ദ࢞࿨ช

Studies on the molecular mechanisms that
regulate lifespan in budding yeastड़լ߮฾͹थໍΝ੏‫ͤޜ‬Ζ෾ࢢ‫ߑؽ‬͹‫ڂݜ‬ӻଞ ߌ࢚

߁ౣ୉ָ୉ָӅ౹߻ਫ਼ໍՌָ‫ڂݜ‬Ռ

2023 ೧ 3 ݆

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1ɿक࿨ช
Studies on the molecular mechanisms that regulate lifespan in budding yeastʤड़լ߮฾͹थໍΝ੏‫ͤޜ‬Ζ෾ࢢ‫ߑؽ‬͹‫ڂݜ‬ʥӻଞ ߌ࢚2ɿࢂߡ࿨ช
(1) SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae,
Koji Masumura, Sachi Matsukami, Kumiko Yonekita, Muneyoshi Kanai,
Kazunori Kume, Dai Hirata, Masaki Mizunuma,
Bioscience, Biotechnology, and Biochemistry, 83(8), 1473-1476 (2019).
(2) S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects,

Takafumi Ogawa, ‫ڨ‬Koji Masumura, Yuki Kohara, Muneyoshi Kanai,‫ڨ‬Tomoyoshi Soga, Yoshikazu Ohya, T. Keith Blackwell, Masaki Mizunuma,
Aging Cell, 21(5), e13604 (2022).
‫ڨ‬

Takafumi Ogawa and Koji Masumura contributed equally to this work.

3ɿ‫ؖ‬࿊࿨ช
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Contents

General introduction .......................................................................................................1

Chapter 1: SKO1 deficiency extends chronological lifespan in Saccharomyces
cerevisiae ...........................................................................................................................7
1.1 Introduction .................................................................................................................7
1.2 Material and Methods ..................................................................................................8
1.3 Results .......................................................................................................................10
1.4 Discussion..................................................................................................................13

Chapter 2: S-adenosyl-L-homocysteine extends lifespan through methionine
restriction effects methionine restriction .....................................................................20
2.1 Introduction ...............................................................................................................20
2.2 Material and Methods ................................................................................................21
2.3 Results .......................................................................................................................27
2.4 Discussion..................................................................................................................30

General conclusions .......................................................................................................44

Acknowledgments ..........................................................................................................48

References.......................................................................................................................50

General introduction

According to the 2021 statistics, the average life expectancy in Japan has
increased for approximately 10 years in the last half century (81.47 and 87.57 years formen and women, respectively). However, the percentage of the population aged 65 years
or older is approximately 30%, because of which the population is called a “super aging
society.” Consequently, slow economic growth and burden on social security andhealthcare are serious dilemmas faced by Japan. Moreover, the health span, i.e., the lengthof the time for which a person can stay healthy, is 73 and 76 years for men and women,
respectively, which implies that individuals suffer from some kind of disorder for
approximately 10 years, leading to a decline in their quality of life. Therefore, increasing
healthy life expectancy has become an urgent issue in Japan.
Elucidation of the mechanisms of aging and lifespan is one of the most important
issues for understanding the extended human healthspan and lifespan. Research on aging
and lifespan is expected to provide insights not only to the understanding of biologicalaging but also the pathogenesis and treatment of various diseases such as cancer,
cardiovascular disorders, and neurodegenerative diseases associated with aging. So far,common molecules and mechanisms involved in lifespan regulation have been identified
in yeasts, nematodes, flies, mice, and other organisms that can be used as models forhumans (Kenyon, 2010). For instance, dietary restrictions (Mair and Dillin, 2008; Sutphin
and Kaeberlein, 2008; Fontana et al., 2010) such as calorie restriction (CR) andmethionine restriction (MetR) (Orentreich et al., 1993; Miller et al., 2005; Wu et al.,2013), inactivation of the mechanistic target of rapamycin complex ᶙ (mTORC1)
pathway (Wullschleger et al., 2013), and activation of adenosine monophosphateactivated protein kinase (AMPK) (Hedbacker and Carlson, 2008) have been reported toextend the lifespan of many organisms. Particularly, the mechanism of CR to extend
lifespan was first revealed in yeast, and the sirtuin gene Sir2, an NAD+-dependent histone
deacetylase, was identified as a key player in CR (Imai et al., 2000). Furthermore, a searchfor compounds that induce longevity has been conducted using yeast, and resveratrol, atype of polyphenol, was discovered as a compound that activates Sir2 (Howitz et al.,2003). Interestingly, resveratrol extended the lifespan of yeasts, nematodes, and flies
without CR (Wood et al., 2004). Thus, although several signaling pathways and molecularmechanisms were involved in the regulation of lifespan, the detailed molecularmechanisms of and the relationship between lifespan and signaling pathways remain
unclear.
The budding yeast Saccharomyces cerevisiae is a single-cell model organism,and the mechanisms that regulate its fundamental life processes are similar to those ofhigher organisms. The concept of lifespan also exists in the yeast and the yeast has twolifespan models, namely, the replicative lifespan (RLS), which is the number of divisions
in the lifetime of a single mother cell, and the chronological lifespan (CLS), which is theperiod during which a cell can survive under limited nutritional conditions after it stops
dividing after the stationary phase (Mortimer and Johnston, 1959; Fabrizio and Longo,
2003). Since RLS is the number of daughter cells that a mother cell can produce before
aging, it provides a model of aging for regularly dividing cells, such as human fibroblasts
and cells of the blood cell lineage. In addition, since the regulatory mechanisms of CLS
are similar to those of aging in higher organisms, the insights gained from the saidmechanisms are vital to understand human aging. The advantage of using yeast forlifespan analysis is that it is easier to obtain data owing to its shorter lifespan comparedto other model organisms. In addition, yeast is easy to analyze genetically and at themolecular level and is cost-efficient. Therefore, yeast is an ideal tool to study the lifespanowing to its various molecular mechanisms and signaling pathways related to lifespan(Botstein and Fink, 2011).
The calcium ion (Ca2+), as a universal intracellular signaling medium, regulates
diverse biological processes, such as fertilization, development, cell proliferation,
hormone secretion, and memory (Aramburu et al., 2000). Previously, Mizunuma et al.
(1998) analyzed the physiological functions of Ca2+ signaling and discovered calcineurin,a Ca2+-dependent serine/threonine phosphatase in budding yeast. Afterward, it was foundthat Ca2+ signaling pathways inhibit the onset of mitosis through the activation of Swe1,
a negative regulator of the G2/M phase, via activation of calcineurin and Mpk1, an MAP
kinase.
To obtain novel regulators of the cell cycle via Ca2+ signaling, Mizunuma et al.
(2001) screened for suppressers of the sensitivity of CaCl2 exhibited by strains deleted
for the cell cycle regulator of ZDS1. The zds1' cells exhibited calcium sensitivity,
abnormal polar bud growth, and delayed G2 phase. As a result, scz1–14 mutations wereobtained from zds1' cells. Interestingly, scz14 was allelic to the SIR3 locus involved inaging. The SIR3 prolongs the RLS of budding yeast, suggesting a link between the scz
mutant strain and lifespan. Furthermore, Tsubakiyama et al. (2011), using the zds1' strain
with shortened RLS, found that calcineurin activation was associated with shortened RLS
in the zds1' strain. These findings suggest that Ca2+ homeostasis regulates lifespan.
Among scz mutants, sah1/scz7 is a mutant of the S-adenosylhomocysteine (SAH)hydrolase SAH1 (Mizunuma et al., 2004). In the sah1 mutants, the cellular accumulationof SAH, a substrate of SAH1, was accompanied by remarkable accumulation of S
adenosyl-L-methionine (SAM). SAM is synthesized from methionine (Met) and ATP
using SAM synthetase and is utilized in the majority of biological methylation reactions(Thomas and Surdin-Kerjan, 1997). On demethylation, SAM is converted into SAH, apotent competitive inhibitor of SAM-dependent methyltransferases. Therefore, SAH1,
encoding SAH hydrolase, is an essential gene for cell growth. Indeed, the sah1 mutants
exhibited a high accumulation of SAH and impaired proliferation, suggesting a link
between Met metabolism and lifespan. Moreover, Ogawa et al. (2016) measured thelifespan of sah1 mutants and found that they had shorter CLS and RLS than wild-type(WT) cells. Therefore, to obtain novel regulators of lifespan extension, we screened formutants that inhibit growth retardation of sah1 mutants and obtained 116 suppressor
mutants. Of these, 15 strains were suppressor mutations within SAH1. The remaining 101
strains were mutations in other genes, all of which were located at one locus anddesignated spontaneous suppressor growth delay in the sah1 (SSG1). The SSG1 mutant
had a high intracellular SAM accumulation and prolonged CLS instead of RLS. Ogawa
et al. (2016) found that this prolonged lifespan was attributed to the activation of SAM
synthesis, reducing the amount of ATP (a substrate of SAM) and activated AMPK.
Interestingly, WT cells, the supplementation of SAH in the medium increased
intracellular SAM and activated AMPK, thereby extending the CLS (Ogawa et al., 2016).
Furthermore, the WT cells accumulated SAH upon severe CR (0.05% Glucose), andaccumulation of SAM and extension of maximum CLS were observed. It is likely thatthe effects of SAH also overlap with CR. However, the mechanism underlying the effectof SAH remains unclear.
Previously, Saito and Posas (2012) have found that strain deletion in HOG1, astress-responsive MAP kinase, exhibits sensitivity to Ca2+ and osmotic pressure. Toinvestigate the relationship between Ca2+ signaling and lifespan, Kobayashi et al. (2008)
screened for mutants that suppress the Ca2+ sensitivity exhibited by hog1' cells. As aresult, the sgh1–6 mutations were obtained from the hog1' cells. Among these mutations,sgh3–6 suppressed not only the Ca2+ sensitivity but also the osmotic sensitivity in hog1'
cells. Particularly, sgh4 is a mutant allele of Whi3, a positive regulator of cell size control.
Mizunuma et al. (2013) showed that whi3 is phosphorylated by the Ras/cAMP-dependent
protein kinase A (PKA), involved in numerous cellular processes such as metabolism,
stress resistance and proliferation, and PKA-dependent regulation. sgh3, sgh5, and sgh6
are mutations in TUP1, SKO1, and CYC8, respectively. Sko1 forms a complex with Tup1–
Cyc8 to inhibit gene transcription induced by hyperosmotic and oxidative stress; however,when responding to stress, the Sko1–Tup1–Cyc8 complex is phosphorylated by Hog1 toactivate transcription (Rep et al., 2001; Proft and Struhl, 2002). Osmotic stress activates
hyperosmotic signaling. Previously, it has been reported that activation of hyperosmotic
signaling extends RLS through an increase in NAD+ and activation of SIR2, a componentof CR, via glycerol synthesis (Kaeberlein et al., 2002). However, the relationship between
CLS and hyperosmotic signaling remains unclear.
Thus, both the hyperosmotic signaling pathway and SAH, which are predicted
to be associated factors of the Ca2+ signaling pathway, were expected to extend lifespanrelated CR. Therefore, I focused on the molecular mechanisms that regulate lifespan as
represented of CR in budding yeast to understand the molecular mechanisms involved inhuman aging and lifespan, and to prevent aging and diseases associated with aging. So,
in Chapter 1, I investigated that inhibition of Sko1 function, a regulator of the highosmolarity glycerol (HOG) pathway, suppressed the oxidative stress susceptibility andshortened CLS of hog1' cells. In Chapter 2, I explored the mechanism of how SAHadministration to cells extends their CLS.



