Mini Review

Mitochondria, reactive oxygen species, and chronological aging: A message

from yeast
Yong Pan
Gladstone Institute of Virology and Immunology, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, United States
a b s t r a c t a r t i c l e i n f o
Article history:
Received 1 April 2011
Received in revised form 22 July 2011
Accepted 15 August 2011
Available online 22 August 2011
Section Editor: T.E. Johnson
Keywords:
Chronological lifespan
Mitochondrial theory of aging
Mitohormesis
Hormetic ROS
Oxidative phosphorylation
Target of rapamycin
Reactive oxygen species
Saccharomyces cerevisiae
As a major intracellular source of reactive oxygen species (ROS), mitochondria are involved in aging and life-
span regulation. Using the yeast chronological aging model, researchers have identied conserved signaling
pathways that affect lifespan by modulating mitochondrial functions. Caloric restriction and a genetic mimetic
with reduced target of rapamycin signaling globally upregulate the mitochondrial proteome and respiratory
functions. Recent discoveries support the notion that an altered mitochondrial proteome induces mitohormesis.
Mitohormesis involves a variety of ROS during several growth stages and extends lifespan in yeast and other
organisms. Here we recap recent advances in understanding of ROS as signals that decelerate chronological
aging in yeast. We also discuss parallels between yeast and worm hypoxic signaling. In sum, this mini-review
covers mitochondrial regulation by nutrient-sensing pathways and the complex underlying interactions of
ROS, metabolic pathways, and chronological aging.
2011 Elsevier Inc. All rights reserved.
1. Introduction: Mitochondria and aging
Mitochondria serve many essential functions, but their primary
role is to generate ATP from nutrients. They also participate in signal
transduction, metabolite conversion, and the biosynthesis of organic
molecules. In addition, they are the major intracellular source of reac-
tive oxygen species (ROS), which damage cellular components and
accelerate aging of the organism. Since mitochondrial ATP production
and ROS generation involve oxidative phosphorylation (OXPHOS),
active regulation by conserved signaling pathways is warranted.
Here, we reviewrecent advances in understanding howmitochondria
contribute to aging, with an emphasis on the regulation of OXPHOS
by the target of rapamycin (TOR) pathway and its effects on the chro-
nological lifespan (CLS) of yeast.
The OXPHOS system consists of protein complexes that transfer
high-energy electrons from NADH and FADH2 to molecular oxygen.
Potential energy is harvested to establish a proton gradient across
the inner mitochondrial membrane. This so-called mitochondrial
membrane potential is dissipated through ATP synthase (OXPHOS
Complex V) to generate ATP. Normally, donation of electrons to
oxygen is catalyzed by Complex IV, or cytochrome oxidase. However,
electrons may react prematurely with oxygen in nonenzymatic reac-
tions, yielding ROS.
The mitochondrial theory of aging, rst proposed by Harman, has
garnered support over the years (Harman, 1972). According to Harman,
cellular damage caused by ROS generated in mitochondria is the major
force driving the aging process. Subsequent research conrmed that
mitochondria generate several types of ROS, including superoxide,
hydrogen peroxide, and hydroxyl radicals. Because the chemical prop-
erties of the ROS are different, their preferred cellular targets differ
(D'Autreaux and Toledano, 2007). The highly reactive hydroxyl radical
is indiscriminate. Negatively charged superoxide primarily targets
iron-sulfur clusters, and neutrally charged hydrogen peroxide reacts
preferably with cysteine thiols. ROS also collaborate with metals in
multi-step reactions, yielding high-molecular-weight products, such
as carbonylated protein aggregates (Nystrm, 2005). ROS-induced
damage is complex and frequently irreversible, and it further impairs
mitochondrial function, rendering them prone to further ROS genera-
tion. This positive feed-forward relationship between mitochondrial
ROS generation and damage was hypothesized to be responsible for
oxidative stress and accelerated aging (Harman, 1972).
Because ROS damage cellular components, eukaryotes have evolved
detoxifying enzymes, such as superoxide dismutases and catalases.
Consistent with the mitochondrial theory of aging, transgenic mice
over-expressing catalase in various cellular compartments have a
longer lifespan than controls (Schriner et al., 2005). Interestingly,
most lifespan gain is achieved by catalase over-expressed in mitochon-
dria, conrming that ROS generated in mitochondria are a major
Experimental Gerontology 46 (2011) 847852
E-mail address: yong.pan@gladstone.ucsf.edu.
