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Roles of SIRT1 in neuronal oxidative damage and brain function

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Roles of SIRT1 in neuronal oxidative damage and brain function

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ROLES OF SIRT1 IN NEURONAL OXIDATIVE DAMAGE
AND BRAIN FUNCTION

by

Ying Li
________________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

December 2008

Copyright 2008 Ying Li
ii

Acknowledgements

My gratitude goes to my family and friends whose love and support over the years have
lit up my way in this foreign land distant from my home country. My advisor, Dr. Valter
D. Longo, has consistently inspired, supported and supervised me throughout my
academic quest and none of the work would have been possible without his insights. His
influence of having big ideas, thinking out of the box and believing in self will continue
to benefit me in the years to come. I am greatly obliged to Dr. Michel Baudry for
tremendous advice and help in both of my research projects. Dr. Christian Pike has
persistently encouraged and helped me throughout my graduate study. Dr. Stephen
Madigan has provided me with valuable advice. I am also indebted to the past and present
members in the Finch, Baudry, Pike and Davies laboratories for all the technical and
intellectual help bestowed upon me. Drs. Min Wei, Paola Fabrizio, Federica Madia and
Edoardo Parrella in Longo laboratory have given me vast support and guidance over the
years. The warmth and assistance offered by my colleagues Jia Hu, Sangeeta Cook,
Stavros Gonidakis, Abdoulaye Galbani, Priya Balasubramanian and Changhan Lee have
made each day a memorable one.

The experiments in chapter 2 were also made possible by the work of Dr. Wei Xu and Dr.
Michael McBurney. The experiments in chapter 3 were also a result of collaborative
efforts from the laboratories of Dr. Michel Baudry, Dr. Rafael de Cabo and Dr. David A.
iii

Sinclair. Of note, Meng-Hsiu Chou, Dr. Shaday Michan and Joanne Allard contributed
important experiments to the project.
iv

Table of Contents

Acknowledgements ................................................................................................................... ii

List of Figures ............................................................................................................... ............ vi

Abstract ...................................................................................................................... ............... ix

Chapter 1. Background and significance ................................................................................... 1
1.1. Oxidative Stress and Aging .............................................................................................. 1
1.1.1. Oxidative stress and aging .......................................................................................... 1
1.1.2. Pro-aging roles of insulin/IGF-I system ...................................................................... 3
1.1.3. Calorie restriction and aging ....................................................................................... 4
1.2. Sir2 in Oxidative Stress and Aging ................................................................................... 6
1.3. Long Term Potentiation in Learning and Memory ........................................................... 8
1.4. Oxidative Stress and ERK1/2 signaling in Normal Brain Function and Brain
Disease ..................................................................................................................................... 10

Chapter 2. Role of SIRT1 in IGF-I/ERK signaling and neuronal oxidative stress .................. 13
2.1. Summary ......................................................................................................................... 13
2.2. Introduction ..................................................................................................................... 13
2.3. Methods and Materials .................................................................................................... 17
2.4. Results ............................................................................................................................. 23
2.4.1. Inhibition of SIRT1 increases oxidative stress resistance in neurons ....................... 23
2.4.2. SIRT1 inhibition does not alter antioxidant enzyme expression in cultured
neurons ..................................................................................................................................... 30
2.4.3. SIRT1 does not alter the activation of Akt or CREB in cultured neurons ................ 31
2.4.4. SIRT1 inhibition decreases Ras/ERK activation in culture and in vivo ................... 34
2.4.5. Subcellular localization of SIRT1 in the brain .......................................................... 39
2.4.6. SIRT1 regulates Ras/ERK activity through deacetylating IRS-2 ............................. 43
2.4.7. MEK/ERK1/2 inhibition protects neurons against oxidative stress .......................... 46
2.4.8. Reduced oxidative brain damage and life span of SIRT1 knockout mice ................ 50
2.5. Discussion ....................................................................................................................... 54

Chapter 3. Role of SIRT1 in synaptic plasticity, learning and memory .................................. 62
3.1. Summary ......................................................................................................................... 62
3.2. Introduction ..................................................................................................................... 63
3.3. Methods and Materials .................................................................................................... 66
3.4. Results ............................................................................................................................. 76
3.4.1. SIRT1 deficiency does not alter gross brain anatomy .............................................. 76
3.4.2. SIRT1 is essential for normal cognitive capabilities in mice .................................... 79
3.4.3. Effect of SIRT1 overexpression on learning and memory ........................................ 89
3.4.4. Impaired LTP in SIRT1 knockout hippocampus ...................................................... 98
3.4.5. Overexpression of SIRT1 in brain has no effect on long-term potentiation ........... 102
3.5. Discussion ..................................................................................................................... 106

v

Summary and Conclusions .................................................................................................... 112

References .............................................................................................................................. 121
vi

List of Figures
Figure 1. SIRT1 inhibitors increase oxidative stress resistance in neurons ..............25
Figure 2. Effect of SIRT1 inhibitors alone on neuronal viability .............................26
Figure 3. Effect of high doses of nicotinamide on neuronal viability when
exposed to oxidative stress........................................................................................27
Figure 4. Inhibition of SIRT1 deacetylase increases oxidative stress
resistance in neurons .................................................................................................29
Figure 5. Inhibiting SIRT1 deacetylase does not protect against certain forms
of stress in neurons....................................................................................................30
Figure 6. Inhibiting SIRT1 deacetylase does not alter MnSOD or catalase
expression in cultured neurons ..................................................................................31
Figure 7. SIRT1 deacetylase does not alter the activation of Akt, S6 kinase or
CREB in cultured neurons ........................................................................................33
Figure 8. SIRT1 inhibitors decrease Ras/ERK1/2 activation in cultured
neurons ......................................................................................................................35
Figure 9. Inhibition of SIRT1 deacetylase decreases ERK1/2 activation in
cultured neurons and in vivo .....................................................................................36
Figure 10. SIRT1 inhibitor decreases IGF-I-induced, but not PMA-induced
ERK1/2 activation in HEK293 cells .........................................................................38
Figure 11. Subcellular localization of SIRT1 in hippocampus CA3 ........................40
Figure 12. Subcellular localization of SIRT1 in the forebrain ..................................41
Figure 13. Nucleus-cytosol shuttling of SIRT1 in cultured neurons ........................42
Figure 14. SIRT1 regulates Ras/ERK1/2 signaling via deacetylation of IRS-2 .......44
Figure 15. Representative blots showing the effect of SIRT1 on IRS-2
deacetylation in neurons ...........................................................................................45
Figure 16. Representative blots showing the effect of resveratrol on the
acetylation level of IRS-2 .........................................................................................46
Figure 17. Inhibiting MEK/ERK1/2 protects neurons against oxidative stress ........48
vii

Figure 18. ERK1/2 inhibition primarily attenuates necrosis ....................................49
Figure 19. A model for SIRT1 regulation of IGF-I signaling...................................50
Figure 20. Reduced oxidative damage in SIRT1 knockout mouse brain .................51
Figure 21. SIRT1 inhibition decreases survival in paraquat induced oxidative
stress ..........................................................................................................................53
Figure 22. Reduced life span of SIRT1 knockout mice under ad-lib and
calorie restriction conditions .....................................................................................54
Figure 23. SIRT1 deficiency does not change gross brain anatomy .........................79
Figure 24. SIRT1KO mice exhibit impaired immediate memory in Y maze
test .............................................................................................................................81
Figure 25. SIRT1KO mice exhibit impaired contextual fear ....................................83
Figure 26. SIRT1KO mice exhibit decreased conditioned fear to auditory cue .......84
Figure 27. SIRT1KO mice show impaired spatial learning in Barnes maze
test .............................................................................................................................86
Figure 28. Differential search strategies employed by WT and SIRT1KO
mice in Barnes maze test...........................................................................................88
Figure 29. SIRT1 protein levels in the hippocampus of NeSTO mice .....................90
Figure 30. SIRT1 overexpression in hippocampal neurons of NeSTO mice ...........91
Figure 31. Overexpression of SIRT1 does not alter spontaneous exploratory
and locomotor activities ............................................................................................93
Figure 32. SIRT1 overexpression in the brain does not alter associative
memory .....................................................................................................................94
Figure 33. Effect of SIRT1 overexpression on performance in 14-unit T-maze
test .............................................................................................................................96
Figure 34. Effect of SIRT1 overexpression on performance in Morris water
maze test....................................................................................................................97
Figure 35. SIRT1 deficiency has no effect on input/output properties at
Schaffer collateral CA1 synapses .............................................................................98
viii

Figure 36. Absence of SIRT1 impairs long-term potentiation .................................99
Figure 37. Absence of SIRT1 impairs long-term potentiation but not short-
term potentiation .....................................................................................................100
Figure 38. SIRT1 loss does not affect burst responses or paired pulse
facilitation in field CA1 of hippocampal slices ......................................................101
Figure 39. SIRT1 overexpression in the brain alters input/output properties at
Schaffer collateral CA1 synapses ...........................................................................103
Figure 40. Effect of SIRT1 overexpression on long term potentiation
recordings in field CA1 of hippocampal slices .......................................................104
Figure 41. Overexpression of SIRT1 does not affect long or short term
potentiation in field CA1 of hippocampal slices .....................................................105
Figure 42. Effect of SIRT1 overexpression on burst responses and paired-
pulse facilitation ......................................................................................................106
ix

Abstract

Aging is a common phenomenon of multiple organisms. In humans aging is frequently
accompanied by cognitive decline and occurrence of neurodegenerative diseases which
reduce the quality of life and impose financial stress on society. Delaying the aging
process, extending life span and decreasing the occurrence of age-related brain function
deficit have always been aspirations of human kind. Extensive research has advanced our
understanding of the mechanisms underlying aging, among which is the ability of calorie
restriction to increase longevity, and the pivotal regulatory roles of insulin/IGF-1
signaling pathway. Some recent studies identified silent information regulator 2 (Sir2;
SIRT1 is the mammalian homolog) as a key mediator of the beneficial effects of calorie
restriction and this prompted development of SIRT1 activators for human consumption to
delay aging and accompanying cognitive decline. However, our laboratory previously
showed in yeast that Sir2 can increase stress sensitivity and limit life span extension
under certain conditions, calling for more detailed characterization of SIRT1. In the
research described in this dissertation I extended this study to the mammalian system and
focused on the role of SIRT1 on the health of neurons and brain functions, especially
learning and memory.

This dissertation consists of three chapters. In chapter 1 I briefly review some recent
progress on aging, oxidative stress, insulin/IGF-1 signaling pathway and learning and
memory with emphasis on the involvement of SIRT1 in these processes. In chapter 2 I
x

focused on the role of SIRT1 in oxidative stress in neurons and its mechanisms. I found
that SIRT1 inhibition increased resistance to oxidative damage and this effect is partially
mediated by a reduction in IGF-I/IRS-2/Ras/ERK1/2 signaling. In chapter 3 I studied the
functions of SIRT1 in learning and memory. The experiments showed that deletion of
SIRT1 impairs a certain form of synaptic plasticity and reduce performance in several
different learning and memory tasks while overexpressing SIRT1 did not substantially
affect learning and memory.