Chapter 1: SKO1 deficiency extends chronological lifespan in Saccharomyces
cerevisiae

1.1 Introduction
Cells of organisms are exposed to various stress stimuli such as osmotic pressure,oxidation, heat, heavy metals, and ultraviolet radiation. In response to such stress, tomaintain proliferation, growth, and function, organisms have various mechanisms tosense these stresses and adapt to them. The HOG is a stress-responsive MAPK pathwaythat is activated to regulate hyperosmolarity. Its downstream factor Hog1 (MAP kinase)regulates various survival responses such as hyperosmolarity, oxidative stress, and cell
cycle progression (Saito and Posas, 2012). Previously, Kobayashi et al. (2008) showedthat the growth defect in hog1' cells with high osmolarity and high concentrations of
CaCl2 was restored by an additional mutation of the tup1-484, sko1-18, or cyc8-389 gene.
Sko1 is a repressor that mediates HOG pathway-dependent regulation in association with
Tup1-Cyc8/Ssn6 and functions as a general repressor of the transcription of genes
involved in a wide variety of processes, such as responding to osmotic and oxidative stress(Rep et al., 2001; Proft and Struhl, 2002). Alternatively, when cells perceive diverse
stresses, the Sko1 complex is phosphorylated by Hog1, which activates transcription(Proft and Struhl, 2002). Previously, sko1' cells exhibited resistance to oxidative stress,
and the sensitivity to oxidative stress exhibited by hog1' cells is suppressed by the lossof Sko1 function (Rep et al., 2001). In addition, hog1' cells exhibited shorter CLS,
suggesting that the HOG pathway is a critical factor for cell survival. Herein, I analyzed
the role of Sko1, a factor that suppresses the stress sensitivity of hog1' cells, in regulating
lifespan.
1.2 Material and Methods
1.2.1. Yeast strains and media
Yeast strains used in this study are listed in Table 1. All yeast strains were derivatives ofW303. Gene disruption was performed by a standard PCR-based method (Longtine et al.,1998). Rich medium (YPD) consisted of 1% yeast extract (Oriental Yeast Co., Ltd.), 2%
polypeptone (Shiotani M.S.), 2% glucose (Nacalai Tesque), 0.04% adenine hemisulfate(Sigma) and 0.02% uracil (Sigma).

1.2.2. Stress-resistance assay
Oxidative and osmotic stress-resistance assays were measured by spot assays. Cells
suspended in water (5 x 107 cells/ml) were spotted onto YPD plates containing various
concentrations of hydrogen peroxide (Santoku Chemical Industry) or KCl (Kanto
Chemical). The cells were then grown at 25℃ for 3 or 4 days before visualization.