0531-5565/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.exger.2011.08.007
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lifespan-limiting factor. However, the relationship between detoxica-
tion enzymes and lifespan is non-linear. For example, neither global
reduction nor over-expression of manganese superoxide dismutase
(Sod2) alters lifespan even though such variation of the enzyme levels
signicantly affects resistance against paraquat (Jang et al., 2009; Van
Remmen et al., 2003). These observations highlight a complex relation-
ship between ROS, detoxication enzyme and lifespan in higher organ-
isms. Nevertheless, the reductionist approach to use simpler model
organisms has shed new light on the roles of ROS in aging.
2. Yeast models for the mitochondrial theory of aging
The budding yeast, Saccharomyces cerevisiae, is a well-established
model for studying mitochondrial function and aging. Despite evolu-
tionary divergence from metazoans, multiple molecular pathways that
regulate mitochondria and aging are conserved in yeast and higher
organisms. Unlike many higher organisms, budding yeast can endure
lack of oxygen and do not require mitochondria for ATP production.
Thus, it is an ideal system for exploring mitochondria's important
roles besides energy production. Inglucose-richenvironments, yeast fa-
vors sugar fermentationover respiration, resulting indistinct features of
upregulation of respiratory gene expression upon sugar depletion.
Genomic tools to better understand changes in expression revealed
patterns of yeast mitochondrial proteins during the switch to respira-
tion (i.e., the diauxic shift). By probing yeast expression proles at
different growth stages, Brown and colleagues revealed a major change
in yeast gene expression during the diauxic shift in glucose-based YPD
media (DeRisi et al., 1997). Specically, the tricarboxylic acid (TCA)
cycle was globally upregulated, and glycolysis was globally downregu-
lated, consistent with metabolite data. This expression pattern sug-
gested that the direction of metabolic ow during the post-diauxic
phase is reversed from that during log growth. Consistent with
increased expression of OXPHOS, the respiratory chain regulator HAP4
was one of the two genes encoding transcription factors that are upregu-
lated during diauxic shift.
Two aging models, replicative aging and chronological aging, have
been developed to model different aspects of yeast aging. Replicative
lifespan measures the number of times a yeast mother cell divides
before reaching senescence (Steinkraus et al., 2008). In contrast, CLS
registers the percent viability of yeast culture in the stationary
phase (Fabrizio and Longo, 2007). Using the yeast CLS system,
researchers conrmed the importance of mitochondrial ROS in
aging. As we shall discuss in depth in the following sections, several
genetic mutations inuence ROS levels in various yeast growth stages
and subsequently affect CLS. Like other aging models, ROS detoxifying
enzymes have important but complicated roles in CLS. [Mitochondrial
ROS have equally critical effects on replicative aging, a topic reviewed
in this journal (Ugidos et al., 2010)].
3. Caloric restriction and its genetic mimetics
Among a handful of manipulations that extend lifespan, caloric
restriction (CR) shows high levels of evolutionary conservation. CR,
typically in the forms of less frequent feeding or a reduction in the
quantity or quality of food, aims at inducing under-nutrition without
malnutrition (Mair and Dillin, 2008). In mammals, typical experi-
ments decrease calorie intake by 2050%, which extends median life-
span by 3080%. In yeast, a common CR protocol uses 0.5% glucose
upon inoculation (a 75% reduction in sugar content from standard
media) without alterations in concentrations of other nutrients.
Compared to the standard 2% glucose medium, CR medium strongly
stimulates oxygen consumption (Lin et al., 2002). The increase in
oxygen consumption is recapitulated by HAP4 over-expression. Com-
parison of gene expression proles further revealed that 55 of 124
genes differentially regulated by CR are similarly regulated by HAP4
over-expression. In addition to glucose, restriction of several other
easily fermentable sugars strongly affects CLS (Smith et al., 2007).
Intriguingly, yeast in complex sugar- or non-fermentable carbon
source-based media are longer-lived than that in a glucose-based
medium, but restrictionof these carbonsources leads to more attenuated
or entirely absent CLS extension (Smith et al., 2007). This observation is
consistent with the fact that metabolizing complex sugars or non-
fermentable carbon sources already extensively involves respiration.