Together, my studies reveal that SIRT1 exacerbates neuronal oxidative damage but is
essential in learning and memory, indicating that SIRT1 plays multiple roles in aging and
brain functions and that caution should be exercised in designing anti-aging or
therapeutic approaches that involve targeting SIRT1.
1

Chapter 1. Background and significance
1.1. Oxidative Stress and Aging
1.1.1. Oxidative stress and aging
Reactive oxygen species (ROS) can be generated at many cellular sites with respiratory
chain being the major source. Mitochondrial complex I and III are the two sites for
oxygen radical generation. By comparing short-lived rat with the long-lived pigeon
(although the body size and basal weight-specific metabolic rate are similar in both
species, rats have a maximum life span of 4 years whereas pigeons live up to 35 years), it
was shown that the generation of oxygen radical by complex I is closely related to aging
(Barja and Herrero, 1998). The free radical generation is not at a fixed percentage of total
electron flow through complex I; instead, it can be different in different animals in
relation to their longevity. Therefore, ROS is not just a byproduct of mitochondrial
respiration but rather its generation is a regulated process. Each cell has ROS scavenging
machinery composed of antioxidant enzymes and low-molecular-weight antioxidants.
The major antioxidant enzymes include superoxide dismutases (SOD), glutathione
peroxidases and catalases. The major antioxidants include glutathione, tocopherols and
vitamin C et cetera. Oxidative stress is generated when ROS exceeds the clearing
capacity of antioxidant machinery.

2

Free radical theory of aging was proposed as early as 1956 (Harman, 1956). Since many
ROS, such as peroxides, which are not free radicals, also play a role in oxidative damage
to cells, this theory was modified to the oxidative stress theory of aging. During the past
few decades evidence has accumulated in support of this theory. Firstly, the level of
oxidative damage becomes more apparent with aging. A profound increase of lipid
peroxidation (over 20 fold) was shown in aged rats (Roberts and Reckelhoff, 2001). A
broad range of proteins are also subjected to oxidative damage during aging, such as
mitochondrial aconitase (Yan et al., 1997), glucose-6-phosphate dehydrogenase (Agarwal
and Sohal, 1993) and Na
+
, K
+
-ATPase (Chakraborty et al., 2003). In addition to lipids
and proteins, oxidative damage to DNA has also been observed in aging. By measuring
the levels of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA, Hamilton ML et al. showed a
marked increase in oxidative modification of nuclear and mitochondrial DNA in aged
animals (Hamilton et al., 2001). Secondly, decreasing the generation of ROS or
increasing the repair of oxidative damage was shown to delay aging. Selective
overexpression of the MSRA gene (methionine sulfoxide reductase A, which catalyzes
the repair of oxidized methionine in proteins) in the nervous system markedly extends the
lifespan of Drosophila (Ruan et al., 2002). Thirdly, manipulations that increase life span
often reduce the age-related increase in oxidative damage. A strain of Drosophila selected
for their resistance to paraquat showed extended longevity (Vettraino et al., 2001).
Similarly, dwarf mice exhibited an increased life span compared to their wild type
littermates and they showed reduced levels of DNA and protein oxidation (Hauck and
Bartke, 2001).
3

Despite accumulating evidence supporting the oxidative stress theory of aging, little is
known about how the oxidative stress leads to aging. The redox status of cells is
determined by both ROS generation and elimination mechanism. But ROS generation
appears to play more crucial roles than ROS elimination. It was shown that the anti-
oxidant did not decrease with aging (Barja, 2004). The long-lived species seemed to have
less oxidant, suggesting that generation of ROS is less in these animals (Perez-Campo et
al., 1998). In addition, experimentally elevating the antioxidants in mammals had little
effects on the maximum lifespan (Orr et al., 2003).

1.1.2. Pro-aging roles of insulin/IGF-I system
A conserved method to consistently extend lifespan is the downregulation of insulin/
insulin-like growth factor-1 (IGF-I) like

signaling pathway in organisms ranging from
lower organisms to mice (Kenyon, 2001; Longo and Finch, 2003). In C. elegans,
mutations in daf-2, a gene encoding an insulin/IGF-1 receptor ortholog, doubled the
lifespan of the animal (Kenyon et al., 1993). Mutations in insulin-like receptor (InR) or in
CHICO, a downstream insulin receptor substrate-like protein, increase the lifespan of
Drosophila (Tatar et al., 2001). In mammals, IGF-I is a key mediator of growth hormone
(GH) action and acts systemically under GH control as well as locally at sites of its
production. Mice deficient in IGF-I receptor, insulin receptor in the fat, or insulin
receptor substrate 2 (IRS2) in the brain live longer (Bluher et al., 2003; Holzenberger et
4

al., 2003; Taguchi et al., 2007). Mutant dwarf mice that have reduced or absent GH, or
IGF-1, or high GH but low IGF-1, all have a longer lifespan (Bartke and Brown-Borg,
2004). Overexpression of Klotho, a circulating hormone, extends longevity in mice,
possibly through repressing intracellular insulin/IGF-1 signaling (Kurosu et al., 2005).

1.1.3. Calorie restriction and aging
Food restriction was shown to retard growth and elongate lifespan of rodents in 1930s by
McCay and colleagues. Further studies indicated that in the components of food, it is the
restriction of calorie intake that accounts for the delay of aging (Masoro et al., 1982). The
commonly adopted protocol of calorie restriction in rodents is to feed animals with
calorie intake of 20–40% less than that consumed ad libitum, without sacrificing
micronutrients. The beneficial effects of calorie restriction on lifespan were later
expanded to a broad range of species, including yeast, worms, flies and cows (Ingram et
al., 2001; Lin et al., 2000). These studies also suggest a conserved mechanism for aging.
Not only does CR elongates lifespan, it also delays the appearance of some markers of
aging process (Roth et al., 2002) and decreases the occurrence of the age-related diseases,
such as Parkinson's disease (Duan and Mattson, 1999)

or Alzheimer's disease (Zhu et al.,
1999). Some beneficial effects of CR are also exhibited in human subjects. CR was
reported to lower human blood glucose, total leukocyte count, cholesterol as well as
blood pressure (Walford et al., 1992). The study on humans with 2 year low-calorie
consumption in Biosphere2 showed physiological, hematologic, hormonal and
5

biochemical changes resembling those observed in CR rodents and monkeys. In addition,
although the participants had a marked reduction in weight, they exhibited excellent
health and a high level of physical and mental activity (Walford et al., 2002). It was also
reported that in the Japanese people on the island of Okinawa with lower calorie intake
than average have lower rates of death

due to cerebral vascular disease, malignancy and
heart disease (Kagawa, 1978).

Despite the long history of studying CR, how exactly CR delays aging and elongate
lifespan remains unclear. CR induces a broad range of physiological changes in tested
animals, such as lower body temperature, lower blood glucose and insulin levels, lower
level of NADH within cells, higher levels of the adrenal steroid dehydroepiandrosterone
sulfate and reduced body fat and weight among others. CR normally reduces the size of
some organs except that of brain. In the past few years, several hypotheses have been
proposed. One theory proposes that the reduction

in protein turnover may result in the
accumulation

of aberrant proteins and aging and that CR promotes the degradation

of
proteins for energy metabolism and therefore delays aging (Aksenova et al., 1998). In
another theory, CR benefits were related to the reduction of AGE (advanced glycation

end products), which represent the covalent modifications of proteins by derivatives of
glucose and are linked to age-related pathologies (Cefalu et al., 1995; Lee and Cerami,
1992). Most of the theories have gathered some experimental support but remain
controversial. Among them, reduction in oxidative stress in CR is one of the most
influential ones (Ingram et al., 2001). Firstly, oxidative damage to proteins (Aksenova et
6

al., 1998), lipid (Pieri et al., 1992), and DNA (Hamilton et al., 2001) is reduced by CR.
Secondly, CR-induced reduction in oxidative damage appears to come from a decrease in
the generation of mitochondrial ROS (de Cabo et al., 2003; Gredilla et al., 2001).
Another influential hypothesis has to do insulin/IGF-I signaling pathway. In organisms
ranging from yeast to rodents, both calorie restriction and mutations in insulin/IGF-1
signaling pathway extend life span. Although reduced insulin/IGF-I signaling does not
appear to mediate the CR effect in C. elegans, as DAF-16 was not required for CR
lifespan extension (Houthoofd et al., 2003), long-lived dwarf mice share many
phenotypic characteristics with long-lived CR mice, like decreased body weight and
temperature, reduced plasma glucose, insulin and IGF-I, increased insulin sensitivity,
reduced oxidative damage and increased antioxidant defenses (Bartke and Brown-Borg,
2004). As a reduction in plasma IGF-1 appears to account for a major portion of the
lifespan extension in dwarf mice, it is possible that CR is mediated in part by a reduction
in IGF-I. Since CR and dwarf mutation have additive effects on lifespan, CR may act on
other pathways as well (Tatar et al., 2003).

1.2. Sir2 in Oxidative Stress and Aging
Sirtuins, or Sir2 family proteins, are NAD-dependent class III histone deacetylases, and
are conserved from bacteria to humans (Frye RA, 2000). The mammalian homolog of
Sir2 is termed SIRT1. Sir2 catalyzes deacetylation of substrate proteins in a NAD-
dependent manner. Small molecule Sir2 inhibitors including nicotinamide, sirtinol,
7

splitomicin and Sir2 activators including resveratrol have been developed (Howitz KT et
al., 2003; Kaeberlein M et al., 2005).

Sir2 was considered as an anti-aging protein. In yeast overexpression of Sir2 extended
replicative lifespan and deletion of Sir2 shortened the lifespan (Kaeberlein M et al.,
1999). In C. elegans Sir2 homolog extended longevity (Tissenbaum HA and Guarente L.,
2001), possibly through regulating daf-16 (Tissenbaum HA and Guarente L., 2001;
Berdichevsky A et al., 2006). In Drosophila overexpression of dSir2 (Sir2 homolog)
ubiquitously or in neurons alone increased lifespan.

Sir2 was also thought to mediate calorie restriction (CR) induced lifespan extension. This
was supported by studying yeast replicative lifespan (Lin SJ et al., 2000) and diet
restriction in Drosophila (Rogina B and Helfand SL, 2004; Rogina B et al., 2002). In
mammals, SIRT1 expression was induced in CR rats or fasting (Cohen HY et al., 2004).
Under CR conditions, SIRT1 knockout mice did not show increased locomotor activity as
wild type mice did (Chen et al., 2005).

However, recent studies put the anti-aging role of Sir2 to question. Our lab showed in
yeast that lack of Sir2 deacetylase activity further extended the lifespan of long-lived
Sch9 or Ras2 deleted mutants, suggesting that under certain conditions Sir2 is actually
pro-aging (Fabrizio F et al., 2005). Evidence is also accumulating suggesting that Sir2 is
not required for CR mediated lifespan extension. In yeast calorie restriction was found to
8

extend lifespan also in a Sir2-independent manner (Kaeberlein M et al., 2004; Lamming
DW et al., 2005). Our lab has shown that Sir2 actually blocks the extreme chronological
longevity extension caused by severe calorie restriction (Fabrizio F et al., 2005). In
mammals, SIRT1-null mice resemble CR mice in several aspects: smaller stature, lower
blood glucose, lower insulin. SIRT1-null mice show increased IGFBP-1 and slightly
decreased IGFBP-3, which is consistent with the changes during prolonged fasting and
severe nutrient restriction (Smith WJ et al., 1995).

