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Regulation of Longevity and Stress Resistance by Sch9 in Yeast


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The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans. We screened for long-lived mutants in nondividing yeastSaccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold. Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension. These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.
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DOI: 10.1126/science.1059497
, 288 (2001); 292Science
et al.Paola Fabrizio,
Sch9 in Yeast
Regulation of Longevity and Stress Resistance by (this information is current as of January 13, 2009 ):
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Regulation of Longevity and
Stress Resistance by Sch9 in
Paola Fabrizio,
Fabiola Pozza,
Scott D. Pletcher,
Christi M. Gendron,
Valter D. Longo
The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling
in mammals and functions in a pathway that regulates longevity and stress
resistance in Caenorhabditis elegans. We screened for long-lived mutants in
nondividing yeast Saccharomyces cerevisiae and identified mutations in ade-
nylate cyclase and SCH9, which is homologous to Akt/PKB, that increase re-
sistance to oxidants and extend life-span by up to threefold. Stress-resistance
transcription factors Msn2/Msn4 and protein kinase Rim15 were required for
this life-span extension. These results indicate that longevity is associated with
increased investment in maintenance and show that highly conserved genes
play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.
Mutations that extend life-span in C. elegans,
Drosophila melanogaster, and mice are asso-
ciated with increased resistance to oxidative
stress (1, 2). However, the mechanisms that
regulate aging in these multicellular organ-
isms are poorly understood. As in higher
eukaryotes, the unicellular yeast Saccharo-
myces cerevisiae undergoes an age-depen-
dent increase in cell dysfunction and mortal-
ity rates (3, 4 ). Aging in yeast is associated
with an enlargement of the cell and a slowing
in the budding rate, and is commonly mea-
sured by counting the number of buds gener-
ated by a single mother cell (replicative life-
span) (5, 6 ). The replicative life-span of yeast
is regulated by the Sir2 protein, which medi-
ates chromatin silencing in a nicotinamide
adenine dinucleotide– dependent manner (6,
7). However, yeast can also age chronologi-
cally as a population of nondividing cells (2,
4, 6). Saccharomyces cerevisiae grown in
complete glucose medium [synthetic com-
plete (SC) medium] stop dividing after 24 to
48 hours and survive for 5 to 7 days while
maintaining high metabolic rates (2, 8, 9), a
situation more akin to their experience in
nature where they are likely to survive as
nondividing populations exposed to scarce
nutrients. For these reasons, and to avoid
extended growth and entry into the hypo-
metabolic stationary phase induced by incu-
bation in the nutrient-richer yeast extract/
peptone/dextrose (YPD) medium (10), our
studies were performed exclusively in SC
medium. The survival of nondividing yeast is
shortened by null mutations in either or both
superoxide dismutases (SODs) (2, 11, 12)
and is modestly extended by overexpressing
the antiapoptotic protein Bcl-2 (8).
To understand the molecular mechanism
that regulates yeast longevity, we transposon-
mutagenized yeast cells and isolated long-lived
mutants (13). Because of the association be-
tween stress resistance and longevity in higher
eukaryotes, we screened for mutants that sur-
vived both a 1-hour heat stress at 52°C and a
9-day treatment with the superoxide-generating
agent paraquat (1 mM). From 2 billion cells
screened, we isolated 4000 thermotolerant col-
onies and 40 paraquat-resistant colonies carry-
ing transposons. From the 4040 stress-resistant
mutants, we isolated nine that were able to
survive to day 9, when most of the wild-type
cells are dead. The only two long-lived mutants
isolated independently in both the paraquat and
heat shock selections, designated Tn3-5 and
Tn3-24, were also the longest lived (Fig. 1A),
suggesting that resistance to multiple stresses is
associated with increased longevity. Allele res-
cue of the mutants revealed that transposons
had integrated in the promoter region of the
Sch9 protein kinase gene (sch9::mTn) (Tn3-5)
(33 base pairs upstream of the start codon) and
in the NH
-terminal regulatory region of ade-
nylate cyclase (cyr1::mTn) (Tn3-24) (between
codon 208 and 209). The mean life-spans of
sch9::mTn and cyr1::mTn were extended by 30
and 90%, respectively. Transformation of
Tn3-5 cells with wild-type SCH9, and of Tn3-
24 cells with CYR1, abolished the survival ex-
tension, strongly suggesting that the decreased
expression or activity of Sch9 and Cyr1 extends
survival (not shown).