Complex sugars and non-fermentable carbon sources, like restriction of
easily fermentable sugars, extend CLS via enhancing mitochondrial
functions.
Manipulation of genetic mimetics of CR produces CLS extension
similar to that of CR. Such mimetics typically reduce evolutionarily
conserved, nutrient-sensing pro-growth signals. Ras and TOR are
two well-studied signaling pathways (Table 1). Reduced activity of ei-
ther pathway by knockout or loss-of-function mutation in Ras or TOR
renders strains longer-lived than wildtype controls (Powers et al.,
2006; Wei et al., 2008). Like yeast under CR, ras2 and tor1 strains
have enhanced resistance to oxidative stress as they enter the station-
ary phase (Wei et al., 2008). Despite different signaling circuitries,
both Ras and TOR inuence key anti-stress regulators, such as
Rim15, Msn2/4, and Gis1. These factors directly or indirectly increase
the levels of detoxication enzymes (e.g., Sod2) and protein chaper-
ones (e.g., heat shock proteins).
Besides regulating stress-resistance genes, Ras directly regulates
mitochondrial respiration and ROS production. Strains over-
expressing constitutively active Ras2
val19
have reduced respiration
(Hlavata et al., 2003). Furthermore, Ras2
val19
engages yeast in an
altered respiratory mode with elevated mitochondrial membrane
potential and failed ATP production. Interestingly, supplementation
with a chemical uncoupler or expression of uncoupling protein
restores respiratory function and reduces ROS accumulation. Evidently,
blocking potential dissipation through ATP synthase increases ROS
production. Not surprisingly, Ras2
val19
-expressing yeast accumulates
excessive oxidative damage products. In agreement with the mitochon-
drial theory of aging, this yeast strain has a much shorter CLS than wild-
type (Fabrizio et al., 2003).
4. TOR signaling and mitochondria
In budding yeast, nutrient-sensing TOR signaling involves TORC1
(Loewith et al., 2002). TORC1 is a well-conserved complex in evolu-
tion, with yeast and mammals sharing homology in its constituents.
In yeast or mammalian systems, TORC1 is specically inhibited by
rapamycin. Furthermore, yeast TORC1 directly phosphorylates
Sch9p, a yeast homolog of ribosomal S6 kinase 1 (S6K1), the target
of mammalian TORC1 (Urban et al., 2007). Two kinases, Tor1p and
Tor2p, constitute TORC1 in yeast; Tor1p is not essential for viability,
although it is present in the complex. For this reason, deletions of
TOR1 (tor1) have been generated to study the effects of reduced
TORC1 signaling.
Like the Ras pathway, the TOR pathway regulates mitochondria
and controls CLS. Tor1 was identied in a genetic screen as a long-
lived mutant (Powers et al., 2006). CLS extension by deletion of TOR1
was recapitulated by treatment of wildtype yeast withrapamycin. Dele-
tion of TOR1 increases respiratory activity and expression of OXPHOS
proteins (Bonawitz et al., 2007). Enhanced respiration is required for
CLS extension in tor1yeast, since CLS in wildtype and tor1is indistin-
guishable in respiration-incompetent cells. The necessity of respiration
in CLS extension is conrmed using strains with reduced respiration.
Over-expression of Puf3, a suppressor of mitochondrial ribosomal bio-
genesis, reduces but does not eliminate respiration (Chatenay-Lapointe
and Shadel, 2011). The over-expression partially abolishes lifespan
extension by deletion of TOR1. Similar to tor1, increased respiration
and extended CLS were found in yeast devoid of SCH9, the major medi-
ator of the regulation of expression of OXPHOS proteins and CLS by
TORC1 (Lavoie and Whiteway, 2008; Pan and Shadel, 2009). Although
848 Y. Pan / Experimental Gerontology 46 (2011) 847852
enhanced respiration is required for CLS extension, it alone is insuf-
cient. Deletion of PUF3 increases mitochondrial translation and
oxygen consumption in both wildtype and tor1 without changes
in CLS (Chatenay-Lapointe and Shadel, 2011).