Sir2 plays roles in stress resistance. In yeast lack of Sir2 deacetylase activity induces
resistance to heat and oxidative stress and deletion of Sir2 further increased stress
resistance in long-lived Sch9 ∆ mutants (Fabrizio F et al., 2005). In mammalian cells
SIRT1 deacetylase regulates several stress-responsive factors, such as p53 tumor
suppressor, forkhead transcription factors and NF- κB (Vaziri H et al., 2001; Langley E et
al., 2002; Yeung F et al., 2004). This suggests that SIRT1 plays important roles in
mammalian stress resistance.

1.3. Long Term Potentiation in Learning and Memory
Synaptic plasticity, the ability for synapses to change their strength in synaptic
transmission has long been hypothesized as one of the major cellular mechanisms
underlying learning and memory. Long term potentiation (LTP) is an extensively studied
model of synaptic plasticity. It can be induced in vivo (in living animals) or in vitro on
9

brain slice preparations. It is commonly studied in the Schaffer collateral pathway
between CA3 pyramidal neurons and CA1 area in the adult hippocampus. When a high
frequency stimulation (HFS, for example, 100 Hz lasting 1 second) or theta burst
stimulation (TBS) is applied the amplitude of excitatory postsynaptic potential (EPSP) or
the slope of the rising phase of EPSP becomes enhanced compared to that recorded
before the delivery of this induction stimulation. This “potentiation’ of synaptic strength
can last for hours, days or even longer depending on the preparations and induction
protocol used, hence the term long term potentiation (Bliss and Collingridge, 1993).

The induction of classical LTP depends on NMDA receptors that act as a coincidence
detector. Following HFS, glutamate, the excitatory neurotransmitter released by
presynaptic terminals, bind to AMPA receptors on the postsynaptic neurons, which in
turn allow sodium (Na
+
) ions to enter the cells and depolarize the membrane to such an
extent that magnesium (Mg
2+
) ions, which usually block the NMDA receptors, are
expelled. The simultaneous removal of magnesium and binding of glutamate to NMDA
receptors during HFS thus trigger an influx of calcium (Ca
2+
) into the cells. The Ca
2+
influx leads to a complicated cascade of signaling events at postsynaptic sites which are
still not well understood. Based on published studies, the activation of multiple protein
kinases are critical to this process, including protein kinase C (PKC), protein kinase A
(PKA), extracellular signal-regulated kinases (ERK1/2) and phosphoinositide 3-kinases
(PI3K), et cetera. As a result, AMPA receptors are modified and inserted onto the
postsynaptic membranes. The increase in the number of AMPA receptors on postsynaptic
10

memebranes thus produces the potentiation of synaptic strength (reviewed in (Malinow
and Malenka, 2002). While the mechanisms of the induction and the expression of LTP is
pretty well understood, how LTP is maintained for hours or even longer remains unclear.
Some recent studies suggest that a sustained kinase activity may be involved (Sacktor,
2008). It is also suggested by other studies that gene transcription and local protein
synthesis may be required for this process (Sutton and Schuman, 2006).

Making LTP an appealing candidate mechanism for learning and memory are several
properties: 1) LTP is input specific, which maximizes brain capacity for information
storage; 2) the associativity of LTP somehow resembles classical conditioning; 3) LTP
can be rapidly induced and be long-lasting, which are also the characteristics of memory
(Bliss and Collingridge, 1993). Accumulating evidence suggests that LTP is indeed
involved in learning and memory. On the one hand, LTP can be induced “naturally” in
learning processes (Tye et al., 2008; Whitlock et al., 2006). On the other hand, blocking
the induction (Morris et al., 1986), expression (Rumpel et al., 2005) or maintenance
(Pastalkova et al., 2006; Shema et al., 2007) of LTP all significantly impair learning or
memory.

1.4. Oxidative Stress and ERK1/2 signaling in Normal Brain
Function and Brain Disease
Reactive oxygen species appear to be necessary for normal brain function, especially
learning and memory. For example, superoxide, can serve as a cellular messenger during
11

long-term potentiation (LTP, a cellular model believed to underlie learning and memory)
and scavengers of superoxide blocked LTP (Klann E, 1998). Hippocampus-dependent
memory is impaired in young Cu/Zn-SOD or EC-SOD overexpressed mice (Serrano F et
al., 2004). But aged EC-SOD transgenic mice exhibited better hippocampus-dependent
spatial learning (Hu D et al., 2006).

Activation of p44/42 MAPK (i.e. ERK1/2) signaling pathway is involved in learning and
memory. ERK was found to be rapidly activated after LTP and this activation was
required for the induction of LTP in rat hippocampus (English JD et al., 1997). Systemic
administration of an inhibitor of MEK1 (activator of MAPK) caused impairment in
contextual and auditory fear conditioning (Atkins CM et al., 1998). Mice expressing
dominant-negative MEK1 in the forebrain showed decreased ERK activation in the
hippocampus and impaired spatial memory and impaired contextual fear conditioning
while auditory fear conditioning remained intact (Kelleher RJ et al., 2004).

Oxidative stress is involved in several neurodegenerative diseases. Oxidative stress is
thought to play a significant role in both the onset and progression of Alzheimer's disease.
Oxidation of lipids, protein and nucleic acids are found in AD patients and in animal
models (Smith MA et al., 2000). Actually, oxidatively modified macromolecules precede
A beta plaque deposition in transgenic mouse models of AD. Decreased expression of
MnSOD, a major antioxidant enzyme, in APP mutant mice led to increased Abeta plaque
(Li F et al., 2004). On the other hand, antioxidant vitamin E reduced Abeta deposition in
12

transgenic animals (Frank B et al., 2005). Oxidative stress may contribute to the
pathogenesis of AD by several mechanisms. It may initiate neuronal apoptosis, activate
kinases such as GSK3 which may phosphorylate tau, and may also cause microglia
activation which in turn aggravates cellular insults (Culmsee C et al., 2006; Nunomura A
et al., 2006). Oxidative damage is also evident in stroke and other neurodegenerative
diseases like Parkinson's disease.
13

Chapter 2. Role of SIRT1 in IGF-I/ERK signaling and
neuronal oxidative stress

2.1. Summary
Sirtuins have been shown to protect cells and delay aging, but our laboratory showed that
yeast Sir2 can also increase stress sensitivity and limit life span extension. Here we
provide evidence for a role of the mammalian Sir2 ortholog SirT1 in the activation of a
pathway that sensitizes neurons to oxidative damage. Inhibition of SirT1 increased
acetylation and decreased phosphorylation of IRS-2, reduced Ras activation and reduced
ERK1/2 phosphorylation, suggesting that SIRT1 may enhance IGF-I signaling in part by
deacetylating IRS-2. Inhibition of either SIRT1 or Ras/ERK1/2 was associated with
resistance to oxidative damage. Consistently, both markers of oxidized proteins and lipids
were reduced in the brain of old SIRT1 deficient mice. However, the life span of the
homozygote knockout mice was shorter under both normal and calorie restricted
conditions, probably due to other beneficial effects of SIRT1. These results are in line
with findings in yeast and other model organisms suggesting that mammalian sirtuins can
play both protective and pro-aging roles.

2.2. Introduction
Sirtuins, or Sir2 family proteins, are NAD
+
-dependent class III histone deacetylases
conserved from bacteria to humans (Frye, 2000). Sir2 modulates aging and life span in
14

yeast, C. elegans and Drosophila (Fabrizio et al., 2005; Kaeberlein et al., 1999; Rogina
and Helfand, 2004; Tissenbaum and Guarente, 2001). Though earlier studies showed the
anti-aging effect of Sir2 and its function in calorie restriction (CR) in lower eukaryotes,
later studies found that Sir2 is not required for CR-induced life span extension in yeast
(Kaeberlein et al., 2004) or worms (Hansen et al., 2007; Kaeberlein et al., 2006; Lee et
al., 2006). Notably, our lab showed that lack of Sir2 in yeast further extended the lifespan
of calorie restricted cells, or long-lived Sch9 (Fabrizio et al., 2005) or of mutants with
deficiencies in the Ras/cAMP pathway suggesting that Sir2 can also promote aging
(Fabrizio et al., 2005). Sch9 is homologous to both mammalian S6 kinase and Akt.
Expression of mammalian Akt was shown to partially rescue defects in yeast lacking
SCH9 but a more recent paper suggests that Sch9 may be more similar to S6 kinase
because of its pattern of phosphorylation by TORC1 (Urban et al., 2007). Notably both
Akt and S6 kinase have been implicated in promoting aging in higher eukaryotes. SIRT1,
the mammalian ortholog of yeast Sir2, has been shown to regulate numerous
physiological processes including glucose metabolism, DNA repair and apoptosis
(Bordone et al., 2006; Cohen et al., 2004; Luo et al., 2001; Moynihan et al., 2005;
Rodgers et al., 2005; Sun et al., 2007). In mammalian cells SIRT1 regulates several
stress-response factors, such as p53 tumor suppressor (Langley et al., 2002; Vaziri et al.,
2001), forkhead transcription factors (Brunet et al., 2004; Motta et al., 2004), and NF- κB
(Yeung et al., 2004) yet it remains unclear if/how SIRT1 regulates resistance to oxidative
stress. Notably, SIRT1 is expressed at a high level in the brain compared to other organs
(Michishita et al., 2005).
15

Genetic manipulations which reduce insulin-like signaling extend the life span of C.
elegans, Drosophila and mammals (Kenyon, 2001; Longo and Finch, 2003). Reduction
of insulin/IGF-I signaling also extends life span in mice (Bluher et al., 2003;
Holzenberger et al., 2003; Taguchi et al., 2007). Work in C. elegans and Drosophila
point to one major longevity regulatory pathway which includes IGF-I-like receptor, Akt
and FOXO stress resistance transcription factors (Hwangbo et al., 2004; Kenyon et al.,
1993). These studies and others have shown that reduced insulin/IGF-I-like signaling also
protects against oxidative damage and other forms of stress in mice (Holzenberger et al.,
2003). Furthermore, Sir2/SIRT1 has been linked to insulin/IGF-1 signaling pathway: in C
elegans Sir2.1 interacts with 14-3-3 proteins to activate DAF-16, which is also part of the
insulin-like pathway (Berdichevsky et al., 2006; Brunet et al., 2004; Wang and
Tissenbaum, 2006). Our studies of the chronological life span of yeast have revealed a
similar longevity and stress resistance regulatory pathway in which glucose, instead of
IGF-I, activates the serine threonine kinase Sch9 which causes the down-regulation of the
downstream stress resistance kinase Rim15 (Cheng et al., 2007; Fabrizio et al., 2001).
Others have shown a similar effect of Sch9 in the regulation of the replicative life span
(Kaeberlein et al., 2005). Our studies have also pointed to a second pro-aging pathway
which includes Ras, adenylate cyclase, PKA and the stress resistance transcription factors
Msn2/Msn4 (Fabrizio et al., 2003; Fabrizio et al., 2004), which is also implicated in the
regulation of the yeast replicative life span (Kaeberlein et al., 2005; Medvedik et al.,
2007). Recently, the down-regulation of the adenylate cyclase/PKA pathway by deletion
16

of 5 adenylyl cyclase was shown to extend the life span of mice and to protect from
reduced bone density and aging-induced cardiomyopathy (Yan et al., 2007). Analogously
to our findings in yeast (Fabrizio et al., 2003; Fabrizio et al., 2001), this study in 5
adenylyl cyclase deficient mice showed increased levels of MnSOD and stress resistance
(Yan et al., 2007). These studies support the hypothesis that the mechanisms of life span
regulation are conserved and that S. cerevisiae chronological life span can point to
additional pathways and mechanisms important for mammalian aging and diseases.