To investigate further the role of SCH9 in
chronological survival, we deleted the SCH9
gene (14). The sch9 mutants grew slowly, but
survived three times longer than wild-type cells
(Fig. 1B). To determine whether the protein
kinase activity of Sch9 accelerates mortality in
nondividing yeast, we transformed mutants
with either wild-type SCH9 or with forms of
SCH9 bearing kinase-inactivating mutations:
and sch9
(15). Transformation
of sch9 with wild-type SCH9 reversed the
life-span extension, whereas transformation
Division of Biogerontology, Andrus Gerontology
Center, and Department of Biological Sciences, Uni-
versity of Southern California, Los Angeles, CA
90089 0191, USA.
Max Planck Institute for Demo-
graphic Research, Rostock, 18057 Germany.
*To whom correspondence should be addressed. E-
Fig. 1. Mutations in CYR1 and in SCH9
increase chronological life-span of S.
cerevisiae. Survival of (A) the wild
type (DBY746), and transposon-mu-
tagenized cyr1::mTn (Tn3-24) and
sch9::mTn (Tn3-5); (B) the wild type
and sch9;(C) sch9 transformed
with vector alone wild-type SCH9
or with a mutated sch9 encoding
for a catalytically inactive proteins
, Sch9
). Cell viability
was measured every 2 days starting at
day3(14). Experiments were repeat-
ed between three and seven times
with two or more samples per exper-
iment with similar results. The aver-
age of all experiments is shown. The
mean life-span increase in cyr1::mTn
(90%), sch9::mTn (30%), and sch9
(300%) is significant [P 0.05, anal-
ysis of variance (ANOVA)].
13 APRIL 2001 VOL 292 SCIENCE www.sciencemag.org288
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with the genes encoding for the inactive
or Sch9
kinases did not (Fig.
Both Sch9 and Cyr1 function in path-
ways that mediate glucose-dependent sig-
naling, stimulate growth and glycolysis,
and decrease stress resistance, glycogen ac-
cumulation, and gluconeogenesis (16). The
COOH-terminal region of Sch9 is highly
homologous to the AGC family of serine/
threonine kinases, which includes Akt/
PKB, whereas the NH
-terminal region
contains a C2 phospholipid and calcium-
binding motif. The 327–amino acid serine/
threonine kinase domain of yeast Sch9 is,
respectively, 47 and 45% identical to that
of C. elegans AKT-2 and AKT-1, which
function downstream of the insulin-recep-
tor homolog DAF-2 in a longevity/diapause
regulatory pathway (14, 17, 18). In this
domain conserved from yeast to mammals,
Sch9 is also 49% identical to human AKT-
1/AKT-2/PKB, which are implicated in bi-
ological functions including insulin signal-
ing, the translocation of glucose transport-
er, apoptosis, and cellular proliferation
The CYR1 gene encodes for adenylate
cyclase, which stimulates cyclic adenosine
monophosphate (cAMP)–dependent protein
kinase (PKA) activity required for cell cycle
progression and growth. The catalytic sub-
units of PKA are also 35 to 42% identical to
C. elegans and human AKT-1/AKT-2, al-
though PKA belongs to a different family of
serine/threonine kinase. The inactivation of
the Ras/cAMP/PKA pathway in S. cerevisiae
increases resistance to thermal stress, in part,
by activating transcription factors Msn2 and
Msn4, which induce the expression of genes
encoding for several heat shock proteins,
catalase (CTT1), and the DNA damage induc-
ible gene DDR2 (14, 16). MnSOD also ap-
pears to be regulated in a similar manner (20).
To determine whether MSN2/MSN4 mediate
survival extension, we deleted both genes in
the cyr1::mTn mutants. The absence of both
transcription factors abolished the life-span
extension conferred by cyr1::mTn, but did
not affect the survival of wild-type cells (Fig.
2A). By contrast, the deletion of MSN2/
MSN4 did not reverse the survival extension
in sch9 cells (Fig. 2B).
The protein kinase Rim15 regulates genes
containing a PDS ( postdiauxic shift) element
AT involved in the induction of
thermotolerance and starvation resistance by
a Msn2/Msn4-independent mechanism (21).