Genomic approaches have been used to study TOR-regulated tran-
scriptional change. For instance, in rapamycin-treated yeast cultures,
glycolytic enzymes are downregulated and TCA cycle proteins are upre-
gulated, as during the diauxic shift (Hardwick et al., 1999). Similarly,
reduced TOR signaling increases respiration during exponential growth
and extends CLS, and microarray analyses of sch9 strains during expo-
nential growth in SD medium conrmed that many mitochondrial
electron transport components are upregulated (Lavoie and Whiteway,
2008). However, another microarray analysis of sch9strains at various
growth stages showed downregulation of many mitochondria-
associated genes (Wei et al., 2008). These discrepancies suggest that
growth stage and strain specicity contribute to gene expression and
indicate that other complementary methods are needed to better
understand how TOR regulates the OXPHOS system.
Since mRNA abundance does not always reect protein abundance,
proteomic studies have been conducted. The Grifn group treated yeast
with rapamycin during the robust growth phase (Bandhakavi et al.,
2008). Among more than 100 differentially expressed proteins, the
OXPHOS units Atp15p, Inh1p, Qcr2p, and Qcr7p were upregulated.
Putative regulators of mitochondrial gene expression and DNA replica-
tion, including Mnp1p, Abf2p, and Pet10p, were also upregulated.
Similarly, Schreiber and colleagues combined chemical genetics and
genomic and proteomic tools to study growth inhibition by rapamycin
(Huang et al., 2004). They identied small molecules that reverse the
effects of rapamycin on cells and discovered a new TOR signaling com-
ponent, Nir1p. To compare the mitochondrial proteomes of wildtype
and tor1, two-dimensional difference gel electrophoresis was used
(Pan and Shadel, 2009). The result conrmed the global upregulation
of OXPHOS components in tor1 yeast.
The regulation of mitochondrial function and lifespan by the TOR
pathway is remarkable (Table 1), as knockout of a single gene, such
as TOR1 or SCH9, leads to a well-orchestrated reconguration of
metabolism and extension of CLS. The global upregulation of OXPHOS
is a complex process involving both the mitochondrial and nuclear
genomes. Coordinating the two genomes is paramount, as imbalanced
OXPHOS is prone to extreme ROS generation. A master regulator, in
the form of transcription or translation factors, may act downstream
of TOR1/SCH9 to ensure the proper global upregulation. Sch9 may
directly phosphorylate transcription factors such as Hap4, which acti-
vates OXPHOS biogenesis. Knockout of Hap4 reduces the increase in
cytochrome c expression in sch9 strains (Lavoie and Whiteway,
2008). However, evidence of a direct physical interaction between
Hap4 and Sch9 will be required to establish Hap4 modication by Sch9.
An intriguing alternative to an activator is the preferential translation
of mRNAs. With reduced TOR signaling, translation rates do not decrease
equally for all mRNAs. A well-characterized example is the regulation of
translation by translation initiation factor eIF4E (Koromilas et al., 1992).
Under physiological conditions, the supply of eIF4E is limited, and
mRNAs must compete for it. Transcripts with complex 5 UTRs rely
heavily on eIF4E for effective translation. Nutrient scarcity alters the
translation prole, favoring mRNAs with simple 5 structures.
Nucleus-encoded mitochondrial proteins in Drosophila share a simple
5 UTR, and their expression is increased upon TOR inactivation (Zid
et al., 2009). The same mechanism may apply to regulation of mito-
chondrial biogenesis by TOR in yeast. Reduced protein translation was
recently identied to extend yeast replicative lifespan (Steffen et al.,
2008). Deletion of several 60S subunit-encoding genes extends lifespan
in a Gcn4-dependent manner. Whether disruption of the same array of
genes changes expression of OXPHOS proteins and CLS remains an un-
answered but intriguing question.
