due to initial oxidative damage and disturbed fission), it may be, as mentioned above, less likely to be autophagocytosed and recycled. This will lead to further mitochondrial damage by ROS, a decrease in ATP production, and additional growth as mitochondrial proteins synthesized by nuclear genes are continuously imported into existing mitochondria. Gradually, more and more mitochondria become enlarged and dysfunctional. These ‘giant’ mitochondria, as well as lipofuscin deposits, may remain within postmitotic cells forever. In a number of myopathies, cardiomyopathies and neurodegenerative diseases, mutant mitochondria also progressively accumulate and gradually replace normal mitochondria [48,49]. Furthermore, as is the case in the aging postmitotic cell, enlarged mitochondria are found in many cases of such disorders [50–52]
De Grey has proposed an alternative explanation for why defective mitochondria have a low rate of degradation [53]. His hypothesis, called SOS (for ‘survival of the slowest’), suggests that as mitochondria with mutant DNA have a reduced proton gradient, less HO2· (a more reactive, protonated form of O2·−) would form within the intermembranous space, despite an increased overall production of O2·−. As a result, the membranes of defective mitochondria may suffer less oxidative damage than those of normal mitochondria, may be less targeted for autophagocytosis and therefore preferentially survive. This hypothesis requires evidence – currently lacking – that mitochondria are selected for autophagocytosis based on the amount of membrane damage. This may, however, be the case. Although it was long believed that macroautophagy is a nonselective process [22], a recent report shows that sperm mitochondria are tagged with ubiquitin, providing for their selective degradation after fertilization [54]. It is still not clear, however, whether ubiqutin also may label oxidatively damaged mitochondria.
Increased replication of defective mitochondria
A number of congenital mitochondrial neuropathies, myopathies and cardiomyopathies begin, in young age, as discrete heteroplasmic mtDNA mutations. It is well documented that in many such conditions, there is age-dependent somatic selection which favours the replication and/or persistence of mitochondria carrying the mutation, explaining why the neurologic and muscular effects of these diseases may appear only later in life [48,55]. Based on these facts, it was hypothesized that defective mitochondria have a replicative advantage over normal mitochondria [56,57]. Analogous selection for dysfunctional mitochondria may also occur in the case of aging; Wanagat et al. recently reported that atrophic muscle fiber segments from old rats contain mtDNA deletions and have depressed cytochrome c oxidase activity [57]. This suggests a clonal expansion of defective mitochondria; however, because mitochondria of old muscle fibers are usually enlarged (see above), it is not possible to exclude decreased autophagocytosis. Moreover, homoplasmic mtDNA mutations have also been found in tumours (but not in normal host tissues) [58]. Overall, these observations suggest that clonal expansion of defective mitochondria may occur in some cases.
Failure of cellular functions and death
Even in young postmitotic cells, autophagocytosis is imperfect, thereby initiating the age-related accumulation of garbage. Given this, it is reasonable to expect a further decrease in autophagocytotic capacity at old age and this may be related to the progressive accumulation of indigestible garbage itself.
Lipofuscin is generally thought to be an innocent end-product of oxidation which has no significant influence on cellular activities [59,60]. However, this does not seem to be the case. Lipofuscin deposits gradually enlarge to occupy an increasing portion of the lysosmal compartment. Lipofuscin-loaded lysosomes constantly receive new hydrolytic enzymes from the Golgi complex, but these imported enzymes are wasted because degradation of the pigment is impossible. Thus, most of the lysosomal enzymes in aged postmitotic cells may be produced unnecessarily, only to be lost within lipofuscin-loaded lysosomes. Apparently, cells have a limited capacity to produce lysosomal enzymes, and as lipofuscin-containing lysosomes act as sinks for newly synthesized enzymes, they are not available to functional (lipofuscin-free) lysosomes. The diminished ability to degrade newly autophagocytosed debris, in turn, promotes further oxidation and accelerating lipofuscin formation from this material. Therefore, in the aging cell, lysosomal enzymes, essential for autophagocytotic recycling of damaged cellular constituents, are in short supply, forcing senescent cells to use their worn-out and poorly operating macromolecules and organelles. Consistent with this hypothesis, schematically outlined in Fig. 2, we have found that lipofuscin loading of fibroblasts does decrease their autophagocytotic capacity [61].
