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Keywords:

  • anaplerotic metabolism;
  • cell death;
  • cellular aging;
  • fatty acid oxidation;
  • hormesis;
  • longevity;
  • mitochondrial retrograde signaling;
  • organelle biogenesis and function;
  • peroxisome;
  • protein degradation;
  • reactive oxygen species;
  • signal transduction

Abstract

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References

The essential role of peroxisomes in fatty acid oxidation, anaplerotic metabolism, and hydrogen peroxide turnover is well established. Recent findings suggest that these and other related biochemical processes governed by the organelle may also play a critical role in regulating cellular aging. The goal of this review is to summarize and integrate into a model the evidence that peroxisome metabolism actually helps define the replicative and chronological age of a eukaryotic cell. In this model, peroxisomal reactive oxygen species (ROS) are seen as altering organelle biogenesis and function, and eliciting changes in the dynamic communication networks that exist between peroxisomes and other cellular compartments. At low levels, peroxisomal ROS activate an anti-aging program in the cell; at concentrations beyond a specific threshold, a pro-aging course is triggered.

Aging of multicellular and unicellular eukaryotic organisms is a highly complex, multifactorial biological phenomenon (1). At the cellular level, declining function of organelles and their elaborate communication systems constitute both a cause and target of aging events (2–6). Emergent studies have revealed that the peroxisome, an organelle known for its essential role in lipid and anaplerotic metabolism, as well as the production and scavenging of hydrogen peroxide and other reactive oxygen species (ROS) (7,8), can also function as an intracellular signaling compartment and as an organizing platform that orchestrates important developmental decisions from inside the cell (9–12). Moreover, peroxisomal dysfunction has been shown to be associated with cellular aging as well as with molecular pathologies that result in the development and progression of specific age-related degenerative diseases (5,13–16). Here, we summarize growing evidence in support of an essential role for the peroxisome in regulating age-related reactions within eukaryotic cells and critically evaluate several molecular mechanisms underlying its involvement in longevity-defining processes.

Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References

The well-established abilities of peroxisomes to oxidize fatty acids to acetyl-coenzyme A (CoA) (7,8) and to use it then in anaplerotic reactions to replenish tricarboxylic acid (TCA) cycle intermediates destined for mitochondria (17,18) are integrated into several longevity regulation pathways within a cell. Such integration involves dynamic communication between peroxisomes and other organelles and is governed by the mechanisms outlined here.

Role of non-esterified fatty acids and diacylglycerol

In yeast grown in glucose-containing media and in fat storage tissues of multicellular eukaryotic organisms, the non-esterified (‘free’) fatty acids that are oxidized in peroxisomes originate mainly from triacylglycerols (TAG) and steryl esters (19–21). These neutral lipids are synthesized in the endoplasmic reticulum (ER), and are then deposited in the hydrophobic core of lipid bodies (21–23). In yeast, the lipolytic conversion of neutral lipids to free fatty acids within lipid bodies and the subsequent import of these fatty acids by peroxisomes are driven by an extensive physical contact between the two organelles (19). In fact, peroxisomes are able to invade the hydrophobic core of these stores of neutral lipids through synthesis of ‘pexopodia’(19). Recently, we proposed a mechanism that links yeast longevity to lipid dynamics in peroxisomes, lipid bodies, and the ER (4,5). This mechanism may underlie the life-extending effect of caloric restriction (CR), a low-calorie diet that increases life span in various organisms and delays the onset, or reduces the incidence, of many age-related diseases in rodent models (24). In the proposed mechanism (Figure 1), short-lived non-CR yeast accumulate ethanol, a product of glucose fermentation. By repressing the synthesis of Fox1p, Fox2p and Fox3p, all of which are the core enzymes of fatty acid β-oxidation in peroxisomes (25), ethanol suppresses peroxisomal oxidation of free fatty acids that originate from TAGs synthesized in the ER and deposited within lipid bodies (5). This leads to the accumulation of electron-dense arrays of free fatty acids (called ‘gnarls’) (19) within lipid bodies, and the initiation of several negative feedback loops. By reducing lipolysis of TAGs in lipid bodies and their biosynthesis in the ER, these feedback loops promote both the accumulation of TAG in lipid bodies and the buildup of diacylglycerol and free fatty acids in the ER (5). Because of such remodeling of lipid dynamics, which is driven by the diet-dependent changes in the efficiency of peroxisomal fatty acid β-oxidation, yeast placed on a calorie-rich diet age and die prematurely. The peroxisome-modulated premature death of non-CR yeast appears to be caused by their (i) necrotic death, triggered by the inability of peroxisomes to oxidize fatty acids which amass (26,27); (ii) caspase- and mitochondria-independent lipoapoptotic death, initiated by the accumulation of free fatty acids and diacylglycerol (28); and (iii) response to a diacylglycerol-induced reorganization of the protein kinase C-dependent signal transduction network, known to affect multiple stress response- and longevity-related processes (29,30).