5. Mitohormesis: That which does not kill yeast makes it long-lived
How do alterations in expression of OXPHOS proteins translate into
extended lifespan? Increasing evidence suggests that mitohormesis is a
contributor, pointing the mitochondrial theory of aging in a new direc-
tion. Hormesis, a phenomenon by which a low dose of toxic stimulus
induces resistance against ensuing stress of similar nature, has a well-
known role in aging. Worms exhibit extended lifespan in response to
a wide variety of environmental stress including heat, hyperbaric
oxygen and a ROS generator (Cypser and Johnson, 2002). Built on hor-
metic extension of lifespan, the mitohormesis theory posits that ROS
production by mitochondria under glucose restriction enhances stress
resistance, thereby extending lifespan (Schulz et al., 2007). The authors
established that lifespan extension by CR in worms requires oxidative
hormetic signals. The respiratory inhibitor azide and the superoxide
inducer paraquat both generate CR-like lifespan extension, which is
eliminated by antioxidants. Similarly, disruption of Complex I subunit
NUO-1 and Complex III subunit ISP-1 induces production of ROS
(Yang and Hekimi, 2010). In particular, both nuo-1 and isp-1 mutations
induce mitochondrial superoxide without elevating overall ROS, indi-
cating that superoxide serves as a hormetic ROS signal in these mutant
strains (Yang and Hekimi, 2010). In agreement with superoxide's signal-
ing role, pro-oxidant paraquat mimics lifespanextensioninnuo-1 andisp-
1 worms while anti-oxidant n-acetyl cystein (NAC) abolishes lifespan ex-
tension in these mutants.
Mitohormesis is conserved as a lifespan-extending mechanism in
yeast. CR in yeast increases hydrogen peroxide in the stationary
phase (Mesquita et al., 2010). A high peroxide level stimulates Sod
activity and thus extends CLS. In agreement with mitohormesis, CLS
is extended by deletion of peroxisomal or cytosolic catalase and
shortened by its over-expression (Mesquita et al., 2010). In tor1
yeast by comparison, superoxide generated during growth serves as
the hormetic signal (Pan et al., 2011). Reduction of TOR signaling
during growth promotes coupled respiration and boosts OXPHOS elec-
tron ow, thereby temporarily elevating mitochondrial membrane
Table 1
Cited yeast genes that alter cellular ROS levels and chronological lifespan.
Genes Mammalian orthologue Manipulation Phenotype Reference
TOR1 mTOR Knock-out Mild increase in superoxide during growth;
lower ROS in stationary phase. Extended lifespan
Bonawitz et al., 2007; Pan et al., 2011
SCH9 S6K Knock-out Lower ROS in stationary phase. Extended lifespan Lavoie and Whiteway, 2008;
Pan and Shadel, 2009
RAS2 RAS Knock-out Reduced damage to mitochondrial aconitase;
enhanced resistance to paraquat. Extended lifespan
Fabrizio et al., 2003
RAS2 RAS Expression of constitutive
val19 allele
Reduced respiration; failed mitochondrial ATP production;
high ROS. Shortened lifespan
Hlavata et al., 2003; Fabrizio et al., 2003
SOD2 SOD2 Knock-out No major impact on lifespan of wildtype;
reverted lifespan extension by SCH9 deletion.
Fabrizio et al., 2003
HAP4 n.a. Knock-out Reduced oxygen consumption;
Reverted lifespan extension by SCH9 deletion
Lavoie and Whiteway, 2008
849 Y. Pan / Experimental Gerontology 46 (2011) 847852
potential and enhancing superoxide generation. The superoxide
peak suppresses ROS production in stationary phase and prolongs
CLS (Pan et al., 2011). In agreement with superoxide's hormetic
effects, superoxide inducer menadione during growth effectively
prolongs CLS while expression of SOD2 prevents a full lifespan
extension.
The apparent negative roles of ROS detoxication enzymes in the
last two examples are noteworthy. These observations, although super-
cially contradicting Harman's mitochondrial theory of aging, provide
an explanation on non-linearity between levels of anti-oxidant en-
zymes and lifespan. In yeast, Longo and colleagues observed little life-
span change by either over-expression or knock-out of Sod2 (Fabrizio
et al., 2003). Only in combination with over-expression of Sod1 do
high levels of Sod2 extend CLS. Perhaps, the benets of low-level super-
oxide dismutase in tackling stress resistance are canceled out by the
enzymes' negative roles in initiating hormesis. The dichotomy of detox-
icationenzymes ininuencing lifespanmay alsoexplainwhy inhigher
organisms Sod2 levels can vary by fourfold without dramatic change in
lifespan (Jang et al., 2009; Van Remmen et al., 2003).