Here we studied primary rat cortical neurons and other mammalian cells to test the role of
SIRT1 in stress resistance. Because we have shown that the down-regulation of either the
Ras or Sch9 pathway extends longevity and increases stress resistance in yeast, and
because homologs of these signal transduction proteins are major components of the pro-
aging insulin/IGF-I-like pathways, we focused on the potential role of SIRT1 in
regulating mammalian Ras and Akt/S6k signaling. In agreement with our results in S.
cerevisiae, we found that inhibition of SIRT1 increased resistance to oxidative stress in
neurons. Our data indicate that this effect is mediated in part by the deacetylation of IRS-
2 and up-regulation of insulin/IGF-1R/IRS-2/Ras/ERK1/2 signaling. Although we also
show that mice lacking SIRT1 have reduced levels of markers of oxidative damage in the
brain, these cellular effects did not translate into a longer life span and in fact the mice
lacking SIRT1 displayed severe developmental defects and were short lived under both
ad lib and calorie restricted conditions. These results indicate that the role of
17

SIRT1deficiency in protecting aginsta oxidative damage is eclipsed by the important role
of this deacetylase in many normal functions.

2.3. Methods and Materials

Materials Antibodies against SIRT1 (07-131), CREB (clone NL904; 05-767), IRS2 (06-
506) and phosphotyrosine clone 4G10 (05-321), Ras activation assay kit (17-218) and
SIRT1 deacetylase (17-370) were obtained from Upstate. Anti-GAPDH (ab9484)
antibody was from Abcam (Cambridge, MA). Antibodies for NeuN (MAB377), GluR2/3
(AB1506) and MAP2 (AB5622) were from Chemicon. Antibodies against Phospho-Akt
(S473) (AF887) and Akt (MAB2055) were from R&D Systems (Minneapolis, MN).
Anti-V5 (R960-25) antibody was from Invitrogen (Carlsbad, CA). Antibodies against
phospho-CREB (Ser133) (87G3) (9198), phospho-p44/42 MAP Kinase (Thr202/Tyr204)
(9101), p44/42 MAP Kinase (9102), IRS-1 (2382), IRS-2 (4502), acetylated-lysine (Ac-
K-103) (9681), phospho-p70 S6 Kinase (Thr389) (9234) and p70 S6 Kinase (9202) were
purchased from Cell Signaling Technology (Danvers, MA). Nicotinamide (N0636),
sirtinol (S7942) and insulin-like growth factor-I (IGF-1) (I3769) were from Sigma-
Aldrich (St. Louis, MO). SL 327 (1969) and U0126 (1144) were from Tocris Bioscience
(Ellisville, MO). U6Pro-SIRT1-siRNA was from Dr. David A. Sinclair encoding siRNA
targeting GAAGTTGACCTCCTCATTGT. SirT2DN (pEGFP-C1-hSIRT2 N168A) was
from Dr. Eric Verdin. V5-SIRT1 was from Dr. Marty W. Mayo.

18

Cell culture and transfection Neurons from E18 Sprague-Dawley (SD) rat cerebral
cortices were dissociated in neurobasal medium supplemented with 0.5 mM L-glutamine,
25 µM L-glutamic acid and 2% B-27 and plated at 3x10
4
/well onto 96-well plates for
viability assays, at 10
5
/well onto 6-well plates for immunostaining or 5x10
5
for
immunoblotting, at 3x10
6
onto 10 cm dishes for activity assay or immunoprecipitation.
Neurons were incubated at 37°C in 5% CO
2
and fed twice per week with neurobasal
medium supplemented with B-27 and 0.5 mM L-glutamine until ready for transfection on
5-7 DIV or other experiments on 10-14 DIV. Neurons for MTT were pretreated with
SIRT1 inhibitor for 48 hr prior to exposure to H
2
O
2
or menadione for 24 hr in Eagle’s
minimal essential medium supplemented with 21 mM glucose, 5% fetal bovine serum
and 5% horse serum.

Negative control and SIRT1 siRNA were obtained from Qiagen and the target sequence
for SIRT1 is AAGTGCCTCAAATATTAATAA. The sequence specificity of siRNA
against rat SIRT1 was verified by a genome-wide search against the rat build four-
genome data base (8014 sequences; 5,387,086,425 total letters, NCBI). At least 2 nt
differences were found in the nearest match, and no predicted rat sirtuins (SIRT2-7) were
in the top 20 hits. On 5 DIV rat cortical neurons were transfected with 25 nM control or
SIRT1 siRNA using HiPerFect Transfection Reagent (Qiagen, USA). 2 days later cells
were treated with chemicals or collected for assays. Neurons were transfected with
corresponding plasmids using calcium phosphate precipitation method with Clontech
CalPhos™ Mammalian Transfection Kit as previously described (Jiang et al., 2004).
19

HEK293 cells were cultured in DEME supplemented with 10% fetal bovine serum.
HEK293 cells were transfected with corresponding plasmids using Lipofectamine LTX
reagent (Invitrogen, Carlsbad, CA).

SIRT1 deacetylase activity was measured using a fluorometric SIRT1 Assay Kit
(CS1040, Sigma, St. Louis, MO) according to manufacturer’s instructions except that 1
μM trichostatin A was also added to the reaction. Fluorescence intensity at 444 nm (exc.
355 nm) was recorded and normalized to μg of protein and all values were represented as
% of control.

MTT survival assay Neuronal cultures

grown in 96-well plates were washed once with
HBSS and their mitochondrial viability

was determined by 3-(4,5-Dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, MTT was added to the
culture at 0.5 mg/ml for 4 hr at 37°C and formazan crystals were dissolved overnight
with lysis solution containing 50% of dimethylformamide and 15% SDS. Absorption

was
read at 570 nm and normalized to controls. 3-4 wells were used for each condition in
each experiment. Data from 3 independent experiments were averaged and shown as
mean±SEM.

20

LDH assay was performed with CytoTox 96® Non-Radioactive Cytotoxicity kit
(Promega, Madison, WI) per manufacturer’s instructions. Data from 3 independent
experiments were averaged and shown as mean±SEM and normalized to control.

Apoptosis was measured with Cell Death Detection ELISA
PLUS
kit (Roche, Indianapolis,
IN) per manufacturer’s instructions. Data from 10 wells from 3 independent experiments
were averaged and shown as mean±SEM and normalized to control.

Live/Dead Assay (L3224, Invitrogen) was also used to assess cell viability. Neurons
were stained with 2 μM calcein AM (green fluorescent due to esterase in living cells) and
2 μM ethidium homodimer 1 (EthD-1, red fluorescent once binding to DNA) for 10 min
at 37°C followed by washing with HBSS and observation under the microscope. In each
experiment 2 coverslips were used for each condition and 4 random fields captured for
each coverslip. Live (green) and dead (red) cells were counted from 3 independent
experiments. A portion of a representative image is shown for each condition.

Immunocytochemistry Neurons were fixed with 4% formaldehyde in PBS for 10 min at
room temperature (RT) and permeabilized with 0.5% Triton X-100 for 5 min. After
blocking with 5% goat serum for 1 hr the cells were incubated with primary antibodies in
2% goat serum for 2 hr at RT and then incubated with a corresponding fluorescence-
tagged secondary antibody for 45 min.

21

Western blotting and immunoprecipitation (IP) Cultured cells or brain tissues were
homogenized in lysis buffer (1% SDS, 10 mM Tris, pH 7.4, protease inhibitor cocktail, 1
mM sodium orthovanadate, 1 mM sodium fluoride), followed by boiling at 95°C for 5
min and centrifugation at 14,000 rpm at 4°C for 30 min. Equal amounts of supernatant
protein were resolved on SDS-PAGE and immunoblotted with indicated antibodies for
ECL detection. The blots were quantified using NIH image and band intensities were
normalized to that of control. For coimmunoprecipitation, cells were lysed in modified
radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl , pH 7.4, 150 mM NaCl, 1%
NP-40, protease inhibitor cocktail, 1 mM sodium orthovanadate, 1 mM sodium fluoride).
Equal amounts of supernatant were incubated with 5 µg of anti-V5 or control IgG for 1 h
at 4°C with constant agitation and then also with protein A agarose for 15 hrs. The
immune-complex was washed five times and eluted off beads in Laemmli sample buffer.
For IP cells were lysed in modified RIPA buffer containing 1 µM TSA and 5 mM
nicotinamide and incubated with anti-IRS1 or anti-IRS2 antibodies and the proteins were
pulled down with protein A agarose.

In vitro deacetylation IRS-2 was immunoprecipitated from HEK293 cells and equal
amount of IRS-2 was incubated with different amounts of SIRT1 deacetylase (0, 1, 2 µg)
and 1 mM NAD in assay buffer (25 mM Tris pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl
2
, 0.2 mM DTT) for 1.5 hr at RT. The reaction was stopped by addition of
denaturing buffer and subjected to SDS-PAGE and immunoblot.

22

Ras Activation Assay Ras activation assay (Upstate 17-218) was performed according to
manufacturer’s instructions. Briefly, Ras binding domain of Raf-1 (bound to glutathione
agarose) was used to immunoprecipitate Ras-GTP from cell lysates. The IP complex as
well as whole cell lysates were resolved on SDS-PAGE and immunoblotted for Ras.

Animals Wild-type C57BL/6J male (3 days and 2 months old) mice were used for brain
fractionation and oxidative stress resistance studies. SIRT1+/+, SIRT1+/- and SIRT1-/-
genotypes have been described previously (McBurney et al., 2003). Mice were deeply
anesthetized with isoflurane followed immediately by decapitation and brains (and
hippocampus) were collected right away. In life span studies the animals were kept in a
12/12 hour light-dark cycle. For ad libitum (AL) group mice were fed NIH-31 standard
feed. For CR mice were fed daily with NIH31/NIA fortified food containing 60% of
calories consumed by the AL animals. Water was available ad libitum for both groups.
For oxidative stress resistance experiment, oxidative stress was induced by
intraperitoneal paraquat injection (70 mg per kg body mass). Then the survival of mice
was checked every hour (nicotinamide experiment) or every 2 hrs (SIRT1 knockout
experiment) and the test was censored at 100 h.