image

Figure 1. Peroxisomal fatty acid β-oxidation acts as a system controller regulating cellular aging by modulating levels of non-esterified fatty acids and diacylglycerol. In yeast, the life-extending effect of a CR diet is due, in part, to a mechanism that links longevity to lipid dynamics in peroxisomes, lipid bodies, and the ER. Please see text for details. Figure adapted from Goldberg et al.(5).

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Peroxisome metabolism and the mitochondrial retrograde signaling pathway

In response to mitochondrial dysfunction, yeast cells activate the mitochondrial retrograde (RTG) signaling pathway (31,32). Controlled by a distinct set of regulatory proteins, this signaling pathway responds to defects in mitochondria by activating transcription of a cassette of nuclear genes (Figure 2). The protein products of these genes cause a specific remodeling of carbohydrate and nitrogen metabolism, activate peroxisome proliferation, promote peroxisomal fatty acid β-oxidation and anaplerotic reactions, stimulate stress responses, and enhance the stability of nuclear and mitochondrial genomes (31–34). The resulting changes in cell physiology compensate for the mitochondrial dysfunction that prompted the response (31,32,35).

image

Figure 2. Peroxisomal fatty acid oxidation and anaplerotic reactions drive the RTG signaling pathway of cellular aging regulation. Functionally compromised mitochondria trigger the RTG pathway in replicatively aging yeast. Gene transcription ensues, altering cell metabolism, stimulating peroxisome function, and maintaining cell viability. Please see text for details.

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The RTG pathway may be triggered in yeast cells by inducing a complete loss of mitochondrial DNA (mtDNA), introducing large deletions into mtDNA, or deleting the nuclear COX4 gene encoding one of the subunits of mitochondrial cytochrome c oxidase (36–38). The resulting loss of mitochondrial respiratory chain activity blocks the TCA cycle at succinate dehydrogenase, a four-subunit component of the chain (Figure 2) (32). Consequently, the first three reactions of the cycle—all downstream of the block—cannot convert pyruvate and acetyl-CoA to α-ketoglutarate, the direct precursor of glutamate (32). To meet the demand of nitrogen supply from glutamate to most of the biosynthetic reactions, yeast cells need to replenish acetyl-CoA, oxaloacetate and α-ketoglutarate. To achieve this, they activate the RTG signaling pathway by a yet-to-be-established mechanism. A collaborative effort of multiple protein regulators in this pathway promotes relocation of the Rtg1p–Rtg3p heterodimeric transcriptional factor from the cytosol to the nucleus, where it activates transcription of RTG-target genes (Figure 2) (31,32,35). Among these are genes for the first three enzymes in the TCA cycle, cytosolic enzymes involved in oxaloacetate and acetyl-CoA biosynthesis, a mitochondrial transporter for carnitine-dependent transfer of acetyl-CoA from the cytosol and peroxisomes to mitochondria, mitochondrial transporters for citrate and α-ketoglutarate, and plasma membrane transporters for carnitine and glutamate. In addition, RTG-target genes include a number that encode proteins required for peroxisome biogenesis and function (31–34). Among these are the Pxa1p subunit of a heterodimeric peroxisomal ATP-binding cassette transporter complex required for import of long-chain fatty acids into peroxisomes; Fox1p, Fox2p and Fox3p, the core enzymes of fatty acid β-oxidation in peroxisomes; Pex11p, a major positive regulator of peroxisome proliferation; and the citrate synthase Cit2p and acetyl-carnitine synthase Cat2p—enzymes required for the conversion of acetyl-CoA, a product of fatty acid oxidation, to citrate and acetyl-carnitine, respectively (Figure 2) (33,34). By promoting fatty acid oxidation and anaplerotic reactions in peroxisomes that replenish TCA cycle intermediates to be delivered to mitochondria, these peroxisomal proteins are vital for the ability of the RTG pathway to maintain viability of yeast cells harboring dysfunctional mitochondria (31,32). In support of this notion, the signaling pathway also responds to mitochondrial respiratory deficiency (but not to the rate of mitochondrial ATP synthesis per se) by greatly increasing peroxisome size and number under conditions that normally do not induce peroxisome assembly and proliferation (33). Moreover, in yeast cells deficient in mitochondrial respiration, the RTG pathway is mandatory for growth on oleate and for oleate-induced expression of genes whose protein products play key roles in peroxisome biogenesis and function (39,40).