1.2.3 CLS assay
CLS analysis was performed in liquid Synthetic complete media SD, that adjusted to pH
6.0, as previously described (Fabrizio et al., 2003). SD contain 0.17% Yeast Nitrogen
Base without Amino Acid (DIFCO), 0.5% ammonium sulfate (Sigma), amino acid (20mg/1 contains adenine hemisulfate, 20 mg/1 uracil, 20 mg/1 tryptophan (Kanto Chemical),
20 mg/1 histidine (Sigma), 20 mg/1 arginine (Kanto Chemical), 20 mg/1 methionine(Nacalai Tesque), 30 mg/1 tyrosine (Sigma), 30 mg/1 leucine (Sigma), 30 mg/l
isoleucine (Sigma), 30 mg/1 lysine (Sigma), 150 mg/1 valine (Sigma) and 60 mg/1
phenylalanine (Kanto Chemical), finally), 2% of glucose. Viability was measured byplating cells onto YPD plates and monitoring CFUs starting from day 3, which wasconsidered to be the initial survival (100%). All data were represented as the average ofthree independent experiments conducted at the same time.

1.2.4 Quantitative real-time PCR analysis
Cells were grown to log phase (2 x 106 cells/ml) in liquid SD medium at 25℃. Total RNA
was isolated from cells using RNeasy Mini Kit (QIAGEN) and quantitative real-time PCR(qRT-PCR) was performed with One Step SYBR Prime Script RT-PCR Kit (Takara) using
Light Cycler (Roche). PCR primers for ACT1 as control were 5’-TTGGATTCCGGTGATGGTGTTACT-3’ and 5’-TGAAGAAGATTGAGCAGCGGTTTG-3’. PCR primers
for GPD1 were 5’-GTATCTGTAGCCAATTGAAAGGTC-3’ and 5’-CATTGAATACCTAGTTCCTCAGTG-3’. PCR primers for GPD2 were 5’-GACCATCCTATCAGAAGATCGGAC-3’ and 5’-CTCTGGCTCGAAGATATGGGAATG -3’ (Invitrogen).

1.2.5 Statistical analysis
All experiments were repeated at least twice with similar results each time. Data represent
biological replicates. Appropriate statistical tests were used for every figure. GraphPad
Prism 6 (GraphPad Software) was used for comparison of CLS, and P values were derived
from a two-way ANOVA with time and strain used as independent factors. The qRT-PCR
data were analyzed appropriate statistical tests (GraphPad Prism 9) as indicated in the
figure legends.



1.3 Results
1.3.1 Effects of loss of function of repressors mediating HOG pathway-dependent
regulation
Previously, we have obtained the sko1-18, tup1-484, and cyc8-389 mutants,
which suppress the Ca2+ sensitivity exhibited by hog1' cells, and we have created double
mutants with hog1' cells to examine the growth phenotype of yeast cells. As previously
reported (Rep et al., 2001), the sko1-18 mutants suppressed the hydrogen peroxide
sensitivity of hog1'cells, and the tup1-484 or cyc8-389 mutants partially suppressed it(Fig. 2A).
Extended lifespan is often correlated with increased tolerance to various stresses,
including oxidative stress (Fontana et al., 2010). So, I measured the CLS of hog1' cells,
which are sensitive to osmotic pressure and oxidative stress, and found that hog1' cells
exhibited a shortened lifespan, suggesting that the HOG pathway is essential for cell
survival. Next, I measured CLS to determine whether the sko1-18, tup1-484, or cyc8-389
mutants could also suppress the shortened CLS of hog1' cells. As a result, sko1-18, tup1484, or cyc8-389 mutants suppressed the shortened lifespan of hog1' cells and extended
its lifespan to the same level as the wild-type strain (WT) (Fig. 2B). Loss of function due
to deletion of Sko1 also suppressed the shortened lifespan of hog1' cells. Furthermore,
the hog1' sko1' double mutant cells had a significantly longer lifespan than WT cells(Fig. 2C), suggesting that the Sko1 protein might play a role in lifespan.

1.3.2 Sko1 deficiency exhibits resistance to oxidative stress and lifespan extension
via glycerol synthesis system
Since Sko1 was predicted to be involved in the regulation of lifespan, I next

examined whether a single sko1' cells would extend CLS. Strikingly, sko1' cells
significantly extended CLS more than WT cells (Fig. 3A). The Sko1 protein forms acomplex with Tup1-Cyc8 when the HOG pathway is inactive, inhibiting the transcriptionof hyperosmolarity and oxidative stress-inducible genes (Rep et al., 2001; Proft andStruhl, 2002). The extended lifespan exhibited by the sko1' cells suggests that the
repression of target genes of Sko1 is derepressed. Previous reports have shown that
hyperosmolarity extends RLS, which requires glycerol biosynthesis (Kaeberlein et al.,2002). To determine whether CLS extension by sko1' cells was mediated by synthesized
glycerol, I measured CLS by deleting GPD1 and GPD2, which encode glycerol-3phosphate dehydrogenase. CLS of sko1' gpd1' gpd2' triple mutants were equivalent to
CLS of WT cells (Fig. 3A), suggesting that glycerol synthesis is essential for the lifespanextension in the sko1' cells.
Next, I tested the resistance to stress in sko1' gpd1' gpd2' triple mutants to
examine whether the beneficial effect of deficient Sko1 function was due to enhanced
glycerol synthesis (Fig. 3B). Resistance to osmotic (KCl) and hydrogen peroxide (H2O2)stress of sko1' cells was abolished by deletion of GPD1 and GPD2, and the stresssensitivity of sko1' gpd1' gpd2' triple mutants were comparable to that of gpd1' gpd2'
double mutants (Fig. 3B). Overall, glycerol synthesis may be an important downstream
factor mediating the effects of sko1' cells on CLS extension and stress tolerance.
Therefore, I measured the transcript levels of GPD1 and GPD2 by quantitative real-time
PCR in sko1' cells to see if the loss of Sko1 function derepressed the glycerol synthesis
gene (Fig. 3C, D). The amount of GPD1 transcript level in the sko1' cells was increased
approximately 1.8-fold change compared to WT cells, suggesting that loss of Sko1
function released the repression of GPD1 expression. On the other hand, there was nochange in the amount of GPD2 transcription, indicating that Sko1 was expected to morestrongly inhibit GPD1 transcription.

1.3.3 Lifespan extension of Sko1 deficiency is an alternative pathway to osmotic
stressDuring hyperosmotic stress, cells activate the HOG pathway and phosphorylate
the Sko1-Tup1-Cyc8 complex, thereby suppressing Sko1 function (Proft and Struhl,
2002). Since hyperosmolarity is known to extend CLS (Smith et al., 2007), I analyzed
whether the extension of CLS in sko1' cells is associated with hyperosmolarity.
Consistent with a previous report (Smith et al., 2007), I found that treatment withhyperosmolarity (sorbitol) appears to extend CLS in WT cells (Fig. 4). I also noted that
hyperosmolarity significantly increased the CLS of sko1' cells early and mid-life (up to
20 days) compared to the CLS of WT cells under osmotic stress conditions (Fig. 4). These
data indicated that CLS extension by sko1' cells or osmotic stress acts via a branching
pathway.