Intriguingly, the aforementioned yeast studies entertain the idea
that different hormetic ROS affect CLS through effects on distinct phases
of the yeast life cycle (Fig. 1). While superoxide induces hormesis dur-
ing growth, hydrogen peroxide does so in stationary phase. A critical
next question to ask is whether and where the two hormetic ROS con-
verge upon the same signaling pathway to promote CLS. In the hormetic
superoxide example, the adaptive phase (when mitochondrial ROS sig-
nals are initiated) and the effective phases (when death occurs) appear
to be separable (Fig. 1). Early alterations of mitochondrial functions in
yeast and worms inuence survival in stationary phase and adulthood
respectively. Respiratory carbon sources, such as glycerol, during
growth pre-adapt yeast and extend its lifespan (Piper et al., 2006).
Even though during the stationary phase the culture is switched to
water, effects on lifespan extension persist. Similarly in worms, a
reduction in expression of OXPHOS proteins during robust growth can
signicantly extend lifespan (Dillin et al., 2002). In contrast, a similar
alteration in adulthood has no effect on lifespan. This work, along
with Hekimi's study (Yang and Hekimi, 2010), implies that
superoxide has an adaptive window during growth to affect worm
lifespan. The lifespan extension by enhanced respiration in yeast and
by reduced respiration in worm highlights that hormetic superoxide,
not oxygen consumption per se, is responsible for lifespan extension.
The examples also illustrate that separation between the adaptive and
effective phases may be evolutionarily conserved. Possibly during
growth, organisms have abundant resources available to ration
between pro-growth and pro-longevity pathways.
Hormetic ROS signals may account for the lifespan extension asso-
ciated with hypoxia. As hypoxia promotes ROS production, one would
predict that hypoxia has the same effect on CLS as ROS. Indeed, during
log growth and early stationary phase, hypoxia extends lifespan, and
hypoxic treatment deep into the stationary phase shortens lifespan
(Bonawitz et al., 2007). The effects of hypoxia highly resemble that
of superoxide inducer menadione with similar treatment windows
(Pan et al., 2011). Furthermore, hypoxia signicantly extends the
CLS of wildtype, but has limited effects on already long-lived tor1.
These observations can be explained by shared hormetic superoxide
during growth in both TOR1 deletion and hypoxia.
Hypoxia extension of yeast CLS nds a mechanistic parallel in
Caenorhabditis elegans. As discussed in preceding paragraphs, nuo-1
and isp-1 produce hormetic superoxide, thereby extending wormlife-
span (Yang and Hekimi, 2010). Kenyon and colleagues further identify
the hypoxia-inducible factor (HIF-1) as the convergence point of
hormetic ROS and hypoxic signals (Lee et al., 2010). A loss-of-function
mutant allele or RNAi knock-down of HIF-1 decreases the lifespan of
the long lived isp-1 mutant, but has no inuence over that of wildtype.
Likewise, superoxide inducer paraquat extends wormlifespan, which is
partially abolished by HIF-1 mutation. Although RNAi against respirato-
ry chain prolongs lifespan only if knockdown is initiated at the larval
stages (Dillin et al., 2002), HIF-1 function is required through adulthood
to ensure lifespan extension (Lee et al., 2010; Mehta et al., 2009). While
consistent with the proposed model, the worm data suggest the
Fig. 1. Conceptual model of yeast ROS signaling and CLS. The blue solid line in the middle designates the articial boundary of adaptive and effective phases. Three representative
cellular ROS scenarios of the adaptive phase are depicted in different colors on the left. Block arrows point to corresponding cellular ROS levels and their impacts on oxidative dam-
age and CLS in the effective phase on the right. Flow charts depict signaling events responsible for respective ROS scenarios and changes of cellular ROS levels between the adaptive
and effective stages: constitutively active nutrient sensors or loss of key stress-resistant pathways cause high ROS in adaptive phase, thereby initiating a vicious cycle of ROS pro-
duction (top left); nutrient sensor with reduced activity, non-fermentable media or probably hypoxia promotes a hormetic response (middle left); wildtype, grown in fermentable
medium, lacks hormetic response (bottom left).