To test the role of Rim15 in survival, we
generated sch9 rim15 mutants. The life-
span of the double mutant was decreased
compared to sch9 (Fig. 2B). The deletion of
RIM15 also abolished the life-span extension
in cyr1::mTn cells (Fig. 2A). However, it is
difficult to establish whether Rim15 mediates
the survival extension in these mutants, be-
cause rim15 single mutants are short-lived
(Fig. 2A).
To test whether the long-lived strains
were stress-resistant, we exposed the mutants
to hydrogen peroxide, menadione, or heat.
All mutants were resistant to a 1-hour heat
shock treatment at 55°C (Fig. 3A). Similarly,
3- to 5-day-old mutants were resistant to a
30-min treatment with 100 mM hydrogen
peroxide (Fig. 3B) or with the superoxide/
-generating agent menadione (20 M)
(Fig. 3C).
In yeast sod2 mutants, superoxide specif-
ically inactivates aconitase and other [4Fe-4S]
cluster enzymes and causes the loss of mito-
chondrial function and cell death (11, 12). To
investigate further the role of superoxide toxic-
ity in aging, we monitored the activity and
reactivation of mitochondrial aconitase, which
can also serve as an indirect measure of super-
oxide concentration (22). In agreement with the
pattern of resistance to superoxide toxicity (Fig.
3C), aconitase specific activity decreased by
50% in wild-type cells, and by 30% in
cyr1::mTn mutants, but did not decrease in
sch9::mTn and sch9 mutants at day 7 com-
pared to day 3 (14 ). The percent reactivation of
aconitase was lowest in the long-lived sch9
mutants and highest in wild-type cells (Fig. 4A)
and correlated with death rates (Fig. 4B), sug-
gesting that cyr1 and sch9 mutants increase
survival, in part, by preventing superoxide tox-
icity. However, the overexpression of both
SOD1 and SOD2 only increases survival by
30% (9), indicating that additional systems, reg-
ulated by Msn2, Msn4, and Rim15, are respon-
sible for the major portion of chronological
life-span extension in cyr1::mTn and sch9
There are many phenotypic similarities
between long-lived mutants in S. cerevisiae,
C. elegans, Drosophila, and mice (1, 2). Cae-
Fig. 2. Transcription factors Msn2,
Msn4, and protein kinase Rim15 are
required for the chronological life-
span extension of cyr1::mTn and
sch9 mutants. (A) Survival of the
wild type and cyr1::mTn mutants lack-
ing either the stress-resistance genes
MSN2/MSN4 or RIM15.(B) Survival of
the wild type and sch9 mutants
lacking either MSN2/MSN4 or RIM15.
Experiments were repeated between
three and seven times with two or
more samples per experiment with
similar results. The average of all ex-
periments is shown.
Fig. 3. Heat-shock and oxidative stress resis-
tance are increased in long-lived mutants. (A)
Serial dilutions (1:1 to 1:1000, left to right) of
cells removed from day 1 postdiauxic phase
cultures were spotted onto YPD plates and
incubated at 30°C (control) or 55°C (heat-
shocked) for 1 hour. Pictures were taken after a
4-day incubation at 30°C. The experiment was
performed twice with two or more samples per
experiment with similar results. Cells removed
from days 3 or 5 in the postdiauxic phase were
(B) diluted to an OD
(optical density at 600
nm) of 1 in expired medium and incubated with
hydrogen peroxide (100 mM) for 30 min or (C)
diluted to an OD
of 0.1 in potassium phos-
phate buffer and treated with 20 Mofthe
-generating agent menadione
for 60 min. Viability was measured by plating
cells onto YPD plates after the treatment. The
experiments were performed twice with similar
results. The average of the two experiments is
on January 13, 2009 www.sciencemag.orgDownloaded from
norhabditis elegans age-1 and daf-2 mutations
extend the life-span in adult organisms by 65 to
100%, by decreasing AKT-1/AKT2 signaling
and activating transcription factor DAF-16 (14,
18, 23). These changes are associated with the
induction of superoxide dismutase (MnSOD),
catalase, and the heat shock proteins HSP70
and HSP90 (14, 17). A role for oxidants in the
aging of C. elegans was confirmed by the ex-
tended survival of wild-type worms treated
with small synthetic SOD/catalase mimetics
(24). Thus, the yeast Gpr1/Cyr1/PKA/Msn2/4-
Sch9/Rim15 and the C. elegans DAF-2/AGE-
1/AKT/DAF16 pathways play similar roles in
regulating longevity and stress resistance (14 ).