1.4 Discussion
In this chapter, I analyzed the role of Sko1 in suppressing the stress sensitivityof hog1' cells, and the high survival rate of the sko1' cells was dependent on glycerol
synthesis. Moreover, the resistance to stress and high survival rate exhibited by the sko1'
cells were suppressed in sko1'gpd1' gpd2' triple mutants (Fig. 3A, B). Therefore,
glycerol synthesis is critical for the high survival rate of sko1' cells. However, becauseof the negative feedback in the HOG pathway (Yamamoto et al., 2010), it is expected that
constant transcriptional activation by Sko1 deletion would also have a negative effect on
the lifespan. Thus, the regulation of lifespan requires not only simple gene activity but
also elaborate regulation of gene activity. In addition, quantitative PCR results showedthat GPD1 transcription was increased in the sko1' cells (Fig. 3C). These results suggestthat the GPD gene is critical for lifespan extension and resistance to stress due to the lossof Sko1 function. The target genes of Sko1 are GRE2, AHP1, SFA1, GLR1, and YML131W,
which protect against oxidative stress and are repressed by Sko1 in non-stressed
conditions (Rep et al., 2001). However, further analysis is needed to determine if thelifespan extension of the sko1' cells depend on these factors.
Curiously, although the sko1' cells suppressed the growth defect of hog1' cells
under hyperosmotic stress conditions (Rep et al., 2001), sko1' cells did not exhibit clear
resistance to osmotic stress conditions (Fig. 3B). Thus, there is little beneficial effect of
sko1' cells on the WT background of nutrient growth under osmotic stress conditions.
This is consistent with the results of partial extension of lifespan when hyperosmoticpressure was applied to the sko1' cells (Fig. 4), suggesting that the response to
hyperosmolarity and the regulation of gene expression by deletion of SKO1 are divergent
pathways in part.
Herein, the tup1-484/scz3 and cyc8-389/scz6 mutations were also factors
suppressing Ca2+ sensitivity in hog1' cells; however, because both hog1' tup1' andhog1' cyc8' double mutants are highly aggregative (data not shown), they were notavailable for further analysis.
Identification of the actual effector mechanisms by which loss of SKO1 protects
against chronological aging is a critical future challenge. During ethanol fermentation,
yeast cells encounter osmotic, oxidative, and ethanol stresses. Thus, the findings in thisstudy might be useful to produce strains that are tolerant to these fermentation-associatedstresses.



Fig. 1 Regulation of the Sko1-Tup1-Cyc8 complex by Hog1.
(1) Under normal conditions without stress, the Sko1-Tup1-Cyc8 complex binds to CRE(cyclic-AMP responsive element), upstream of stress-inducible genes, and represses
transcription. (2) When cells sense hyperosmotic stress, Hog1 migrates to the nucleus andphosphorylates Sko1. Then, the phosphorylated Sko1-Cyc8-Tup1 complex is repressed
and mobilizes RNA polymerase II (Pol II) to activate transcription




A

B

C

Fig. 2 Deletion of SKO1 suppressed hog1' sensitivity to oxidative stress and extended
lifespan.
(A) Growth of various strains on solid YPD medium containing the indicatedconcentrations of hydrogen peroxide. Ten-fold serially diluted cells of WT, hog1', hog1
' tup1-484, hog1' sko1-18, and hog1' cyc8-389 were spotted onto YPD solid medium
containing various concentrations of hydrogen peroxide. The cells were then grown at
25ºC for 3 days before visualization. (B) CLS curves for WT, hog1', hog1' tup1-484,
hog1' sko1-18, and hog1' cyc8-389 are shown. (C) CLS curves of WT, hog1', and
hog1' sko1 are shown.




A

C

D

B

Fig. 3 Glycerol Synthesis System is required for resistance to oxidative stress andlifespan extension in sko1'(A) CLS curves for WT, sko1', gpd1' gpd2', and sko1' gpd1' gpd2' are shown. (B)Osmotic (KCl) (left panel) and hydrogen peroxide (right panel) test. Ten-fold seriallydiluted cells of WT, sko1', gpd1' gpd2', and sko1' gpd1' gpd2' were spotted onto
solid medium containing KCl or hydrogen peroxide at 25ºC and incubated for 3 days. (C,D) Relative expression level of (C) GPD1 and (D) GPD2 in WT, sko1' measured byreverse transcription Real-Time PCR. Mean ± SD (n = 3), two-sided unpaired t-test; ns,
not significant.

Fig. 4 Lifespan extension of sko1' is a partially separate pathway from that inducedby hyperosmolarity.
CLS curves for WT and sko1' in the presence or absence of hyperosmolarity (0.75 M
sorbitol) are shown.

Table 1 Strains used in Chapter 1
Source ofReference

StrainName

Genotype

W303-1A

WT

MATa ; trp1-1 leu2-3,112 ade2-1 ura3-1 his3-11,15 can1-100 NBRP/YGRPYFK100hog1 '

MATa ; hog1 '::TRP1

Lab. StockYFK71

hog1 'sko1-18

MATa ; hog1 '::TRP1 sko1-18

Lab. Stock

YFK66

hog1 'tup1-484

MATa ; hog1 '::TRP1 tup1-484

Lab. StockYFK75

hog1 'cyc8-389

MATa ; hog1 '::TRP1 cyc8-389

Lab. Stock

YFK73

hog1 ' sko1 'MATa ; hog1 '::TRP1 sko1 '::kanMX4Lab. Stock

YAL12

sko1 '

MATa ; sko1 '::kanMX4Lab. Stock

YAL18

gpd1 ' gpd2 'MATa ; gpd1 '::kanMX4 gpd2 '::kanMX4Lab. Stock

YAL26

sko1 ' gpd1 ' gpd2 ' MATa ; sko1 '::kanMX4 gpd1 '::kanMX4 gpd2 '::kanMX4Lab. Stock

Table 2 P value for CLS analysis in Chapter 1
FigureStrain A

Strain B

P -value against B

2B

WT

hog1 '<0.001hog1 '

hog1 'sko1-18<0.001hog1 '

hog1 'tup1-484<0.001hog1 '

hog1 'cyc8-389<0.001WT

hog1 ' sko1 '<0.001hog1 '

hog1 ' sko1 '<0.001

WT

sko1 '<0.001WT

gpd1 ' gpd2 '0.0686

sko1 '

sko1 ' gpd1 ' gpd2 '<0.001WTWT (Glycerol 0.75 M)<0.001

sko1 '

sko1 ' (Glycerol 0.75 M)<0.001

2C
3A

4

P value were derived from a two-way ANOVA with time and strain used as independent
factors.

Chapter 2: S-adenosyl-L-homocysteine extends lifespan through methionine
restriction effects methionine restriction

2.1 Introduction
As people live longer, research has turned to finding ways to extend life while
avoiding the diseases and infirmities of advanced age, particularly through the study ofdiet at a basic level. Dietary restriction such as MetR is an effective strategy to promotelongevity and counteract age-related morbidities (Ables and Johnson, 2017; Parkhitko etal., 2019). So far, genetic manipulation or pharmacological inhibition of Met metabolic
pathways (Annibal et al., 2021; Hepowit et al., 2021; Johnson and Johnson, 2014; Obata
and Miura, 2015; Ogawa et al., 2016; Ruckenstuhl et al., 2014) and a Met-restricted dietextend lifespan (Orentreich et al., 1993; Wu et al., 2013); however, it is not practical toapply these interventions in humans. Previously, Ogawa et al. (2016) have found that
supplementation with SAH, an endogenous Met derivative, activates the energy sensor
AMPK and extends lifespan in yeast (Fig. 5). SAH treatment triggered high accumulationof SAM, which may be attributed to the consumption of ATP and the induction of AMPK
activation. However, the details of the mechanism of lifespan extension using SAH are
unknown. Here, I discovered that the intake of SAH reduces intracellular Met and induces
benefits that mimic MetR in budding yeast Saccharomyces cerevisiae. In addition,
treatment with SAH activates AMPK and mimics some of the benefits of MetR.