It should be stressed that replicatively aging yeast gradually activate the RTG signaling pathway to compensate for the age-associated decline in their mitochondrial function which is manifest by a progressive deterioration of mitochondrial respiratory chain activity, loss of mitochondrial membrane potential, and accumulation of mutations in mtDNA (38,41–43). Importantly, the extent to which the RTG pathway is activated in response to the accumulation of dysfunctional mitochondria in yeast is directly correlated with the resulting increase in their life span (31,35,38,44). Thus, by being essential for the ability of the RTG signaling pathway to maintain viability of yeast cells harboring dysfunctional mitochondria, peroxisomes play a key role in regulating yeast longevity.

Two other nutrient-status-activated signaling pathways impact the peroxisome's ability to modulate the RTG reaction. One is the target of rapamycin (TOR) pathway, which responds primarily to the quality of the nitrogen source (45). The TOR pathway adjusts the RTG response via Lst8p, a subunit of TOR Complex 1 (TORC1) and negative regulator of the mitochondrial pathway (Figure 2) (32). Of note, a potent immunosuppressive drug, rapamycin, extends the replicative and chronological lifespans of yeast by inhibiting TORC1 (46) and, as a result, activating the RTG pathway (45). In addition, the RTG response is modulated by the Ras2p/protein kinase A signaling pathway, which responds primarily to the quality of the carbon source available (45) and impacts the pathway at Mks1p (Figure 2) (32,35).

The key point here is that the ability of peroxisomes to modulate the RTG pathway is integrated into a nutritional sensing network that regulates yeast longevity.

Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References

By housing a number of ROS-producing oxidases as well as the scavenging enzymes catalase and peroxiredoxin, peroxisomes not only control the intraperoxisomal homeostasis of ROS but can also contribute to the maintenance of extraperoxisomal ROS levels within the entire cell (13,47). Here, we summarize the growing evidence that ROS homeostasis and macromolecular oxidative damage in peroxisomes govern several peroxisome-confined anti-aging processes and control the dynamics of communications between these organelles and other cellular compartments, thereby helping to define the replicative and chronological lifespan of a cell.

Peroxisome senescence

Peroxisomes manifest age-related changes to their metabolism and biogenesis. Studies employing cultured human cells reveal that peroxisomes senesce, with dramatic changes seen to their protein import capacities, their ability to regulate organelle growth and division, their functional integrity, and their capacity to process ROS. Compromised trafficking of the hydrogen peroxide-metabolizing enzyme catalase certainly contributes to the latter effect, and may actually advance all the properties listed (48). A method of re-establishing peroxisomal catalase has been developed and dramatic effects on aging cells observed. Indeed, old cells with newly established, redox-balanced peroxisomes delay appearance of senescence markers, re-establish mitochondrial integrity, and enjoy oxidative equilibrium (15). The effects of altering what is clearly age-related peroxisomal hypocatalasemia may have important health ramifications as there is compelling epidemiological evidence that as the level of catalase is reduced across a population, the incidence of debilitating disease is dramatically increased (49). Thus, it seems as if the intraperoxisomal concentration of ROS cannot be actively maintained below a specific toxic threshold, the organelle is damaged, oxidative balance is lost, and both the organelle and the cell suffer the consequences. It can also be imagined that as peroxisome function declines with age, its ability to participate in the anti-aging processes described above (and outlined in Figures 1 and 2) would be seriously impaired.

Mechanisms for minimizing buildup of oxidatively damaged peroxisomes

Eukaryotic organisms have evolved several mechanisms designed to minimize the buildup of peroxisomal macromolecules damaged by exposure to ROS in cells. These include the selective elimination of oxidatively damaged peroxisomal proteins and lipids, and prevention of potentially damaging inheritance of functionally compromised organelles by daughter cells during mitosis. Examples of these mechanisms at work are considered here.

The insulin degrading enzyme (IDE) in mammalian peroxisomes and the peroxisomal Lon (pLon) protease in yeast peroxisomes carry out the intraperoxisomal degradation of oxidatively damaged proteins (50,51). Interestingly, the mitochondrial form of the Lon protease (mLon) is essential for metabolizing oxidatively damaged (mitochondrial) proteins and its activity declines with age (52). It is tempting to speculate that its peroxisomal counterpart, pLon, also plays a role in cellular aging by eliminating oxidatively damaged proteins in peroxisomes of old or diseased cells.