Analogously, a Drosophila line with a mutation
in the heterotrimeric guanosine triphosphate–
binding protein (G protein)–coupled receptor
homolog MTH gene displays a 35% increase in
life-span and is resistant to starvation and para-
quat toxicity (25). Furthermore, in flies,
aconitase undergoes age-dependent oxidation
and inactivation (26), and the overexpression
of SOD1 increases survival by up to 40% (27,
28). A mutation in a signal-transduction gene
also increases resistance to stress and length-
ens survival in mammals. A knockout muta-
tion in the signal transduction p66
increases resistance to paraquat and hydrogen
peroxide and extends survival by 30% in
mice (29).
We propose that yeast Sch9 and PKA and
worm AKT-1/AKT-2 evolved from common
ancestors that regulated metabolism, stress re-
sistance, and longevity in order to overcome
periods of starvation. Analogous mechanisms
triggered by low nutrients may be responsible
for the extended longevity of dietary restricted
rodents (3). The phenotypic similarities of long-
lived mutants ranging from yeast to mice (1, 2),
and the role of the conserved yeast Sch9 and
PKA and mammalian Akt/PKB in glucose me-
tabolism, raise the possibility that the funda-
mental mechanism of aging may be conserved
from yeast to humans.
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1 February 2001; accepted 15 March 2001
Published online 5 April 2001;
Include this information when citing this paper.
Functional Specialization in
Rhesus Monkey Auditory Cortex
Biao Tian, David Reser, Amy Durham, Alexander Kustov,
Josef P. Rauschecker*
Neurons in the lateral belt areas of rhesus monkey auditory cortex prefer complex
sounds to pure tones, but functional specializations of these multiple maps in the
superior temporal region have not been determined. We tested the specificity
of neurons in the lateral belt with species-specific communication calls pre-
sented at different azimuth positions. We found that neurons in the anterior
belt are more selective for the type of call, whereas neurons in the caudal belt
consistently show the greatest spatial selectivity. These results suggest that
cortical processing of auditory spatial and pattern information is performed in
specialized streams rather than one homogeneously distributed system.
Hearing plays a dual role in the identification
of sounds and in their localization. Although
it is undisputed that auditory cortex partici-
pates in the analysis of spectro-temporal pat-
terns for the identification of complex sound
objects, including speech and music, the neu-
ral basis of auditory spatial perception re-
mains a matter of controversy. Brainstem-
structures play a significant role in the pro-
cessing of binaural cues, which contain im-
portant information for sound localization
(1). However, lesions of auditory cortex also
impair auditory spatial analysis (2, 3). With
the recent discovery of multiple cochleotopic
maps in nonprimary auditory cortex of the
rhesus monkey (4, 5), the question arises
whether neurons in some of these areas show
greater specificity for sound source location
than in others. This could indicate the exis-
tence of a specialized cortical stream for the
processing of auditory space, similar to what
Georgetown Institute for Cognitive and Computa-
tional Sciences, Department of Physiology and Bio-
physics, Georgetown University Medical Center,
Washington, DC 20007, USA.
*To whom correspondence should be addressed. E-
Fig. 4. Mutations in cyr1
and sch9 delay the re-
versible inactivation of
the superoxide-sensitive
enzyme aconitase in the
mitochondria. (A) Mito-
chondrial aconitase per-
cent reactivation after
treatment of whole-cell
extracts of yeast re-
moved from cultures at
day 5 through 7 with
agents (iron and Na
S) able to reactivate superoxide inactivated [4Fe-4S] clusters. (B) Death rate
reported as the fraction of cells that lose viability in the 24-hour period following the indicated day.