850 Y. Pan / Experimental Gerontology 46 (2011) 847852
requirement of active signaling beyond the adaptive phase to maintain
hormesis. In yeast, HIF-1-like factors that mediate hypoxic and hormetic
ROS signaling remain to be identied. In the media pre-adaption example
coveredpreviously, Piper andcolleagues showedthe de-repressionof res-
piration and subsequent lifespan extension can be recapitulated by over-
expression of Hap4 (Piper et al., 2006). The facts that HAP4 resides down-
stream of TOR1 and SCH9 in regulating respiration and lifespan and that
Hap4 contains an oxygen-sensing heme prosthetic group also make it
an appealing candidate of HIF-1 functional analog in yeast.
6. Metabolism and lifespan: Toward a comprehensive picture
Considerable evidence indicates that TOR has pleiotropic effects on
metabolism. In addition to regulating mitochondria, TOR signaling like-
ly regulates other energy production pathways that share metabolic in-
termediates with mitochondrial OXPHOS. Proteomic experiments
suggest that rapamycin represses glycolysis (Hardwick et al., 1999).
The identication of acetic acid as a major contributor to chronological
aging points to the fact that metabolites produced by glycolytic and
other pathways directly affect CLS (Burtner et al., 2009). Medium acid-
ity, as a result of acetic acid production, decreases CLS. Under CR, yeast
cultures produce substantially less acetic acid and are thus exposed a
higher media pH. Long-lived strains, such as sch9, have greater resis-
tance to acetic acid. Elevated acidity increases intracellular ROS levels
in stationary phase, suggesting a close link between metabolites and
mitochondrial functions (Pan et al., 2011). Despite the shared feature
of low ROS in stationary, reduction of acetic acid likely acts indepen-
dently of mitohormesis in promoting CLS. Neutralization or swapping
of media has minimal impact on oxygen consumption during growth
and early stationary phase (Pan et al., 2011). Still, toxic metabolites
likely contribute to a vicious cycle of ROS production in late stationary
phase.
The development of metabolomics adds newtools to achieve a com-
prehensive understanding of metabolism and lifespan. By analyzing
long-lived genetic mutants and longevity-promoting culture condi-
tions, Fukusaki and colleagues identied 87 yeast metabolites associat-
ed with longer replicative lifespan (Yoshida et al., 2010). Enrichment in
the glycolytic intermediates 3-phosphoglycerate and phosphoenol py-
ruvate correlates negatively with lifespan, whereas abundance of the
TCA cycle intermediates malate, succinate, citrate, and fumarate corre-
lates positively with lifespan. The effects of these metabolites on CLS re-
main to be determined. Given the fundamentally different nutritional
environments replicative lifespan and chronological lifespan assays
provide, one should be cautious about jumping to conclusion on a
metabolite's effects on aging (Steinkraus et al., 2008). Nonetheless,
the availability of metabolomic tools allows examination of new
unknowns in CLS. For example, howdo environmental and intracellular
stresses inuence each other and how do they affect various signaling
pathways? Do alterations of other metabolic pathways also possess
adaptive windows as does TOR regulation of mitochondria? The
answers to these questions will help better understand how events
early in life inuence the aging process.
7. Summary
The principles of mitochondrial theory of aging are supported by
decades of experiments, and great progress has been made in reveal-
ing the roles of mitochondria. Past studies established detoxication
of ROS as a major regulatory mechanism by mitochondria regulate
the aging process. Using yeast CLS and other models, researchers
found that several molecular events, such as hypoxia and hormetic
ROS, have an evolutionarily conserved effects on aging. Long before
damage by ROS becomes lethal, cellular signaling is affected either
ensuring better detoxication or initiating events that exacerbate
ROS damage (Fig. 1). Unlike Harman's initial model, genetic factors
play an active role in modulating the production and dissipation of
ROS, and thus aging. Although the molecular picture is far more com-
plex than envisioned by Harman, the mitochondrial theory of aging
remains amazingly resilient and robust even to this day.
Acknowledgments
The author thanks Dr. Gerald Shadel for his support. Part of this
work was supported by grant P01ES-011163 awarded to G.S. The
author would like to acknowledge Dr. Carl Hashimoto, Dr. Sherman
Weissman, Dr. Wenjuan He, Dr. Philip Merksamer, Margaret Wang,
Stephen Ordway and Gary Howard for editorial assistance and con-
structive suggestions. The author would also like to thank Dr. Eric
Verdin for insightful discussions on various topics on aging.
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