2.2 Material and Methods
2. 2. 1. Yeast strains and media
Yeast strains used in this study are listed in Table 3. All yeast strains were derivatives ofW303. Gene disruption was performed by a standard PCR-based method (Longtine et al.,1998). SD contain 0.17% Yeast Nitrogen Base without Amino Acid (DIFCO), 0.5%ammonium sulfate (Sigma), amino acid (20 mg/1 contains adenine hemisulfate, 20 mg/1
uracil, 20 mg/1 tryptophan (Kanto Chemical), 20 mg/1 histidine (Sigma), 20 mg/1
arginine (Kanto Chemical), 20 mg/1 methionine (Nacalai Tesque), 30 mg/1 tyrosine(Sigma), 30 mg/1 leucine (Sigma), 30 mg/l isoleucine (Sigma), 30 mg/1 lysine (Sigma),
150 mg/1 valine (Sigma) and 60 mg/1 phenylalanine (Kanto Chemical), finally), 2% of
glucose.

2.2.2. CLS assay
Described in 1. 2. 3.

2.2.3. Growth conditions for metabolome analysis of yeast
WT cells were cultured in SDC liquid medium. Cells were grown to log phase (OD600
nm = 0.2) in SDC liquid medium at 25℃. SAH was added to SDC medium to 1 mM andcultured for another eight hours (OD600 nm = 0.4). As a control, cells were grown log
phase in SDC medium at 25℃ (OD600 nm = 0.4).

2.2.4. Extraction of the intracellular metabolite from yeast
Metabolite extraction was conducted using the previously described modification (Sogaet al., 2003). Cells were harvested from the culture medium (OD600 nm = 30) and filteredthrough a 0.45 μm pore size filter. The internal cationic and anionic standards weremethionine sulfone and 2-morpholinoethanesulfonic acid, respectively. The lyophilized
samples were dissolved in 50 μl Milli-Q water and subjected to capillary electrophoresistime-of-flight mass spectrometry (CE-TOFMS) analysis.

2.2.5. Method for metabolome analysis
Instrumentation: All capillary electrophoresis mass spectrometry (CE-MS) experiments
were conducted using Agilent 7100 CE capillary electrophoresis (Agilent Technologies,
Waldbronn, Germany), Agilent 6230 LC/MSD TOF system (Agilent Technologies, Palo
Alto, CA, USA), an Agilent1100 series binary HPLC pump, and the G1603A Agilent CEMS adapter- and G1607A Agilent CE-ESI-MS sprayer kit. For anionic metabolite
analysis, the original Agilent stainless Electrospray ionization (ESI) needle was replacedwith the Agilent G7100-60041 platinum ESI needle (Soga et al., 2009). Agilent
MassHunter Workstation was used to conduct system control and data acquisition, anddata analysis was done using the Keio MasterHands software.

Cationic metabolite analysis by CE-MS: Separations were conducted in a fused silica
capillary (50 mm i.d. x 100 cm total length) filled with 1 M formic acid as the electrolyte(Soga et al., 2003; Soga et al., 2006). About 5 nl sample solution was injected at 50 mbar
for five seconds, and 30 kV of voltage was applied. The capillary temperature wasmaintained at 20°C and the sample tray was cooled below 5°C. Methanol-water (50%v/v) containing 0.01 mM Hexakis (2,2-difluoroethoxy) phosphazene was delivered as thesheath liquid at 10 μl/min. ESI-time-of-flight mass spectrometry (ESI-TOFMS) was
performed in the positive ion mode and the capillary voltage was set at 4,000 V. A flowrate of heated dry nitrogen gas (heater temperature 300°C) was maintained at 7 psig. In
TOFMS, the fragmentor-, skimmer-, and Oct RFV voltage were set at 75 V, 50 V, and 500
V, respectively. Automatic recalibration of each acquired spectrum was conducted usingreference masses of reference standards. The 13C isotopic ion of a protonated methanol
dimer ([2MeOH + H]+, m/z 66.0631) and Hexakis(2,2-difluoroethoxy)phosphazene ([M+ H]+, m/z 622.0290) provided the lock mass for exact mass measurements (Satoh et al.,2017).

Anionic metabolite analysis by CE-MS: A commercially available COSMO(+)(chemically coated with cationic polymer) capillary (50 mm i.d. x 105 cm total length)(Nacalai Tesque) was used with a 50 mM ammonium acetate solution (pH 8.5) as theelectrolyte (Satoh et al., 2017; Soga et al., 2009). Sample solution (30 nl) was injected at
50 mbar for 30 sec and -30 kV of voltage was applied. Ammonium acetate (5 mM) in
50% methanol-water (v/v) containing 0.01 mM Hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 μl/min. ESI-TOFMS was conducted in
the negative ion mode; the capillary voltage was set at 3,500 V. For TOFMS, thefragmentor-, skimmer-, and Oct RFV voltage were set at 100 V, 50 V, and 500 V,
respectively. Automatic recalibration of each acquired spectrum was conducted usingreference masses of reference standards, i.e., 13C isotopic ion of deprotonated acetic aciddimer ([2CH3COOH-H]-, m/z 120.0384), and Hexakis + deprotonated acetic acid (m/z
680.03554) provided the lock mass for exact mass measurements.

Statistical analysis: MetaboAnalyst software ver. 5.0 was used for statistical analysis(Pang et al., 2021). Categories that have missing values were excluded. Data scaling wasperformed with mean-centered and divided by the standard deviation of each variable.

2.2.6. Growth conditions with [methyl-13C]Met for metabolome analysis of yeast
WT cells were preincubated in SD-Met liquid medium containing 20 mg/L [methyl13C]Met (Cambridge Isotope Laboratories) at 25℃ overnight. The cells were then grownin a fresh SD-Met liquid medium containing 20 mg/L [methyl-13C]Met liquid medium at
25℃ until the logarithmic growth phase (OD600 nm = 0.2). These procedures were
designed to ensure that the only external source of Met was [methyl-13C]Met. The culture
medium was then split into two, and 1 mM SAH solution was added to one of them, andthe cells were cultured for another eight hours (OD600 nm = 0.4). The other was used asa control and cultured until the logarithmic growth phase (OD600 nm = 0.4). The [methyl13C]Met and [methyl-13C]SAM content were expressed as nmol per mg dry cell weight(DCW).

2.2.7 Extraction of intracellular [methyl-13C]Met and [methyl-13C]SAM from yeast
Extraction of SAM and SAH was conducted as previously described (Christopher et al.,2002). The cells were harvested (total OD600 nm = 15), washed twice with 20 ml cold
water, and then extracted with 1 ml 10% perchloric acid (Fujifilm Wako Chemical) for
one hour at room temperature. The supernatant was diluted with MilliQ-grade water, andthe samples were filtered for CE-TOFMS.