13 APRIL 2001 VOL 292 SCIENCE www.sciencemag.org290
on January 13, 2009 www.sciencemag.orgDownloaded from
... The effect of the deletion of the gene coding for the Ras2 protein on longevity extension and stress resistance allowed the identification of Ras2 as one of the very first longevity regulatory genes in any organism [28]. RAS2 deletion doubles CLS and increases the resistance to heat and oxidative stresses [29,30] (Figure 1). Ras2 codes for one of the two monomeric G-proteins that are capable, when GTP-bound, of stimulating adenylate cyclase, which in turn activates PKA. ...
... Ras2 codes for one of the two monomeric G-proteins that are capable, when GTP-bound, of stimulating adenylate cyclase, which in turn activates PKA. The involvement of PKA as a pro-aging pathway was further confirmed by the observation that mutants with impaired synthesis of cAMP were also long-lived [29]. The Ras pathway appears to have a pro-aging role conserved from yeast to mammals, since in mice maternal uniparental disomy of the Ras exchange factor coding gene RasGRF (which is inactivated during oogenesis) has been associated with increased longevity [30], a hypothesis confirmed by the lifespan extension in mice carrying homozygous deletion of the gene coding for this nucleotide exchange factor [31]. ...
... The role of this pathway in regulating aging has been since confirmed in worms and flies [41,42]. Inhibition of Tor-S6k activity increases both chronological and replicative lifespan [29,35,43] (Figure 1). Decreased Tor signaling increases respiration [44] as well as mitochondrial protein expression [45], and reactive oxygen species are also increased, likely activating an adaptive response that extends CLS. ...
Full-text available
S. cerevisiae plays a pivotal role as a model system in understanding the biochemistry and molecular biology of mammals including humans. A considerable portion of our knowledge on the genes and pathways involved in cellular growth, resistance to toxic agents, and death has in fact been generated using this model organism. The yeast chronological lifespan (CLS) is a paradigm to study age-dependent damage and longevity. In combination with powerful genetic screening and high throughput technologies, the CLS has allowed the identification of longevity genes and pathways but has also introduced a unicellular “test tube” model system to identify and study macromolecular and cellular damage leading to diseases. In addition, it has played an important role in studying the nutrients and dietary regimens capable of affecting stress resistance and longevity and allowing the characterization of aging regulatory networks. The parallel description of the pro-aging roles of homologs of RAS, S6 kinase, adenylate cyclase, and Tor in yeast and in higher eukaryotes in S. cerevisiae chronological survival studies is valuable to understand human aging and disease. Here we review work on the S. cerevisiae chronological lifespan with a focus on the genes regulating age-dependent macromolecular damage and longevity extension.
... The Workman group showed that H3T11 was phosphorylated by both Sch9 and Cka1, and reported that Δsch9 and Δcka1 as well as H3T11A prolonged CL [20]. Although this seems opposite to our result where the phosphorylated mimic H3T11D showed CLE, we note that the Δsch9 mutant strain will facilitate activated expression of Msn2/4 stress response genes associated with CLE [42], and it is not clear if the CLE observed was due to absence of H3T11 phosphorylation or induction of stress response genes. A phosphorylated mimic H3T11D was not tested [20]. ...
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We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the tran-scriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.
... It is worth noting that the CLS analysis begins with the entry of cells into the stationary phase. It has been proposed that, during CLS, two main pathways are activated: the Tor/S6K pathway [63] and the Ras/adenylate cyclase/PKA pathway [64]. Interestingly, all of the studied strains showed a longevity comparable to that of the WT. ...
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Precise DNA replication is pivotal for ensuring the accurate inheritance of genetic information. To avoid genetic instability, each DNA fragment needs to be amplified only once per cell cycle. DNA replication in eukaryotes starts with the binding of the origin recognition complex (ORC) to the origins of DNA replication. The genes encoding ORC subunits have been conserved across eukaryotic evolution and are essential for the initiation of DNA replication. In this study, we conducted an extensive physiological and aging-dependent analysis of heterozygous cells lacking one copy of ORC genes in the BY4743 background. Cells with only one copy of the ORC genes showed a significant decrease in the level of ORC mRNA, a delay in the G1 phase of the cell cycle, and an extended doubling time. Here, we also show that the reducing the levels of Orc1-6 proteins significantly extends both the budding and average chronological lifespans. Heterozygous ORC/orcΔ and wild-type diploid cells easily undergo haploidization during chronological aging. This ploidy shift might be related to nutrient starvation or the inability to survive under stress conditions. A Raman spectroscopy analysis helped us to strengthen the hypothesis of the importance of lipid metabolism and homeostasis in aging.