Brain Fractionation Forebrain homogenates were separated into nuclear, cytosol and
membrane fractions through sucrose-gradient centrifugation. Briefly, the forebrain was
homogenized in chilled buffer containing 0.32 M sucrose, 10 mM HEPES pH 7.4, 2 mM
EDTA, protease inhibitors, phosphatase inhibitors and centrifuged at 1000 x g for 15 min
23

to yield the nuclear fraction pellet. The supernatant was then centrifuged at 200,000 x g
for 15 min to yield cytosol in the supernatant. The resulting pellet was resuspended and
centrifuged 4 more times to obtain the membrane fraction.

Determination of Oxidative Stress Half brain minus cerebellum was homogenized in 50
mM phosphate buffer pH 7.4 and centrifuged to yield supernatant, which was then used
to assay for protein carbonyl content with OxiSelect™ Protein Carbonyl ELISA Kit
(CellBiolabs Inc, San Diego, CA) according to manufacturer instructions. For lipid
peroxidation half brain minus cerebellum was homogenized in TBARS homoginization
buffer and subjected to TBARS assay as previously described (Liu et al., 2003).

Statistical analysis Student’s t-test was used to compare 2 groups. One-way ANOVA
was used to compare multiple groups and Turkey’s multiple comparison test was used as
a post-hoc test. Groups were determined significantly different when P<0.05.

2.4. Results
2.4.1. Inhibition of SIRT1 increases oxidative stress resistance in neurons
Previous studies in our lab showed that the deletion of SIR2 increased stress resistance in
S. cerevisiae (Fabrizio et al., 2005). Here, we investigated whether SIRT1, the ortholog
of yeast Sir2, plays a similar role in mammalian cells. Oxidative stress was induced in
cultured neurons by hydrogen peroxide or menadione, (which generates both superoxide
24

and hydrogen peroxide) and cell viability was measured by the MTT assay. 10 days in
vitro (DIV) cortical neurons were incubated with 400 µM of H
2
O
2
or 7.5µM of
menadione for 24 hr, followed by MTT assay. After H
2
O
2
treatment, approximately 30%
of neurons survived, whereas 50% survived the menadione treatment (Fig. 1 A, B, C, D).
We also pretreated neurons with SIRT1 inhibitors, nicotinamide or sirtinol at various
concentrations for 48 hrs and then subjected them to the above oxidative stress protocol.
Nicotinamide dose-dependently increased neuronal survival after H
2
O
2
treatment. 500
µM and 5 mM of nicotinamide pretreatment increased the survival of H
2
O
2
-treated cells

to 52.3 ± 4.7% (P<0.01, H
2
O
2
vs. H
2
O
2
+500 µM nicotinamide, n=3, one-way ANOVA,
Turkey’s test) and 58.7 ± 2.3% (P<0.001, H
2
O
2
vs. H
2
O
2
+5 mM nicotinamide, n= 3, one-
way ANOVA, Turkey’s test), respectively (Fig. 1 A); while 10 µM (P>0.05, H
2
O
2
vs.
H
2
O
2
+ 10 µM nicotinamide, n=3, one-way ANOVA, Turkey’s test) and 100 µM (P>0.05,
H
2
O
2
vs. H
2
O
2
+ 100 µM nicotinamide, n=3, one-way ANOVA, Turkey’s test) of
nicotinamide showed no significant effect. Nicotinamide also dose-dependently rescued
neurons in menadione-induced

oxidative stress (Fig. 1 B). 500 µM and 5 mM
nicotinamide pretreatment increased the survival of menadione

-treated cells

to 77.0 ±2.2
% (P<0.01, menadione

vs. menadione + 500 µM nicotinamide, n=3, one-way ANOVA,
Turkey’s test) and 86.1 ±2.4% (P<0.001, menadione

vs. menadione + 5 mM
nicotinamide, n=3, one-way ANOVA, Turkey’s test), respectively; while 10 µM (P>0.05,
menadione

vs. menadione + 10 µM nicotinamide, n=3, one-way ANOVA, Turkey’s test)
and 100 µM (P>0.05, menadione

vs. menadione + 100 µM nicotinamide, n=3, one-way
ANOVA, Turkey’s test) of nicotinamide showed no significant effect. These values were
adjusted to the effects of nicotinamide alone. Nicotinamide alone was not significantly
different from control (Fig. 2, P>0.05, control vs. nicotinamide at each concentration,
n=4, one-way ANOVA).
H
2
O
2
10 μM 100 μM 500 μM5mM
0
25
50
75
400 μM H
2
O
2
+Nicotinamide
A
***
**
MTT reduction
% of control
H
2
O
2
5 μM25 μM50 μM
0
25
50
75
C
***
400 μM H
2
O
2
+Sirtinol
**
MTT reduction
% of control
10 μM 100 μM500 μM5mM
0
25
50
75
100
Menadione
7.5 μM
Menadione+Nicotinamide
**
***
B
MTT reduction
% of control
5 μM25 μM50 μM
0
25
50
75
100
Menadione
7.5 μM
Menadione+Sirtinol
D
**
MTT reduction
% of control
**

Figure 1. SIRT1 inhibitors increase oxidative stress resistance in neurons
Cortical neurons from E18 rat embryos were cultured onto 96-well plates. On 10-14 DIV neurons were
treated with nicotinamide (A, B) or sirtinol (C, D) at indicated concentrations for 48 hrs. Oxidative stress
was then induced by 400 μM of hydrogen peroxide (A, C) or 7.5 μM of menadione (B, D), respectively. 24
hrs later cell viability was measured by MTT assay. The ODs are normalized to respective controls
(namely: H
2
O
2
/menadione relative to vehicle-treated control; H
2
O
2
+nicotinamide or H
2
O
2
+sirtinol relative
to nicotinamide/sirtinol alone; menadione+nicotinamide or menadione+sirtinol relative to
nicotinamide/Sirtinol alone). Compared with vehicle-treated ctrl, MTT reduction was decreased by sirtinol
alone (97 ± 1.1% at 5 μM, 87.0 ± 1.7% at 25 μM and 78.3±1.8 % at 50 μM), but not by nicotinamide
(102.0±1.5% at 10 μM, 103.0±1.5% at 100 μM and 101.3±1.0% at 500 μM and 103.8±1.4% at 5mM). 3 or 4
wells were used for each condition per experiment and 3 independent experiments were performed. Data
from 3 experiments are shown here as mean+SEM (*P < 0.05, **P < 0.01, and ***P < 0.001 compared
with H
2
O
2
- or menadione- treated groups, respectively, one-way ANOVA).

25

Figure 2. Effect of SIRT1 inhibitors alone on neuronal viability
10-14 DIV neurons were treated with nicotinamide or sirtinol for 48hrs and cell survival was assessed by
MTT assay. Nicotinamide did not change the viability of cells (P>0.05, control vs. Nico at each
concentration, n=4, one-way ANOVA). Compared to vehicle MTT reduction was decreased by sirtinol
alone (97 ± 1.1% at 5 μM, 87.0 ± 1.7% at 25 μM and 78.3±1.8 % at 50 μM; P<0.05, control vs. 50 µM
sirtinol, n=4, one-way ANOVA, Turkey’s test). Data are represented as mean±SEM.

Similar effects were achieved with another SIRT1 inhibitor: sirtinol. 25 µM and 50µM
of sirtinol pretreatment increased the survival of H
2
O
2
-treated neurons to 49.4 ± 3.6%
(P<0.01, H
2
O
2
vs. H
2
O
2
+ 25 µM sirtinol, n=3, one-way ANOVA, Turkey’s test) and 70.4
±1.2% (P<0.001, H
2
O
2
vs. H
2
O
2
+ 50 µM sirtinol, n=3, one-way ANOVA, Turkey’s test),
respectively (Fig. 1 C, D); while 5 µM of sirtinol (P>0.05, H
2
O
2
vs. H
2
O
2
+5 µM sirtinol,
n=3, one-way ANOVA, Turkey’s test) showed no significant effect. Sirtinol also dose-
dependently increased neuronal survival in menadione-induced

oxidative stress. 25 µM
and 50µM of sirtinol pretreatment increased the viability of menadione-treated cells

to
86.7 ± 5.7 % (P<0.01, H
2
O
2
vs. H
2
O
2
+ 25 µM sirtinol, n=3, one-way ANOVA, Turkey’s
test) and 93.4±1.9% (P<0.01, H
2
O
2
vs. H
2
O
2
+ 50 µM sirtinol, n=3, one-way ANOVA,
26

Turkey’s test), respectively, whereas 5 µM of sirtinol (P>0.05, H
2
O
2
vs. H
2
O
2
+ 5 µM
sirtinol, n=3, one-way ANOVA, Turkey’s test) showed no effect. The values were
adjusted to the effects of sirtinol alone as it decreased MTT reduction by 13-21% at 25-
50 µM (Fig. 2, P<0.05, control

vs. 50 µM sirtinol, n=4, one-way ANOVA, Turkey’s test)
compared with control.

Figure 3. Effect of high doses of nicotinamide on neuronal viability when exposed to oxidative stress
On 10-14 DIV neurons were treated with nicotinamide (nico) at 1, 5, 10, 25 mM for 48 hrs. Oxidative
stress was then induced by 400 μM of H
2
O
2
or 7.5 μM of menadione (Md), respectively. 24 hrs later cell
viability was measured by MTT assay. The ODs are normalized to untreated control. At high
concentrations of 10 and 25 mM nicotinamide did not protect against oxidative stress (P<0.001, H
2
O
2
vs.
H
2
O
2
+ nico 1 mM, H
2
O
2
vs. H
2
O
2
+ nico 5 mM; P<0.01, Md vs. Md + nico 1 mM, Md vs. Md + nico 5
mM; n=3, one-way ANOVA, Turkey’s test). Data are represented as mean±SEM.

We also performed a dose response experiment for nicotinamide doses ranging from 1 to
25 mM (Fig. 3). At higher concentrations than those tested before (10 and 25 mM)
nicotinamide did not protect further against oxidative stress.

27

28

To confirm that the stress resistance is caused by reduced SIRT1 activity we transfected
either control or SIRT1 siRNA into 5 DIV neurons and measured survival after hydrogen
peroxide or menadione induced oxidative stress (Fig. 4 A). Knockdown of SIRT1
increased the viability of H
2
O
2
treated cells from 26.2 ± 2.7 % to 57.5 ± 3.7% (P < 0.001,
H
2
O
2
vs. H
2
O
2
+ SIRT1 siRNA, n=4, t-test). Inhibition of SIRT1 also boosted the survival
of menadione-treated cells from 54.2 ± 2.7% to 78.5 ± 3.7% (P < 0.01, menadione vs.
menadione + SIRT1 siRNA, n=4, t-test). Viability of SIRT1 siRNA treated cells did not
differ from that of control.

To confirm that SIRT1 was indeed knocked down by siRNA we initially attempted to
examine protein expression, however, none of the commercial antibodies of rat SIRT1 we
have tried (05-707 Anti-SIRT1 clone 2G1/F7; 07-131 Anti-Sir2; both from Millipore,
Billerica, MA) appear to work for rat neurons; so we carried out SIRT1 deacetylase
activity using a fluorometric kit assay 2 days after transfection (Fig. 4 B). SIRT1 siRNA
decreased SIRT1 deacetylase activity to 53.2 ± 4.2% of control (P < 0.01, n=4, control
vs. SIRT1 siRNA, t-test; due to possible detection of other deacetylases as well the
downregulation effect of SIRT1 siRNA could be even higher). Consistent with our
research in yeast, these results in neurons suggest that inhibiting SIRT1 can protect
against oxidative stress.