2.2.8 GFP-ATG8 processing assay
Cells harboring the GFP-Atg8 expressing plasmid were grown to log phase (OD600 nm= 0.2) in liquid SD medium lacking uracil at 25ºC. SAH solution was added to SDCmedium to 1 mM, and the cells were incubated for another eight hours (OD600 nm = 0.4).
For the control, cells were grown to log phase (OD600 nm = 0.4) in liquid SDC medium
at 25ºC. Cells growth was stopped by adding TCA (Sigma) to about 9% final
concentration and putting on ice for at least five minutes before cells were pelleted. The
cells were obtained by centrifugation, washed twice with cold acetone (Nacalai Tesque),and dried in a speed-vac. Pellets were resuspended in 100 μl of urea buffer (50 mM Tris
(Sigma) [pH 7.5], 1 mM EDTA (Sigma), 6 M urea (Kanto Chemical), 1% SDS (Nacalai
Tesque), 1 mM PMSF (Sigma), 1x complete protease inhibitor cocktail (Roche), and
0.1% Tween 20 (Bio-Rad)) and lysed by vortexing with an equal volume of glass beads
at room temperature, with subsequent heating for ten minutes at 65ºC. Further analysis
was conducted using SDS-PAGE and immunoblotting using anti-GFP antibody (Roche,
1:1000) and anti-PSTAIR antibody (Novus bio., 1:1000). ImageJ was used to quantify
signals for Western blotting results, and GraphPad Prism 9 was used for statistical analysis.

2.2.9 Detection of Rps6 activation
WT cells were grown to log phase (OD600 nm = 0.2) in the SDC liquid medium at 25°C.
SAH was added to SDC medium to 1 mM and cultured for another eight hours (OD600
nm = 0.4). As a control, cells were grown log phase in SDC medium at 25°C (OD600 nm= 0.4). Cells were then harvested, washed with 1ml TEG +PPi buffer (50 mM Tris
(Sigma)-HCl (Nacalai Tesque), 1 mM EDTA (Sigma) [pH 7.5], 10% glycerol (Nacalai
Tesque), 30 mM NaCl (Nacalai Tesque), 10 mM NaF (Fujifilm Wako Chemical), 1 mM
sodium orthovanadate (Na3VO4) (Fujifilm Wako Chemical), 1 mM PMSF (Sigma), 1 mM
DTT (Nacalai Tesque), 1x complete protease inhibitor cocktail (Roche), and 0.1% Tween
20 (Bio-Rad)). The precipitate was frozen in liquid nitrogen and stored at -80°C. Thefrozen precipitate was dissolved on ice and gently resuspended in SDS-PAGE sample
buffer, and boiled for ten minutes. The eluted proteins were resolved by SDS-PAGE anddetected by anti-Phospho-S6 Ribosomal Protein (Ser235/236) antibody (Cell signaling
Tech., 1:1000) and anti- PSTAIR antibody (Novus bio., 1:1000). ImageJ (NIH) was used
to quantify signals for Western blotting results, and GraphPad Prism 9 (GraphPad
Software) was used for statistical analysis.



2.3 Results
2.3.1 Intracellular methionine levels are prominently reduced when cells aresupplemented with SAH
To investigate the basis for the extension of yeast cell lifespan when
supplemented with SAH, I performed a metabolomics analysis of WT with or without
SAH treatment. However, because SAH treated cells have a slower growth rate thanuntreated cells (Fig. 6), the comparison was made with cells cultured to the same OD600
value (about 0.4) rather than cells cultured at the same time. Here, a total of 148
metabolites were identified (Table 4). Of these, 63 metabolites were significantly upregulated and 6 were down-regulated (Fig. 7A-C, Table 4). As previously reported,
administration of SAH increased not only SAH but also SAM (Ogawa et al., 2016).
Because SAH is a potent competitive inhibitor of SAM, the major methyl group donor inorganism, intracellular SAH must be rapidly degraded by the SAH hydrolase SAH1
(Christopher et al., 2002). Therefore, accumulation of SAH has a negative effect on cellproliferation. Previously, Mizunuma et al. (2004) have found that exogenous SAM
significantly improved the growth of sah1, a mutant allele of the SAH1 gene that
accumulates high levels of SAH. Therefore, I speculate that the increase in SAM levels
by SAH supplementation may be the result of up-regulation of SAM synthesis from Met
by an unknown mechanism that acts to counteract the effects of SAH. Since SAM isbiosynthesized from Met and ATP, activation of SAM synthesis would be expected todecrease intracellular Met levels. As expected, only methionine was significantly reduced
among the amino acids in SAH treated cells (Fig. 7D).

2.3.2 SAH promotes SAM synthesis and decreases amount of MetNext, to investigate whether the decrease in intracellular Met was due toaccelerated consumption, I switched the L-Met in the culture medium to L-[methyl13C]Met and the fate of [methyl-13C]Met with or without SAH treatment were chased. As

a result, supplementation with SAH significantly decreased intracellular [methyl-13C]Metand increased [methyl-13C]SAM compared to the vehicle-treated control cells (Fig. 8A,
B). Furthermore, extracellular metabolomic data showed that [methyl-13C]Met levels
after supplementation with SAH were slightly lower than in the medium of control cells,
but were still sufficiently present (Fig. 8C). These results suggest that SAH promotes Met
reduction by converting endogenous Met to SAM, although I cannot exclude thepossibility that SAH inhibits SAM degradation.
The lower Met content in SAH treated cells suggests that longevity that derives
from SAH supplementation may involve a MetR-like state. Because high concentrationsof Met have been reported to shorten lifespan (Wu et al., 2013), if the lifespan extension
effect of SAH is due to Met reduction, it would be expected that excess Met would block
lifespan extension by SAH. Therefore, I evaluated the effect of the excess amount of Met
on the CLS. As expected, high concentrations of Met (10x Met) partially suppressed
lifespan extension when supplemented with SAH (Fig. 8D). Collectively, these results
indicate that SAH extends lifespan by reducing Met.

2.3.3 SAH treatment inactivate mTORC1 and promote autophagy
Given that Met inhibits autophagy and MetR extends CLS (Fabrizio and Longo,
2007) in an autophagy-dependent manner (Plummer and Johnson, 2019; Ruckenstuhl etal., 2014), I hypothesized that SAH might promote autophagy. The GFP-ATG8 cleavage
assay (Nair et al., 2011), which assesses the autophagy-dependent cleavage of GFP-Atg8

within the vacuole, can be used to monitor bulk autophagy progression in SAH treatedcells. The WT strains showed little autophagy activity during the logarithmic phase on
SDC medium (Fig. 9A). On the other hand, bulk autophagy was induced by exposure ofcells to SAH media during the logarithmic phase.
Since mTORC1 inhibits autophagy (Shimobayashi and Hall, 2014), I expected
SAH to inhibit mTORC1 function. Thus, I investigated the effect of SAH treatment on
the phosphorylation level of Rps6 (a homolog of mammalian S6), a downstream target
kinase of mTORC1 (Wullschleger and Hall, 2006). In the logarithmic growth phase of
WT strains, supplementation with SAH reduced the phosphorylation of Rps6, whichcorrelated with its kinase activity (Fig. 9B), suggesting that SAH reduces levels ofmTORC1 activity. Moreover, I examined if SAH extends CLS by mechanisms requiring
mTORC1 and autophagy. The extended CLS of tor1' cells were further enhanced when
SAH was added, but it was like that of WT treated with SAH, suggesting that SAH may
be involved in lifespan extension, at least in part, through inhibition of mTORC1 (Fig.
9C). Furthermore, when the ATG7 gene, which encodes an essential component of the
autophagic machinery, was deleted, SAH decreased the death rate of atg7' cells during
the early period of the lifespan (around 2 days), but overall, the CLS of atg7' cells
reduced (Fig. 9C). This suggests that autophagy is required for the sustained lifespanextension by SAH.