... mTOR kinase is an important regulator of various vital cellular events such as cell division, growth, and metabolism [96][97][98][99][100]. The correlation between mTOR and lifespan was initially demonstrated by Fabrizio and coworkers in invertebrates using gene-editing techniques [101]. mTOR can act as a both negative and positive regulator in the process of neural aging. ...
Aging can lead to changes in the cellular milieu of the brain. These changes may exacerbate, resulting in pathological phenomena (including impaired bioenergetics, aberrant neurotransmission, compromised resilience and neuroplasticity, mitochondrial dysfunction, and the generation of free radicals) and the onset of neurodegenerative diseases. Furthermore, alterations in the energy-sensing pathways can accelerate neuronal aging but the exact mechanism of neural aging is still elusive. In recent decades, the use of plant-derived compounds, including astragaloside IV, to treat neuronal aging and its associated diseases has been extensively investigated. This article presents the current understanding of the roles and mechanisms of astragaloside IV in combating neuronal aging. The ability of the agent to suppress oxidative stress, to attenuate inflammatory responses and to maintain mitochondrial integrity will be discussed. Important challenges to be tacked for further development of astragaloside IV-based pharmacophores will be highlighted for future research.
... Mechanistically, down-regulation of TOR pathway activity has proven to increase both CLS and RLS (Cao et al. 2016). Deletion of the SCH9 kinase, a major TOR effector, also promotes CLS (Fabrizio et al. 2001). TOR1 deletion increases mitochondrial respiration. ...
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Molecular causes of aging and longevity interventions have witnessed an upsurge in the last decade. The resurgent interests in the application of small molecules as potential geroprotectors and/or pharmacogenomics point to nicotinamide adenine dinucleotide (NAD) and its precursors, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide, and nicotinic acid as potentially intriguing molecules. Upon supplementation, these compounds have shown to ameliorate aging related conditions and possibly prevent death in model organisms. Besides being a molecule essential in all living cells, our understanding of the mechanism of NAD metabolism and its regulation remain incomplete owing to its omnipresent nature. Here we discuss recent advances and techniques in the study of chronological lifespan (CLS) and replicative lifespan (RLS) in the model unicellular organism Saccharomyces cerevisiae. We then follow with the mechanism and biology of NAD precursors and their roles in aging and longevity. Finally, we review potential biotechnological applications through engineering of microbial lifespan, and laid perspective on the promising candidature of alternative redox compounds for extending lifespan.
... It should be noted that the absence of trade-offs between lifespan and fitness components was also observed in some long-lived Drosophila mutants, such as Indy (Marden et al., 2003) and E(z) . At the same time, different model organisms longevity was found to be associated with increased resistance to a wide range of stressful conditions, including oxidative stress, heat shock, and starvation (Fabrizio et al., 2001;Johnson et al., 2001;Longo, 2003;Perez et al., 2009). ...
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The gasotransmitter hydrogen sulfide (H2S) is an important biological mediator, playing an essential role in many physiological and pathological processes. It is produced by transsulfuration − an evolutionarily highly conserved pathway for the metabolism of sulfur-containing amino acids methionine and cysteine. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in cysteine metabolism and H2S production. Here we investigated the fitness components (longevity, stress resistance, viability of preimaginal stages, and reproductive function parameters) in D. melanogaster lines containing deletions of the CBS and CSE genes. Surprisingly, in most tests, CSE deletion improved, and CBS worsened the fitness. Lines with deletion of both CBS and CSE demonstrated better stress resistance and longevity than lines with single CBS deletion. At the same time, deletion of both CBS and CSE genes causes more serious disturbances of reproductive function parameters than single CBS deletion. Thus, a complex interaction of H2S-producing pathways and cellular stress response in determining the lifespan and fitness components of the whole organism was revealed.