Figure 4. Inhibition of SIRT1 deacetylase increases oxidative stress resistance in neurons
Cortical neurons from E18 rat embryos were cultured onto 96-well plates (A) or 10 cm dishes (B). (A) On
5 DIV neurons were transfected with either control or SIRT1 siRNA. 2 days later some neurons were
subjected to H
2
O
2
or menadione, followed by MTT survival assay 24 hrs later. ***P < 0.001 compared
with H
2
O
2
-treated, **P < 0.01 compared to menadione-treated groups, respectively, t-test. (B) On 5 DIV
neurons were transfected with either control or SIRT1 siRNA and 2 days later SIRT1 deacetylase activity
was measured (**P < 0.01, n=4, control vs. SIRT1 siRNA, t-test). Data are represented as mean±SEM.

We also tested if SIRT1 inhibition protects against other forms of stress. Inhibition of
SIRT1 did not rescue neurons from either ultraviolet irradiation (UV) or methyl
methanesulfonate (MMS) induced cell damage, suggesting that protection may be
specific for oxidative stress (Fig. 5). This specificity may also in part explain why others
have described protective effects for SIRT1 under different stress conditions.

29

Figure 5. Inhibiting SIRT1 deacetylase does not protect against certain forms of stress in neurons
On 5 DIV neurons were transfected with either control or SIRT1 siRNA. 2 days later some cells were
subjected to either UV irradiation (1 J/m
2
, Stratalinker 254 nm) or methyl methanesulfonate (MMS, 100
µg/ml, 1hr). Viability was assessed with MTT assay 24 hrs later. Data are represented as mean±SEM.

2.4.2. SIRT1 inhibition does not alter antioxidant enzyme expression in
cultured neurons
In a first attempt to determine how inhibition of SIRT1 may help protect neurons against
oxidative stress, we tested if the antioxidant machinery including MnSOD and catalase
etc. were upregulated by inhibition of SIRT1. 10-14 DIV cultured cortical neurons were
treated with SIRT1 inhibitor nicotinamide (Nico) (or in combination with a general
HDAC inhibitor, trichostatin A [TSA]) or vehicle for 48 hr. Some groups of neurons
were also subjected to 100 µM of H
2
O
2
for 24 hr. Nicotinamide alone did not change the
expression level of a superoxide dismutase, MnSOD or catalase compared to control.
H
2
O
2
increased expression of MnSOD, but not of catalase, and this effect was not altered
by nicotinamide (Fig. 6 A, B). These preliminary data suggest that the effect of inhibition
of SIRT1 is unlikely to be mediated by upregulation of antioxidant defenses.
30

Figure 6. Inhibiting SIRT1 deacetylase does not alter MnSOD or catalase expression in cultured
neurons
10-14 DIV rat cortical neurons were pretreated for 48 hrs with 5 mM nicotinamide (Nico), 1 μM trichostatin
A (TSA, general inhibitor for class I and II histone deacetylases) + 5 mM Nico, or vehicle. Some groups of
neurons were also subjected to 100 µM of H
2
O
2
for 24 hr. Total cell lysates were collected and separated
by SDS-PAGE and immunoblotted with anti-MnSOD (A; Stressgen Biotechnologies), anti-catalase (B), or
anti- β-tubulin (H-235; Santa Cruz Biotechnology #sc-9104). The protein expression of MnSOD or catalase
was not altered by SIRT1 inhibitor.

2.4.3. SIRT1 does not alter the activation of Akt or CREB in cultured
neurons
The mammalian IGF-I/Akt pathway has been shown to both protect and sensitize cells
against oxidative damage (Kops et al., 2002; Song et al., 2005). Therefore, we tested
31

32

whether Akt phosphorylation was affected by SIRT1 inhibitors. 10-14 DIV cultured
cortical neurons were treated with SIRT1 inhibitors, 1, 10 or 25 mM of nicotinamide
(Nico) or 30 or 60 µM of sirtinol for 48 hr. Cell lysates were collected for SDS-PAGE
and immunoblotted for phosphorylated Akt (P-Akt) and total Akt to measure the
activation of PI3K/Akt pathway. Neither nicotinamide nor sirtinol changed the levels of
phosphorylated Akt (Fig. 7 A). When cells were challenged to H
2
O
2
pretreatment with
SIRT1 inhibitors also did not alter the activation of Akt (Fig. 7 B). These data suggest
that the effect of inhibition of SIRT1 is unlikely to be mediated by the Akt pathway. In
addition we did not observe any significant difference in the activation of p70 S6 kinase,
another major component downstream of PI3 kinase (Fig. 7 C).

Figure 7. SIRT1 deacetylase does not alter the activation of Akt, S6 kinase or CREB in cultured
neurons
(A) Representative blots showing the effect of SIRT1 inhibitors nicotinamide (Nico) or sirtinol on Akt
signaling. 10-14 DIV neurons were treated with vehicle, nicotinamide, or sirtinol at indicated
concentrations for 48 hrs. Total cell lysates were separated in SDS-PAGE and immunoblotted with anti-
phospho-Akt (S473; R&D Systems #AF887) or anti-Akt (R&D Systems #MAB2055). (B) Representative
blots showing the effect of SIRT1 inhibitors on Akt signaling following H
2
O
2
treatment. 10-14 DIV
neurons were pretreated for 48 hrs with 5 mM nicotinamide (Nico) or 60 µM sirtinol followed by 24 hrs of
100 µM H
2
O
2
treatment. (C) Representative blots showing the effect of SIRT1 inhibitors nicotinamide
(Nico) or sirtinol on the activation of S6 kinase. 10-14 DIV neurons were treated with vehicle,
nicotinamide, or sirtinol at indicated concentrations for 48 hrs. Total cell lysates were separated in SDS-
PAGE and immunoblotted for phospho-p70 S6 Kinase (Thr389) (Cell Signaling #9234) or p70 S6 Kinase
(Cell Signaling, #9202). (D) Representative blots showing the effect of sirtinol on the activation of CREB.
10-14 DIV cortical neurons were treated with sirtinol at indicated concentrations for 48 hrs. Total cell
lysates were separated in SDS-PAGE and immunoblotted for CREB (Upstate #05-767) or phospho-CREB
(Ser133; Cell Signaling # 9198).
33

34

cAMP response element-binding (CREB)-mediated transcription is implicated in
neuronal survival (Finkbeiner, 2000; Mantamadiotis et al., 2002). We therefore tested
whether CREB activation was altered by SIRT1. 10-14 DIV cultured cortical neurons
were treated with the SIRT1 inhibitor sirtinol for 48 hr. Cell lysates were collected for
SDS-PAGE and immunoblotted for phosphorylated CREB and CREB to assess the
activity of CREB. Sirtinol did not alter phosphorylated CREB levels (Fig. 7 D),
suggesting that the effect of inhibition of SIRT1 is not mediated by CREB signaling
pathway.

2.4.4. SIRT1 inhibition decreases Ras/ERK activation in culture and in vivo
Neuronal Ras/ERK (extracellular signal-regulated kinase) is another major pathway
regulating oxidative stress. In our yeast studies, we described the Ras pathway as the
second pro-aging pathway acting in parallel to the Sch9 pathway (Fabrizio et al., 2003).
The deletion of SIR2 in yeast increased further stress resistance in mutants lacking SCH9
but not in mutants with reduced Ras/cAMP signaling (Fabrizio et al., 2005) raising the
possibility that Sir2 may regulate components of the Ras/cAMP pathway. Based on our
yeast results and because mammalian Ras functions downstream of the pro-aging IGF-I
receptor we studied the role of SIRT1 inhibitors on Ras/ERK signaling. We performed a
Ras activation assay on cultured neurons. Neurons cultured in neurobasal media
supplemented with B-27 (containing ~4 µg/ml insulin) showed a high basal activation of
active Ras (GTP-bound Ras), which was reduced by 4 hr treatment of 60 µM of sirtinol
(Fig. 8 A), indicating that SIRT1 modulates Ras Activity.

Figure 8. SIRT1 inhibitors decrease Ras/ERK1/2 activation in cultured neurons
(A) Representative blots showing the effect of SIRT1 inhibitor sirtinol on Ras activation. 10-14 DIV rat
cortical neurons were treated with vehicle or 60 µM sirtinol for 4 hrs and subjected to Ras activation assay
or immunoblotting with anti-Ras. (B, C) Representative blots showing the effect of SIRT1 inhibitors
nicotinamide (Nico, B) and sirtinol (C) on ERK
1/2
signaling. 10-14 DIV rat cortical neurons were treated
with nicotinamide for 48 hrs, or sirtinol for 4 hrs at indicated concentrations. Total cell lysates were
collected for SDS-PAGE and blotted with anti-phospho-ERK
1/2
and anti-ERK
1/2
, respectively. (D)
Quantification of immunoblots showing the effect of sirtinol on ERK
1/2
activation, expressed as ratio of
phosphorylated-ERK
1/2
to ERK
1/2
(P- ERK/ ERK) (*P<0.05, control vs. sirtinol 30 µM; **P<0.01, control
vs. sirtinol 60 µM; n=4).

We also checked if ERK1/2, a major effector of Ras, is altered by SIRT1 inhibition. 10-
14 DIV rat cortical neurons were treated with vehicle, SIRT1 inhibitors nicotinamide
(Nico) for 48 hr, or sirtinol for 4 hr and cell lysates were collected for SDS-PAGE and
35

blotted with anti-phospho-ERK
1/2
(P-ERK1/2) and anti-ERK
1/2
, respectively. 10 mM and
25 mM of nicotinamide decreased phosphorylated ERK1/2 (Fig. 8 B). The effect of
sirtinol was dose-dependent as 30 µM of sirtinol reduced the ratio of P-ERK/ERK by
45% while 60 µM of sirtinol reduced the ratio of P-ERK/ERK by 68% (Fig. 8 C, D) (P
<0.05, control vs. sirtinol 30 µM; P <0.01, control vs. sirtinol 60 µM; n=4, One-way
ANOVA, Turkey's test).

Figure 9. Inhibition of SIRT1 deacetylase decreases ERK1/2 activation in cultured neurons and in
vivo
(A) 7 DIV cortical neurons were transfected with either U6Pro-SIRT1-siRNA + GFP or dominant-negative
GFP-SirT2 (SirT2DN) and 48 hrs later were immunostained with anti-phospho-ERK
1/2
. (B) Quantification
of immunodensity from 12 transfected and 12 nearby non-transfected cells for SIRT1 siRNA and SirT2DN,
respectively (P<0.05, control vs. SIRT1 siRNA, n=12). (C, D) Representative blots (C) and quantification
(D) showing the effect of SIRT1 deacetylase on ERK
1/2
signaling in the hippocampus of mice (*P<0.05,
SIRT1+/+ vs. SIRT1-/-, n=6).