2.4 Discussion
In this chapter, I discussed that supplementation with SAH reduces intracellular
Met levels by promoting SAM synthesis. Supplementation with SAH mimicked thephysiological phenomenon that occurs during MetR, inhibiting the activity of mTORC1,
the major nutrient-sensing pathway of the organism, and promoting autophagy activity.
The effects of SAH can possibly explain how the high accumulation of SAM leads to theconsumption of Met and ATP, which in turn induces the inactivation of mTORC1 and theactivation of AMPK, respectively. Although there seem to be a contradiction between
SAM synthesis consuming Met and ATP and the increase in ATP levels (Fig. 7A, C),previous results by Ogawa et al. (2016) suggested that this was probably the result of theaction of an unknown mechanism by which AMPK compensates for the consumption ofATP. To compensate for consumed ATP, the activation of AMPK by supplementation with
SAH is also suggested to affect mitochondrial function, the major source of ATP in an
organism. In fact, metabolomic analysis suggests that SAH-treated cells have increased
metabolites in the TCA cycle, such as citric acid, fumaric acid, and malic acid, thereby
increasing mitochondrial activity (Fig. 7A, C).
Considering that glucose restriction (0.5% glucose) is associated with MetR
(Zou et al., 2020) and physiological increases in SAH levels have been reported in evenlower (0.05%) glucose-treated yeast cells (Ogawa et al., 2016), it is likely that the effectsof SAH also overlap with CR. Therefore, metabolomic analysis showed that
supplementation with SAH increased NAD+, which activates the sirtuin gene, acomponent of CR (Bonkowski and Sinclair, 2016) (Fig. 7A, Table 4). In addition,
spermidine, a natural polyamine that mimics CR, was increased (Eisenberg et al., 2009)(Fig. 7A, C, Table 4). These results suggest that SAH mimics some effects of CR as well

as MetR (Fig. 10).
While SAH inhibits the methylation reaction of SAM, this study shows that SAH
is also promotes SAM synthesis. However, it unknown how SAH activates SAM
synthesis. Christopher et al. (2002) proposed that the SAM/SAH ratio is important forcell growth, and a decrease in this ratio inhibits growth. Therefore, the supplementationof SAH may be a kind of hormesis to counteract the decrease in the SAM/SAH ratio andpromote SAM synthesis. To apply the benefits of SAH treatment in lifespan extension
more safely and effectively to humans, it is a future issue to investigate in detail themetabolic changes and molecular mechanisms of cells treated with SAH.
Although consuming a Met-restricted diet or the reduction of Met or ATP byinhibiting their respective synthetic enzymes can have benefits such as extending lifespan,
the finding that SAH, a metabolite of organism, affects Met and ATP levels provides new
insights in terms of their consumption. Moreover, Ogawa et al. (2022) found that SAH
treatment extends lifespan in Caenorhabditis elegans, a multicellular model organism, byactivating AMPK and the benefits of MetR. Accordingly, MetR extends the lifespan ofmany species, and exposure to SAH is expected to have various benefits acrossevolutionary boundaries.

Fig. 5 Stimulating SAM synthesis activates AMPK and extends lifespan.
Model for yeast longevity mediated by the stimulation of SAM synthesis by SAH.



Fig. 6 Growth rate of SAH treated cells
Growth rate and cell density of WT cells with (closed circle) or without (opened circle)
SAH treatment is shown. WT cells cultured in an SDC medium to log phase (OD600 nm= 0.2) were further cultured with or without 1 mM SAH. A representative set of data fromtwo biological repeats is shown. Mean ± S.D. (n = 3).




A

B




C

D




Fig. 7 Supplementation of SAH decreased intracellular methionine levels.
(A, B) Heat map (A) or volcano plot (B) showing metabolite levels in WT cells with orwithout SAH treatment. n = 3. FDR < 0.05, two-sided unpaired t-test. See also in Table 4.
(C) Metabolite changes in the presence or absence of SAH in WT cells. Red and blue
show increased and decreased metabolites, respectively. For a complete list, see Table 4.
Mean±S.D. (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided unpaired t-test
assuming equal variance). (D) Values of amino acids in WT cells were set to one,respectively, after which the amino acids values when SAH was added were compared.
Mean±S.D. (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided unpaired t-test
assuming equal variance).




A

B

C

D

SAH
SAH

Fig. 8 SAH promoted SAM synthesis and consumes methionine.
(A-C) Intracellular [methyl-13C]Met (A), Intracellular [methyl-13C]SAM levels (B), and[methyl-13C]Met levels in the medium (C) were assessed using CE-TOFMS.
Mean ± S.D, n = 3, two-sided unpaired t-test. (D) The CLS curve is indicated. ns, notsignificant; ***p < 0.001. Statistical analyses are shown in Table 5.




A

B

Pho85

Pho85

C

Fig. 9 SAH inhibits activity of mTORC1 and mimics the effects of MetR
(A, B) The relative intensity of free GFP (A) or phosphorylated Rps6 (B) normalized to
Cdc28 is shown. Mean ± SD, n = 3, two-sided unpaired t-test. (C) The CLS curve isindicated. ns, not significant; ***p < 0.001. Statistical analyses are shown in Table 5.




NH2
N
- OOC

S

O

N

N
N

NH3+
OH OH

SAH
AMPK

ATP

SAM
Met

TOR1
AutophagyLifespan extension
Fig. 10 SAH mimics MetR by promoting SAM synthesisTable 3 Strains used in Chapter 2
StrainName

Genotype

W303-1A

WT

MATa ; trp1-1 leu2-3,112 ade2-1 ura3-1 his3-11,15 can1-100 NBRP/YGRP

Source of Reference

YMA32

tor1 '

MATa ; tor1 '::kanMX4Lab. Stock

YYK1

atg7 '

MATa ; atg7 '::kanMX4Lab. StockTable 4 Metabolomics data in SAH treated cells
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6DFFKDURSLQH



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

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63

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$FHW\OEXW\UDWH

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

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

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9DO

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1$'3+

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      

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

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 *OXFRVDPLQH

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  *OXWDWKLRQH UHG

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3KH

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*OX*OX

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3\ULGR[LQH

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

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

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,OH



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



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     

   
  