Fasting and fasting mimicking diets extend lifespan and healthspan in mouse models and decrease risk factors for cancer and other age-related pathologies in humans. Normal cells respond to fasting and the consequent decrease in nutrients by down-regulating proto-oncogene pathways to enter a stress-resistant mode, which protects them from different cancer therapies. In contrast, oncogene mutations and the constitutive activation of pathways including RAS, AKT, and PKA allow cancer cells to disobey fasting-dependent anti-growth signal. Importantly, in different tumor types, fasting potentiates the toxicity of various therapies by increasing reactive oxygen species and oxidative stress, which ultimately leads to DNA damage and cell death. This effect is not limited to chemotherapy, since periodic fasting/FMD cycles potentiate the effects of tyrosine kinase inhibitors, hormone therapy, radiotherapy, and pharmacological doses of vitamin C. In addition, the anticancer effects of fasting/FMD can also be tumor-independent and involve an immunotherapy-like activation of T cell-dependent attack of tumor cells. Supported by a range of pre-clinical studies, clinical trials are beginning to confirm the safety and efficacy of fasting/FMD cycles in improving the therapeutic potential of different cancer therapies, while decreasing side effects to healthy cells and tissues.
Small compounds are a large group of chemicals characterized by various biological properties. Some of them also have antiaging potential, which is mainly attributed to their antioxidant activity. In this study, we examined the antiaging effect of 4-N-Furfurylcytosine (FC), a cytosine derivative belonging to a group of small compounds, on budding yeast Saccharomyces cerevisiae. We chose this yeast model as it is known to contain multiple conserved genes and mechanisms identical to that of humans and has been proven to be successful in aging research. The chronological lifespan assay performed in the study revealed that FC improved the viability of yeast cells in a concentration-dependent manner. Furthermore, enhanced mitochondrial activity, together with reduced intracellular ROS level, was observed in FC-treated yeast cells. The gene expression analysis confirmed that FC treatment resulted in the restriction of the TORC1 signaling pathway. These results indicate that FC has antiaging properties.
Fission yeast RNA polymerase II consists of 12 subunits, Rpb1‐Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow‐growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino‐terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.
Diet as a whole, encompassing food composition, calorie intake, and the length and frequency of fasting periods, affects the time span in which health and functional capacity are maintained. Here, we analyze aging and nutrition studies in simple organisms, rodents, monkeys, and humans to link longevity to conserved growth and metabolic pathways and outline their role in aging and age-related disease. We focus on feasible nutritional strategies shown to delay aging and/or prevent diseases through epidemiological, model organism, clinical, and centenarian studies and underline the need to avoid malnourishment and frailty. These findings are integrated to define a longevity diet based on a multi-pillar approach adjusted for age and health status to optimize lifespan and healthspan in humans.
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Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was performed. The mutant line methuselah(mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate–binding protein–coupled seven–transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span.
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Reactive oxygen (RO) has been identified as an important effector in ageing and lifespan determination. The specific cell types, however, in which oxidative damage acts to limit lifespan of the whole organism have not been explicitly identified. The association between mutations in the gene encoding the oxygen radical metabolizing enzyme CuZn superoxide dismutase (SOD1) and loss of motorneurons in the brain and spinal cord that occurs in the life-shortening paralytic disease, Familial Amyotrophic Lateral Sclerosis (FALS; ref. 4), suggests that chronic and unrepaired oxidative damage occurring specifically in motor neurons could be a critical causative factor in ageing. To test this hypothesis, we generated transgenic Drosophila which express human SOD1 specifically in adult motorneurons. We show that overexpression of a single gene, SOD1, in a single cell type, the motorneuron, extends normal lifespan by up to 40% and rescues the lifespan of a short-lived Sod null mutant. Elevated resistance to oxidative stress suggests that the lifespan extension observed in these flies is due to enhanced RO metabolism. These results show that SOD activity in motorneurons is an important factor in ageing and lifespan determination in Drosophila.
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Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.
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The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.
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Yeast lacking copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (SOD), catalase T, or metallothionein were studied using long term stationary phase (10-45 days) as a simple model system to study the roles of antioxidant enzymes in aging. In well aerated cultures, the lack of either SOD resulted in dramatic loss of viability over the first few weeks of culture, with the CuZnSOD mutant showing the more severe defect. The double SOD mutant died within a few days. The severity reversed in low aeration; the CuZnSOD mutant remained viable longer than the manganese SOD mutant. To test whether reactive oxygen species generated during respiration play an important role in the observed cellular death, growth in nonfermentable carbon sources was measured. All strains grew under low aeration, indicating respiratory competence. High aeration caused much reduced growth in single SOD mutants, and the double mutant failed to grow. However, removal of respiration via another mutation dramatically increased short term survival and reversed the known air-dependent methionine and lysine auxotrophies. Our results suggest strongly that mitochondrial respiration is a major source of reactive oxygen species in vivo, as has been shown in vitro, and that these species are produced even under low aeration.