36

37

To confirm that the above effects are due to specific activation or inhibition of SIRT1 a
plasmid encoding siRNA of SIRT1 was co-transfected with GFP into neurons. This
plasmid has been shown to reduce expression level of SIRT1 (Cohen HY et al., 2004). 48
hr after transfection the cells were immunostained with anti-P-ERK1/2 and anti-GFP
(Fig. 9 A). The immunodensity of P-ERK1/2 in transfected cells was significantly lower
than that of surrounding non-transfected cells (P < 0.05, n=12, Student’s t-test). As a
control a plasmid encoding a dominant-negative SirT2, GFP-SirT2, was transfected into
neurons. P-ERK1/2 staining remained the same in SirT2 DN transfected cells compared
to non-transfected ones (P > 0.05, n=12, Student’s t-test) (Fig. 9 B). These data confirm
that SIRT1 inhibition attenuates the activation of ERK1/2. We also checked whether the
same occurs in vivo and indeed a decrease in ERK1/2 activation was found in the
hippocampus of 17-day-old SIRT1 knockout mice (P<0.05, SIRT1+/+ vs. SIRT1-/-, n=6,
t-test) (Fig. 9 C, D). Together, these data suggest that inhibition of SIRT1 can down-
regulate ERK1/2 activity.

We also tested whether the effect of SIRT1 on ERK1/2 is observed in a human cell line
and whether it inhibits IGF-I signaling. HEK293 cells were serum starved for 15 hrs and
treated with IGF-1 (500 ng/ml) for 5 min. Cell lysates were immediately collected for
SDS-PAGE and immunoblotted for P-ERK or ERK (Fig. 10 A, B). Starved cells showed
low levels of phosphorylated ERK1/2, whereas IGF-1 induced a 12-fold increase in
phosphorylated ERK1/2 (P<0.01, control vs. IGF-1, n=3, one-way ANOVA, Turkey’s
test). Pretreatment with 60 µM sirtinol for 4 hrs markedly reduced this effect of IGF-1 on
ERK1/2 phosphorylation (60% decrease, P<0.05, IGF-1 vs. IGF-1+sirtinol, n=3, one-way
ANOVA, Turkey’s test). This indicates that inhibition of SIRT1 downregulates insulin-
IGF-I/ERK signaling in cells other than neurons.

Figure 10. SIRT1 inhibitor decreases IGF-I-induced, but not PMA-induced ERK1/2 activation in
HEK293 cells
Representative blots (A, C) and quantification (B, D) showing the effect of sirtinol on ERK
1/2
signaling in
HEK293 cells. HEK cells were starved for 15 hrs in media containing 0.5% FBS and incubated with 60
µM sirtinol for 4 hrs and then treated with IGF-1 (500 ng/ml) or PMA (1 µg/ml) for 5 min. Total cell
lysates were collected for SDS-PAGE and blotted with anti-phospho-ERK
1/2
or anti-ERK
1/2
, respectively
(*P<0.05, IGF-1 vs. IGF-1+sirtinol, n=3). Quantifications are represented as mean±SEM.

To begin to determine how SIRT1 inhibition affects ERK activation, we applied PMA
(phorbol 12-myristate 13-acetate, a phorbol ester, 1 µg/ml), which is known to activate
ERK1/2 through the PKC/Raf-1/MEK1 pathway (Liebmann, 2001), not involving IRS-
1/2 or Ras. Starved cells were treated with PMA. As expected, it induced a 24-fold
38

39

increase in phosphorylated ERK1/2 (P<0.001, control vs. PMA, n=3, one-way ANOVA,
Turkey’s test) (Fig. 10 C, D). However, pretreatment with sirtinol did not block this
effect (P >0.05, PMA vs. PMA + sirtinol, n=3, one-way ANOVA, Turkey’s test). These
data are consistent with those in neurons and indicate that the target molecule(s) of
SIRT1 lies between insulin/IGF-1R and Ras.

2.4.5. Subcellular localization of SIRT1 in the brain
For SIRT1 to regulate the activation of Ras/ERK, SIRT1 may either regulate the gene
expression of upstream molecules, or act directly upon those molecules through post-
translational modification. SIRT1 had long been considered a nuclear protein until
recently (Tanno et al., 2007). The subcellular localization of SIRT1 in neuronal cells is
poorly understood. We investigated the possibility that SIRT1 acts on non-nuclear
substrates by first examining its localization in mouse brains (Fig. 12). Brains from
postnatal day 3 (P3, Fig. 12 A) and adult (Fig. 12 B) mouse were collected, homogenized
and separated into nuclear, cytosol and membrane fractions through sucrose-gradient
centrifugation. These fractions were then immunoblotted for SIRT1 (Upstate 07-131),
NeuN (as a nucleus marker), GAPDH (as a cytosol marker), and GluR
2/3
(a membrane
associated protein). Anti-SIRT1 antibody recognizes one band at 110 kDa, which is
absent from the brain of SIRT1 knockout mice (Fig. 12 C), confirming the specificity of
this signal. In P3 brains SIRT1 is localized to both nucleus and cytosol with the majority
residing in the cytosol; whereas in the adult brain it is found predominantly in the cytosol
(Fig. 12 A, B).

Figure 11. Subcellular localization of SIRT1 in hippocampus CA3
Frozen brain slices from wild type (WT, upper panel) and SIRT1 knockout (KO, lower panel) mice were
immunostained with SIRT1 antibody (Upstate) and NeuN. A comparison of WT with KO staining reveals
that SIRT1 is localized in both the nucleus and cytosol.

40

Figure 12. Subcellular localization of SIRT1 in the forebrain
(A, B) Forebrains from postnatal 3 days (P3, A) and adult (B) mice were homogenized and separated into
nuclear, cytosol and membrane fractions through sucrose-gradient centrifugation and immunoblotted with
SIRT1, NeuN, GAPDH, GluR
2/3
, respectively. In P3 brains SIRT1 is localized in both nucleus and cytosol,
while it is found predominantly in cytosol in the adult brain. (C) The specificity of SIRT1 immunoblot was
confirmed with SIRT1 knockout mice (SIRT1-/-).

We also examined the localization of SIRT1 in cultured neurons. To overcome the non-
specificity with anti-SIRT1 antibody in immunostaining we transfected cultured cortical
neurons (10 DIV) with FLAG-tagged SIRT1 (SIRT1-Flag). 48 hrs later neurons were
immunostained with anti-FLAG, anti- microtubule-associated protein 2 (MAP2) as a
neuronal marker and counterstained with DAPI for nucleus. Surprisingly, we found that
in cultured neurons exogenously expressed SIRT1 was exclusively localized in the
nucleus (Fig. 13, upper panel). SIRT1’s cytosolic localization in brain tissue and its
nuclear localization in cultured neurons made us to hypothesize that SIRT1 may shuttle
between these two compartments depending on certain situations, such as developmental
stages and environmental changes. To test this hypothesis we first subjected the neurons
to oxidative stress. Cultured neurons were transfected with SIRT1-Flag and treated with
200 µM of H
2
O
2
for 1 hr and fixed right away and then stained as above. We found that
in 9% of neurons (32 out of 352 SIRT1-Flag positive neurons counted) SIRT1 were
41

localized in the cytosol (Fig. 13, middle panel), implicating that SIRT1 can shuttle into
cytosol in response to oxidative stress. We also treated neurons with NMDA, an agonist
for NMDA-subtype glutamate receptor. At excitotoxic concentrations NMDA (100 µM,
1 hr; neurons were fixed right after treatment) showed similar effects to that of H
2
O
2

(SIRT1 was localized in the cytosol in 47 among 426 SIRT1-Flag positive neurons
counted), while it showed no effect at physiological concentrations (5 µM, 5 min
treatment; neurons were fixed one hour after treatment) (Fig. 13, lower panel). These data
suggest that SIRT1 is mainly localized in the cytosol in the brain and it can shuttle
between the cytosol and the nucleus upon oxidative stress or other environmental insults.

Figure 13. Nucleus-cytosol shuttling of SIRT1 in cultured neurons
Cultured rat cortical neurons (10 DIV) were transfected with SIRT1-Flag and treated with H2O2 (200 µM,
1 hr, middle panel), NMDA (100 µM, 1 hr, lower panel), or vehicle (upper panel) 48 hrs after transfection.
The neurons were then fixed and immunostained with MAP2 and FLAG and counterstained with DAPI. In
cultured neurons exogenously expressed SIRT1 was exclusively localized in the nucleus, whereas it
shuttled into the cytosol upon insults including H2O2 or NMDA in ~10% of neurons.
42

43

2.4.6. SIRT1 regulates Ras/ERK activity through deacetylating IRS-2
The localization of SIRT1 to the cytosol could allow direct deacetylation of substrate
protein(s) in the cytosol. The signaling pathway upstream of Ras is composed of
sequential activation of the receptors, insulin receptor substrates (IRS) or Shc, Grb2 and
SOS. Among the signaling molecules between insulin/IGF-1 receptors and Ras,
theinsulin receptor substrate-1 (IRS-1) has been shown to be an acetylated protein (Kaiser
and James, 2004). Therefore, we hypothesized that SIRT1 may regulate insulin/IGF-1
signal transduction by deacetylating IRS. To test this hypothesis we first conducted co-
immunoprecipitation to see whether SIRT1 physically interacts with IRS-1 or IRS-2.
HEK 293 cells were transfected with a plasmid encoding V5-tagged SIRT1 and the cell
lysates were immunoprecipitated with anti-V5 or control IgG and probed with anti-IRS1
or anti-IRS2, respectively (Fig 14. A). IRS-2, but not IRS-1, was coimmunoprecipitated
with V5-SIRT1, suggesting that IRS-2 might be a substrate of SIRT1. To determine
whether SIRT1 can directly deacetylate IRS-2, we set up an in vitro deacetylation
experiment. We purified IRS-2 protein from HEK 293 cells by immunoprecipitation and
incubated equal amounts of the purified IRS-2 with different doses of SIRT1 and 1 mM
of NAD for 1.5 hr and then blotted with anti-acetylated-lysine (AcK) or IRS-2 (Fig 14.
B). This experiment confirmed that IRS-2 is acetylated and that SIRT1 can directly
deacetylate IRS-2 in a cell-free system in agreement with a recent finding (Zhang,
2007b).

Figure 14. SIRT1 regulates Ras/ERK1/2 signaling via deacetylation of IRS-2
(A) Physical interaction between SIRT1 and IRS-2. HEK293 cells were transfected with V5-SIRT1 and 48
hrs later cell lysates were immunoprecipitated with anti-V5 antibody or control IgG and probed with anti-
IRS1 or anti-IRS2, respectively. (B) SIRT1 deacetylates IRS-2 in vitro. HEK293 cell lysates were
immunoprecipitated with anti-IRS2 and equally divided into 3 portions to be incubated with 0, 1 µg, or 2
µg SIRT1 deacetylase for 1.5 hr. They were then subjected to SDS-PAGE and blotted with anti-acetylated-
lysine (AcK). (C, D) Representative blots (C) and quantification (D) showing that inhibition of SIRT1
deacetylase in HEK cells increased acetylation of IRS-2 and decreased phosphorylation of IRS-2 while
having no effect on IRS-1. (C) HEK293 cells were incubated with nicotinamide (5 mM) for 48 hr, sirtinol
(60 µM) for 4 hrs, or vehicle while starving for 15 hrs in media containing 0.5% FBS and then treated with
IGF-1 (500 ng/ml) for 5 min. Cell lysates were immunoprecipitated with anti-IRS1, or anti-IRS2,
respectively, and blotted with anti-AcK or anti-phosphotyrosine (PY). Cell lysates were also
immunoblotted with anti-IRS1 or anti-IRS2 and no change was seen in IRS-1 or IRS-2 protein levels. (D)
Quantification from 4-5 blots (mean±SEM; P<0.05 for PY between IGF1 vs. IGF1+Nico and
IGF1+sirtinol; P<0.001 for AcK between IGF1 vs. IGF1+Nico and IGF1+sirtinol; One-way ANOVA,
Turkey’s test). (E) Quantification of acetylated IRS-1 and total IRS-1. Data are mean±SEM from 4-5 blots.