Table 5 P value for CLS analysis in Chapter 2
FigureStrain A

Strain B

P -value against B

8D

WT, 1×Met

WT, 10×Met<0.001

WT, 1×Met

WT, 1×Met, SAH<0.001

WT, 10×Met

WT, 10×Met, SAH<0.001

WT, 1×Met, SAH

WT, 10×Met, SAH<0.001WT

WT, SAH<0.0001

tor1 '

tor1 '<0.0001

WT, AdoHcy

tor1 ', SAH

0.6151

atg7 '

atg7 ', SAH

0.01349C

General conclusions

Aging is a universal phenomenon common to all organisms (López-Otín et al.,2016), and it is the result of a complex interplay and accumulation of various factors suchas genomic instability, telomere attrition, loss of proteostasis, deregulated metabolic
signals, mitochondrial dysfunction, and senescence of the immune system (de Cabo et al.,2014; López-Otín et al., 2016; Campisi et al., 2019). Thus, although aging is a complex
process, studies of aging and lifespan have been conducted using various modelorganisms and it has become clear that similar to other life phenomena, aging and lifespanexhibit common mechanisms at the genetic level (Kenyon, 2005, 2010; Fontana et al.,2010).
Some lifespan pathways discovered from yeast are highly conserved in higherorganisms. For instance, CR is known to extend the lifespan of many model organisms,
and the molecules involved in CR were first discovered in yeast studies. The effect oflowering the glucose concentration in yeast from 2% to 0.5% in the medium on lifespanextension of CR was found to depend on a sirtuin gene, Sir2 (Imai et al., 2000). Deletionof Sir2 did not result in CR-induced lifespan extension, which was also observed for thesirtuin gene in C. elegans and Drosophila (Tissenbaum and Guarente, 2001; Rogina andHelfand, 2004). In addition, Powers et al. (2006) screened about 4800 essential genes inbudding yeast for identifying factors that extend lifespan and detected many mutations in
the mTORC1 pathway. Moreover, they found that adding rapamycin, an inhibitor ofmTORC1, extended lifespan. The mTORC1 pathway regulates important functions, suchas protein synthesis and autophagy, which degrades and recycles proteins in the cell
(Shimobayashi and Hall, 2014). Therefore, suppression of mTORC1 is critical forlifespan extension by inhibiting protein translation and promoting autophagy. Thus,
starting with yeast research, many pathways and molecular mechanisms that extend
lifespan have been identified so far; however, the detailed mechanisms remain unclear. It
has also become clear that several drugs and metabolites targeting mTORC1 extend
lifespan (Chin, et al., 2014; Hine, et al., 2015); however, the relationship between their
metabolic pathways and regulation of lifespan is not fully understood. In this study, therelated factors in the Ca2+ signaling pathway were predicted to regulate lifespan as related
to CR. Therefore, to search for a novel mechanism of lifespan extension in budding yeast,
I analyzed lifespan extension involving Sko1, a downstream factor of the HOG pathway,
and SAH, one of the Met metabolites.
In Chapter 1, I showed that Sko1 function, a regulator of the HOG pathway,
suppressed the oxidative stress sensitivity and short lifespan of hog1' cells, indicating
glycerol synthesis system-dependent resistance to oxidative stress and lifespan extension.
Currently, abnormal or inappropriate function of MAPK pathway has been identified in avariety of diseases, such as cancer, inflammatory diseases, and diabetes (Kim and Choi,2010). Hence, the lifespan regulation mechanisms involving the HOG pathway and its
downstream target genes identified in this study are expected to lead to the discovery of
new therapeutic agents and understanding of diseases using the stress-responsive MAPK
pathway, which is homologous to the HOG pathway in higher organisms. Furthermore,
yeast cells are exposed to osmotic, oxidative, and ethanol stresses during ethanol
fermentation, and the results of this study analyzing the relationship between stressresponse pathways and survival during aging are expected to be applied to breeding yeast
for fermentation.
In Chapter 2, I analyzed the mechanism of how the addition of SAH, a Metmetabolite, prolongs lifespan. I showed that SAH promotes SAM synthesis and consumes
Met, thereby decreasing intracellular Met concentrations, via metabolomic analysis andL-[methyl-13C]Met tracking. Furthermore, SAH inhibited TORC1 and activatedautophagy, which was expected to mimic the effects of MetR. Met metabolism regulatesa variety of metabolic processes, such as translation rates, gene expression, mitochondrial
function, and autophagy. MetR can extend lifespan and delay the onset of agingassociated pathologies in most model organisms. Although genetic manipulation of Met
metabolism and drug therapies such as metformin have been used as methods of MetR, it
is not easy for us to try these methods. This study and Ogawa et al. (2022) showed thatthe metabolite of organism SAH mimics MetR in both yeast and nematodes, which maylead to safer and more effective lifespan extension. It is expected that a similar effect ofMetR by SAH will be observed in further higher organisms in the future. In addition,
more detailed analyses of the effect of SAH treatment on lifespan extension are expected
to provide novel insights into the mechanisms of lifespan regulation by MetR.
Initially, both Sko1 and SAH were expected to control lifespan via CR.Indeed,promotion of glycerol synthesis, which activates Sir2, a component of CR, was essential
for prolonging the lifespan of sko1' cells. Also, supplementation with SAH increased
NAD+, a component of CR, and spermidine, a natural polyamine that mimics CR.
However, it is not well understood how these factors mediate lifespan extension via Sko1
deficiency and SAH.Therefore, exploring the specific factors of CR that are involved inthese life span extensions is a challenge for future analysis.
In this study, I suggest two novel mechanisms of lifespan extension in budding

yeast, and both of these mechanisms are expected to be highly conserved to otherorganisms, including humans. Therefore, I expect that our findings of the molecularmechanisms that regulate lifespan in budding yeast will be applied to human health, aging
and lifespan, and treatment of diseases.

Acknowledgements

First of all, I would like to express my great thanks to Professor Masaki Mizunuma forhis constant guidance, not only in technical matters, but also in the way of being a
researcher and its aspirations.
I have been greatly indebted to Professor Dai Hirata, Associate Professor Kazunori Kume,
Assistant Professor Makoto Horikawa, Dr. Takafumi Ogawa for their constant discussion,
guidance, and encouragement.
I am grateful to thank for Professor Masaki Mizunuma, Professor Seiji Kawamoto,
Associate Professor Koichi Funato, Associate Professor Kazunori Kume for fruitful
discussion and carefully reviewing this study.
I am deeply grateful to Professor T. Keith Blackwell, Professor Yoshikazu Oya, Professor
Tomoyoshi Soga, Dr. Muneyoshi Kanai, Dr. Takafumi Ogawa, Eri Sanda, Kumiko
Yonekita, Sachi Matsukami, Yuki Kohara for their technical guidance, discussions, andseveral experiments.
I also thanks Mizunuma’s Laboratory 2017-2023, Takako Ezaki, Dr. Takafumi Ogawa,Yuto Takeuchi, Kenji Nishikawa, Haruka Kondo, Yuika Seo, Mayuka Tanetani, Sachi
Matsukami, Saki Ueda, Konami Kawata, Yusaku Nakagaki, Maiko Baba, Chiho Funaki,
Kana Watanabe, Matthew Spence, Miyuki Aohara, Syunya Takeo, Yuki Chikaishi,
Takuya Tsuji, Kei Yoneda, Yuki Kohara, Takahiro Fujimoto, Sota Kanda, Kota Takashima,
Sara Mizogami, Hiroya Matsuzaki, Mayu Tsutsumi, Harumi Hiramatsu, Rikuto
Furuyama, Hiiro Yamashita for their constant guidance, support and help.
I have been a recipient of Japan Society for the Promotion of Science Fellowship (DC1)from April 2020 to March 2023, which I appreciate very much.
Finally, I would like to thank my parents and family from the bottom of my heart for theirrespect and support of my choice and decision.

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