We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems ofCaenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.
SOD2, encoding manganese superoxide dismutase (MnSOD), is essential for stationary-phase survival of yeast cells. In addition, stationary-phase cells are more resistant to oxidative stress than exponential-phase cells. The use of a SOD2::lacZfusion construct in this study shows that transcription of SOD2 increases 6.5-fold as cells enter stationary phase in rich, glucose medium. The increase in SOD2 expression appears to be due to two phenomena - the switch to a non-fermentable carbon source and nutrient limitation. Analysis of SOD2 transcription in mutant Saccharomyces cerevisiae strains showed that the gene was negatively regulated by intracellular cAMP levels which decrease as cells enter stationary phase. Mutation of 'stress-responsive' (STRE) elements in the SOD2 promoter which respond to cAMP levels resulted in the loss of cAMP-dependent expression but only partially reduced the increase in expression as cells entered stationary phase. A putative Yap1p-binding site was found to be inactive and mutation of YAP1 had no effect on the STRE-mediated expression. To fully eliminate the stationary-phase response, it was necessary to mutate a HAP2/3/4/5 complex binding site in addition to the STRE elements. It is postulated that the effects of the STRE sites and the HAP2/3/4/5 complex binding site are additive.
A mutation in the age-1 gene of the nematode Caenorhabditis elegans has been shown to result in a 65 percent increase in mean life-span and a 110 percent increase in maximum life-span at 25 degrees. One of the hallmarks of organismic aging and senescent processes is an exponential acceleration of age-specific mortality rate with chronological age. This exponential acceleration is under genetic control: age-1 mutant hermaphrodites show a 50 percent slower rate of acceleration of mortality with chronological age than wild-type strains. Mutant males also show a lengthening of life and a slowing of the rate of acceleration of mortality, although age-1 mutant males still have significantly shorter life-spans than do hermaphrodites of the same genotype. The slower rates of acceleration of mortality are recessive characteristics of the age-1 mutant alleles examined.
The life spans of individual Saccharomyces cerevisiae cells were determined microscopically by counting the number of buds produced by each cell to provide a measure of the number of cell generations (age) before death. As the cells aged, their generation times increased five- to sixfold. The generation times of daughter cells were virtually identical to those of their mothers throughout the life spans of the mothers. However, within two to three cell divisions after the daughters were detached from their mothers by micromanipulation, their generation times reverted to that characteristic of their own age. Recovery from the mother cell effect was also observed when the daughters were left attached to their mothers. The results suggest that senescence, as manifested by the increase in generation time, is a phenotypically dominant feature in yeast cells and that it is determined by a diffusible cytoplasmic factor(s) that undergoes turnover. This factor(s) appeared to be transmitted by a cell not only to its daughter, but also indirectly to its granddaughter. In separate studies, it was determined that the induced deposition of chitin, the major component of the bud scar, in the yeast cell wall had no appreciable effect on life span. We raise the possibility that the cytoplasmic factor(s) that appears to mediate the "senescent phenotype" is a major determinant of yeast life span. This factor(s) may be the product of age-specific gene expression.
Like other microorganisms, the yeast Saccharomyces cerevisiae responds to starvation by arresting growth and entering stationary phase. Because most microorganisms exist under conditions of nutrient limitation, the ability to tolerate starvation is critical for survival. Molecular analyses have identified changes in transcription, translation, and protein modification in stationary-phase cells. At the level of translation, the pattern of newly synthesized proteins in stationary-phase cells is surprisingly similar to the pattern of proteins synthesized during exponential growth. When limited for different nutrients, yeast strains may not enter stationary phase but opt for pathways such as pseudohyphal growth. If nutrient limitation continues, the end-point is likely to be a stationary-phase cell. Based on the results of recent studies, we propose a model for entry into stationary phase in which G(o) arrest is separable from acquisition of the ability to survive long periods of time without added nutrients.