Plant peroxisomes employ a peroxisome-associated protein degradation (PexAD) system—which appears to function similar to the ER-associated protein degradation (ERAD) system—for the export of oxidatively damaged matrix proteins from the peroxisome to the cytosol where they are subsequently degraded, perhaps by the ubiquitin-proteasome system (53). PexAD involves the peroxisome-bound ubiquitin-conjugating enzyme Pex4p, its membrane tether Pex22p, and the peroxisome-associated ATPase associated with diverse cellular activities (AAA) family member, Pex6p. This system also requires the cycling peroxisomal targeting signal 1 import receptor, Pex5p, although its role in PexAD-driven protein degradation is yet to be delineated (53).

A healthy population of peroxisomes in ‘young’ yeast cells may also be maintained by pexophagy, an autophagy-related process of selective degradation of whole peroxisomes—perhaps, mainly dysfunctional organelles containing oxidatively damaged proteins—following their sequestration by vacuoles (51,54). Importantly, autophagy is mandatory for the life-extending effects of a CR diet, as well as of the anti-aging drugs rapamycin, resveratrol, and spermidine. These interventions all stimulate autophagy in aged cells and modulate the age-related dynamics of ROS (55–60). Moreover, hydrogen peroxide serves as a potent signaling molecule that initiates autophagy through redox-sensitive proteases (61). It would be interesting to know if age-related oxidative damage of the membrane-bound peroxins, Pex3p and Pex14p, increases their affinity for Atg30p, a protein whose Pex3p-dependent phosphorylation on the peroxisomal surface is required for targeting the organelle to the autophagy machinery for degradation (54). The increased size of dysfunctional, oxidatively damaged peroxisomes may also commit them for autophagy through recruitment of two other pexophagy-specific proteins, Atg11p and Atg26p (62).

In plant cells, an ER to peroxisome connectivity system may contribute to the elimination of oxidatively damaged peroxisomal proteins and lipids. It appears that in response to endogenously induced hydroxyl radical stress, plant peroxisomes produce dynamic tubular extensions called ‘peroxules’ which then elongate (63). Both peroxules and elongated peroxisomes extend along path defined by the ER tubules (63). Based on this observation, it has been proposed that plant peroxisomes are continuous with the ER and that the ER-peroxisome connectivity system responds to conditions of oxidative stress by enabling the retro-flow of oxidatively damaged matrix proteins as well as of membrane proteins and lipids to the ER. Such retro-flow would reduce the oxidative stress to peroxisomes (63,64) and would allow undamaged peroxisomal constituents (delivered either from the ER or from the cytosol) to support the battery of peroxisome-confined, anti-aging processes described above.

Finally, mechanisms exist to prevent inheritance of dysfunctional, oxidatively damaged peroxisomes by the daughter cell during mitosis. Indeed, two such pathways have been identified in the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica(65,66).

Peroxisomal ROS may function as anti-aging signaling molecules, akin to mitochondrial ROS

Growing evidence supports the view that in addition to their pro-aging role postulated by the free radical theory of aging—ROS at low, non-toxic levels can function as potent anti-aging signaling molecules that induce ‘stress-response hormesis' (3,4,6,67–73). The molecular mechanisms underlying the anti-aging potential of mitochondrial ROS (13,74) have begun to emerge; they involve communication between mitochondria and the nucleus via several signaling networks (3,6,75–77). Sub-lethal concentrations of hydrogen peroxide—following its diffusion from mitochondria—appear to trigger the c-Jun amino-terminal kinase signaling cascade that ultimately enables the single forkhead box O transcriptional factor to activate expression of critical anti-aging genes in the nucleus. It is also possible that hormetic levels of ROS elicit partial mitochondrial dysfunction, oxidative damage to specific organellar constituents and ultimately trigger the RTG, TOR, or AMPK signaling pathways described above.

Importantly, in some animal tissues—such as rat liver—peroxisomes can produce up to 35% of all cellular hydrogen peroxide (78). In addition to being able to control the synthesis and degradation of ROS within the organelle, peroxisomes also contribute to the maintenance of cellular ROS homeostasis (13,47). It is not unreasonable to speculate that if ROS are maintained below a specific threshold level, peroxisomes could elicit an anti-aging cellular program akin to that seen with mitochondria. In fact, a recent study suggests that CR yeast induce hydrogen peroxide, presumably at non-toxic/pro-hormetic levels, and experience extended chronological lifespans (79). Similar effects are seen with catalase inactivation, even though oxidative damage to macromolecules is increased under such circumstances. Whether or not peroxisomal oxidation/dysfunction caused by low concentrations of ROS actually triggers the RTG, TOR, AMPK or other longevity-defining signaling pathways remains to be determined.

Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References

A model for how peroxisome metabolism impacts cellular aging emerges from our analysis and is depicted schematically in Figure 3. The model includes the notion that in replicatively and chronologically ‘young’ cells, efficient Pex5p-dependent peroxisomal import of the ROS scavenging enzymes catalase and peroxiredoxin enables peroxisomes to minimize the oxidative damage to their proteins and lipids. Quality control of the Pex5p-driven reaction is assured by the receptor accumulation and degradation in the absence of recycling (RADAR) pathway (80). Inside the organelle, the IDE and pLon proteases as well as the PexAD system for the degradation of oxidatively damaged peroxisomal matrix proteins eliminate those molecules that are unable to support the anti-aging processes orchestrated by functionally active peroxisomes.

image

Figure 3. A model for the ROS-driven integration of peroxisomal processes that regulate cellular aging. A) In replicatively and chronologically ‘young’ cells, the intraperoxisomal concentration of ROS is actively maintained below a toxic threshold. In these cells, peroxisomes function both as a cellular compartment housing anti-aging processes (depicted in Figures 1 and 2) and as a signaling platform for activating extraperoxisomal anti-aging processes within the cell. B) In replicatively and chronologically ‘old’ cells, the intraperoxisomal concentration of ROS exceeds a toxic threshold. In these cells, the peroxisome switches from being a platform for activating anti-aging processes to a staging area for the development of a pro-aging program within the cell. Not only is the peroxisome unable to carry out intraorganellar anti-aging processes, but it now releases hydrogen peroxide and related ROS to the cytosol where it contributes to oxidative damage throughout the cell. Please see text for additional details. H2O2 = hydrogen peroxide; CAT = catalase; PRX = peroxiredoxin.

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The efficient Pex5p- and Pex7p-dependent import of Fox1p, Fox2p, and Fox3p into peroxisomes of ‘young’ cells enhances their ability to oxidize fatty acids that originate from TAGs synthesized in the ER and deposited within lipid bodies. Such accelerated peroxisomal fatty acid oxidation enables ‘young’ cells to maintain low levels of non-esterified fatty acids and diacylglycerol, thereby preventing their premature death by impairing necrosis and lipoapoptosis and by maintaining stress resistance through the attenuation of protein kinase C signaling (Figure 1). Moreover, the efficient Pex5p-dependent import of the citrate synthase Cit2p and acetyl-carnitine synthase Cat2p into peroxisomes of ‘young’ cells—in conjunction with the efficient peroxisomal import of Fox1p, Fox2p, and Fox3p—enhances the life-extending ability of peroxisomes to drive the anti-aging RTG signaling pathway of peroxisomes–mitochondria, mitochondria–nucleus and nucleus–peroxisomes communications (Figure 2). The peroxin Pex11p, a major positive regulator of peroxisome proliferation, amplifies the RTG pathway by increasing the number of peroxisomes carrying out fatty acid oxidation and anaplerotic reactions. In our model, peroxisomes in ‘young’ cells not only efficiently import the protein components constituting a cassette of anti-aging processes but may also function as a signaling platform that maintains ROS concentration at a certain optimal level. This level of ROS is insufficient to damage cellular macromolecules but can activate several redox signaling networks (75–77) that induce stress-response hormesis by increasing the abundance or activity of stress-protecting and other anti-aging proteins.

Our model further posits that, over time, peroxisomes senesce and begin to amass hydrogen peroxide and related ROS. Import of critical proteins is compromised, and an imbalance in peroxisomal (and cellular) ROS homeostasis is created. The organelle is oxidatively damaged, metabolically compromised, and the dynamic communication networks that exist between itself and other organelle systems are corrupted. The ability of the peroxisome to function as a regulator of anti-aging metabolic and signaling reactions is thereby dramatically reduced. As a result, the cell adopts a pro-aging program.

The peroxisome is very clearly integrated into an endomembrane system that governs cellular aging. The challenge for the future is to define the nature of these interactions, the basis of their regulation, and how, perhaps, they can be manipulated pharmaceutically to extend longevity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References

VIT research was supported by grants from the CIHR, NSERC of Canada, Canada Foundation for Innovation, and Concordia University Chair Fund. VIT is a Concordia University Research Chair in Genomics, Cell Biology and Aging. S. R. T. was supported by grants from the National Institutes of Health (USA).

References

  1. Top of page
  2. Abstract
  3. Mechanisms Linking Cellular Aging to Fatty Acid Oxidation and Anaplerotic Reactions in Peroxisomes
  4. Mechanisms Linking Cellular Aging to Peroxisomal ROS Homeostasis
  5. Conclusions and a Model for the Integration of Peroxisomal Processes that Regulate Cellular Aging
  6. Acknowledgments
  7. References