Age-related accumulation of damaged mitochondria and lipofuscin inclusions within postmitotic cells. Oxidative injury to mitochondrial DNA and proteins may disturb fission of some mitochondria, resulting in their enlargement. Large mitochondria are likely to be poorly autophagocytosed, which leads to further damage and enlargement. Lysosomes of aging postmitotic cells progressively accumulate lipofuscin pigment. Apparently, lipofuscin-containing lysosomes attract large amounts of newly synthesized lysosomal enzymes which, however, fail to degrade the undegradable pigment. Consequently, it may result in insufficient supply of lysosomal enzymes for autophagocytosis, further lessening mitochondrial recycling. Poorly functioning ‘giant’ mitochondria and lipofuscin-filled lysosomes (acting as sinks for lysosomal enzymes) gradually displace normal organelles, eventually causing decreased ATP production and failing autophagocytosis, resulting in death of postmitotic cells. Oxidatively-damaged mitochondria are indicated by a dotted pattern (more dots = more damage). F, mitochondrial fission. APS, autophagosome. The column charts show age-related trends in the mitochondrial production of ATP, autophagocytotic capacity (APG), lipofuscin content (LF), and formation of reactive oxygen species (ROS).
Although loaded with biological ‘garbage’ such as defective mitochondria and lipofuscin deposits, postmitotic cells do contain normal mitochondria that can divide to compensate for decreased ATP production. This explains why many senescent and severely damaged postmitotic cells continue to function, and is consistent with the fact that such cells usually are larger than corresponding young cells. Cells cannot increase in size indefinitely, however, because larger cells poorly move nutrients to their core. Therefore, this compensatory mechanism finally fails and the senescent postmitotic cell dies for lack of ATP.
Senescent mitochondria are not only deficient in ATP production, but also generate elevated amounts of superoxide and hydrogen peroxide [38,62,63], suggesting that increased oxidative stress also may contribute to the death of old postmitotic cells. It is known that oxidative stress above a certain limit causes lysosomal rupture due to intralysosomal iron-catalyzed peroxidative reactions [25,64]. If large amounts of H2O2 diffuse into lysosomes, the formation of hydroxyl radicals (or, more likely, iron-centered ferryl or perferryl radicals) may be sufficient to jeopardize the stability of the lysosomal membrane and to induce leak into the cytosol of lysosomal lytic enzymes. Moderate release of lysosomal enzymes has been shown to induce apoptosis in a variety of ways (Fig. 3), while more pronounced release of such enzymes results in necrotic cell death [64–66]. Lipofuscin-loaded cells appear highly sensitive to oxidant-induced damage as a result of their enlarged lysosomal compartment and the presence of iron bound to lipofuscin [33,67,68].
Possible pathways of apoptosis induction involving lysosomal enzymes in senescent postmitotic cells. With increasing oxidative stress, intralysosomal Fenton-type reactions become violent enough to jeopardize the stability of lysosomal membranes, resulting in leak of a variety of hydrolytic enzymes into the cytosol. Such lysosomal enzymes may directly activate Bid, pro-caspases, or lytic cytosolic pro-enzymes such as phospholipase A2, while the latter, together with lysosomal enzymes, may attack mitochondria, releasing cytochrome c and, as well, induce further lysosomal breach.
Conclusion
As assumed in our earlier publications [15,25], aging, which mainly affects postmitotic cells and extracellular matrix, results from the inability of an organism to completely remove biological ‘garbage’, which includes oxidatively-damaged and dysfunctional macromolecules and organelles, as well as lipofuscin inclusions. In this review, we further develop the idea that mitochondrial and lysosomal injury and dysfunction play a central role in the aging of postmitotic cells, as well as in aging of the whole organism, considering the particular importance of such cells (including neurons and cardiac myocytes) for life maintenance. Mitochondria are the main source of ROS formation, as well as the main target for free radical attack. The accumulation of defective mitochondria within aging cells suggests that some are not properly autophagocytosed. Aged mitochondria are often enlarged (apparently due to impaired division) which may explain their inefficient autophagocytotic removal. Moreover, macromolecular components of autophagocytosed mitochondria and other cellular structures undergo further oxidative modification within lysosomes, resulting in the formation of an undegradable material called lipofuscin. Heavy lipofuscin loading of postmitotic cells decreases their autophagocytotic capacity, leading to progressively less mitochondrial recycling. Consequently, mitochondrial and lysosomal age-related alterations may amplify each other, eventually causing profound dysfunction and death of postmitotic cells.
Acknowledgements
We thank Ms. Diane Konzen for expert linguistic help and Drs John W. Eaton and John D. Furber for numerous helpful comments. Supported by the Swedish Medical Research Council (grant 4481) and the Linköping Health University/University Hospital Aging Foundation.