Next we examined if SIRT1 could deacetylate IRS-1 or IRS-2 in live cells and if this
deacetylation affects IGF-I signaling. HEK 293 cells were serum starved for 15 hr and
treated with 500 ng/ml of IGF-1 for 5 min. IRS-1 or IRS-2 were immunoprecipitated
from the cells and probed with AcK (Fig. 14 C). In 4 experiments IGF-1 did not
significantly alter the acetylation level of IRS-1 or IRS-2 (Fig. 14 D, E). However,
44

pretreatment with 5 mM of nicotinamide (Nico) for 24 hr or 60 µM of sirtinol for 4 hrs
significantly increased the acetylation level of IRS-2 by 93.5% or 79.7%, respectively
(P<0.01, IGF-1 vs. IGF-1+Nico, n=4; P<0.05, IGF-1 vs. IGF-1+sirtinol, n=4, one-way
ANOVA, Turkey’s test) but had no effect on IRS-1. To study whether the alteration of
the acetylation level of IRS-2 affects its signaling we measured its phosphorylation level.
Serum-starved HEK 293 cells were treated with IGF-1 with or without SIRT1 inhibitors
and IRS-2 was immunoprecipitated from the cells and probed for phosphotyrosine (PY).
IGF-1 induced a 14-fold increase in phosphorylation of IRS-2, which was significantly
diminished by pretreatment with nicotinamide (46.7% decrease, P <0.05, IGF-1 vs. IGF-
1+Nico, n=4, one-way ANOVA, Turkey’s test) or sirtinol (55.3% decrease, P<0.05, IGF-
1 vs. IGF-1+sirtinol, n=4, one-way ANOVA, Turkey’s test).

Figure 15. Representative blots showing the effect of SIRT1 on IRS-2 deacetylation in neurons
On 5 DIV cultured rat cortical neurons were transfected with either control or SIRT1 siRNA. 2 days later
cell lysates were immunoprecipitated with anti-IRS2 and blotted for AcK or phosphotyrosine (PY). Cell
lysates were also immunoblotted with anti-IRS2, anti-phospho-ERK
1/2
and anti-ERK
1/2
, respectively.

To corroborate the results in HEK293 cells we also examined rat cortical neurons
transected with control or SIRT1 siRNA (Fig. 15). Inhibition of SIRT1 increased
acetylation level of IRS-2 and decreased phosphorylation of IRS-2. Concurrently,
45

activation of ERK
1/2
was reduced by SIRT1 inhibition. Taken together these data indicate
that SIRT1 enhances insulin/IGF-I signaling by reducing IRS-2 acetylation and
promoting its phosphorylation.

We also tested whether resveratrol would alter the acetylation level of IRS-2 in neurons
but did not observe any change (Fig. 16). This may be explained by the inability of
resveratrol to activate further neuronal SIRT1 or to change the balance between
acetylation and deacetylation under the culture conditions.

Figure 16. Representative blots showing the effect of resveratrol on the acetylation level of IRS-2
10-14 DIV neurons were incubated with resveratrol (res) at indicated concentrations for 48 hr. Cell lysates
were immunoprecipitated with anti-IRS-2, and blotted with anti-AcK. Cell lysates were also
immunoblotted with anti-IRS2.

2.4.7. MEK/ERK1/2 inhibition protects neurons against oxidative stress
To test if down-regulation of ERK1/2 can account for the effect of SIRT1 inhibition on
stress resistance we applied MEK1/2 (immediately upstream of ERK1/2) inhibitors to
cultured neurons. Oxidative stress was induced with H
2
O
2
or 7.5 µM of menadione for 24
hr. The indicated groups of cells were pretreated with 10 µM of U0126 or 10 µM of
SL327, two MEK1/2 inhibitors, for 4 hr. Cell survival was measured by MTT assay or
live-dead assay. MTT assay suggested that 400 µM of H
2
O
2
killed 71.4 ± 5.4% of
46

47

neurons (Fig. 17 A). Pretreatment with U0126 or SL327 increased the survival to 45.9 ±
4.5% (P >0.05, H
2
O
2
vs. H
2
O
2
+U, n=3, one-way ANOVA, Turkey’s test) or 49.2 ± 5.5%
(P <0.05, H
2
O
2
vs. H
2
O
2
+SL, n=3, one-way ANOVA, Turkey’s test), respectively.
Menadione

(Md) alone reduced the viability to 49.9 ± 5.3% compared to control.
Pretreatment of U0126 or SL327 increased the survival to 73.9 ± 3.7% (P < 0.05, Md

vs.
Md+U, n=3, one-way ANOVA, Turkey’s test) or 68.3 ± 1.5% (P > 0.05, Md

vs. Md+SL,
n=3, one-way ANOVA, Turkey’s test), respectively. Live/Dead assay was also employed
and representative images are shown in Fig. 17 B. 100 µM of H
2
O
2
caused a survival rate
of 57.5 ± 3.3% compared to control (Fig. 17 C). Pretreatment of U0126 increased
viability to 74.4 ± 3.5% (P <0.05, H
2
O
2
vs. H
2
O
2
+U, n=4, one-way ANOVA, Turkey’s
test), and SL327 increased the survival to 74.1±3.6% (P <0.05, H
2
O
2
vs. H
2
O
2
+SL, n=4,
one-way ANOVA, Turkey’s test). Similarly, menadione reduced the viability to 52.1 ±
2.6% compared to control. Pretreatment of U0126 increased viability to 65.8 ± 3.8% (P
>0.05, Md

vs. Md+U, n=4, one-way ANOVA, Turkey’s test), whereas SL327 increased
the survival to 69.4 ± 2.6% (P <0.05, Md

vs. Md+SL, n=4, one-way ANOVA, Turkey’s
test). Thus, inhibition of MEK1/ERK1/2 is sufficient to increase the stress resistance of
cultured neurons, suggesting that SIRT1 can sensitize neurons to oxidative stress at least
in part by up-regulating ERK1/2 activity.

Figure 17. Inhibiting MEK/ERK1/2 protects neurons against oxidative stress
Cortical neurons from E18 rat embryos were cultured onto 96-well plates (A) or glass cover slips in 6-well
plates (B). On 10-14 DIV the neurons were pre-treated with ERK
1/2
inhibitors U0126 or SL327 for 4 hr.
Oxidative stress was induced by hydrogen peroxide or menadione. Cell viability was measured with MTT
survival assay (A) or live/dead assay (B). (C) % live was calculated from the number of live (green) and
dead (red) cells.

48

As both SIRT1 and ERK1/2 are involved in the regulation of apoptosis we studied how
inhibition of SIRT1 or ERK1/2 affects apoptosis in our system. We transfected neurons
with SIRT1 siRNA or treated them with ERK1/2 inhibitor SL327 and then exposed the
cells to 400 µM of H
2
O
2
or 7.5 µM of menadione for 24 hrs. We then estimated necrosis
by measuring LDH release into the media and quantified apoptosis by measuring
cytoplasmic histone-associated mono- and oligonucleosomes with Cell Death Detection
ELISA
PLUS
kit (Fig. 18 A, B). H
2
O
2
and menadione increased LDH release to 5.5 ± 0.7
and 4.4 ± 0.4 fold of control, respectively. Inhibition of SIRT1 or ERK1/2 with SL327
significantly alleviated this increase in LDH release (Fig. 18 A; P < 0.01 compared with
H
2
O
2
, P < 0.05 compared with menadione; n=3, t-test). On the other hand, H
2
O
2
and
menadione induced apoptosis to 1.6 ± 0.2 and 2.0 ± 0.2 fold of control, respectively.
Neither SIRT1 nor ERK1/2 inhibition effectively reduced the apoptosis induced by H
2
O
2

(1.6 ± 0.2 fold of control, P < 0.05, n=10, t-test) but SIRT1 inhibition had a small but
significant effect in reducing apoptosis induced by menadione (Fig. 18 B). These results
suggest that inhibition of SIRT1 and ERK1/2 protect primarily against necrosis induced
by oxidative stress.

Figure 18. ERK1/2 inhibition primarily attenuates necrosis
Neurons were transfected with control or SIRT1 siRNA, or incubated with SL327 before being exposed to
either 400 μM H
2
O
2
or 7.5 μM menadione. 24 hrs later samples were subjected to LDH assay or apoptosis
assay (& P < 0.01 compared with H
2
O
2
; # P < 0.05 compared with menadione, t-test).
49

Life span extension
Insulin/IGF-I
Insulin/IGF-I receptor
IRS1
Ras
SIRT1
Akt
Stress resistance/starvation
response genes
, IRS2
ERK
?
?
FOXO

Figure 19. A model for SIRT1 regulation of IGF-I signaling
Inhibition of SIRT1 upregulates IRS2 acetylation, which in turn curbs IRS-2 activation, causing a reduction
in Ras/ERK signaling.

2.4.8. Reduced oxidative brain damage and life span of SIRT1 knockout
mice
We have shown that in neurons SIRT1 inhibition protects against exogenous oxidative
insults of hydrogen peroxide or menadione. To determine whether SIRT1 can have
similar effects in vivo we measured markers of oxidative damage in the brain of 18 month
old mice. We removed brains (minus cerebellum) from 18-month-old SIRT1 +/+ and
SIRT1 -/- mice and measured protein carbonyl content with an ELISA kit (Fig 20. A) and
lipid peroxidation using TBARS method (Fig 20. B), respectively. Compared to wild type
mice (2.3 ± 0.14 nmol/mg), knockout mouse brain showed 17% decrease in protein
carbonyl content (1.9 ± 0.1 nmol/mg; P < 0.05, n=7, t-test). Lipid peroxidation was also
reduced in knockout mouse brain (3.7 ± 0.2 nmol malondialdehyde/mg), 20% less than
50

that in wild type (4.5 ± 0.2 nmol/mg; P < 0.05, n=7, t-test). This reduction of markers of
oxidative stress in SIRT1 knockout mouse brain is consistent with our findings in culture.
Although a recent report determined that SIRT1 protects the heart against oxidative stress
(Alcendor et al., 2007a) these results suggest that SIRT1 may play a different role in the
brain and other cells.

Figure 20. Reduced oxidative damage in SIRT1 knockout mouse brain
Protein carbonyl content (A) and lipid peroxidation (B) were measured in 18 month old SIRT1 +/+ and -/-
mice brains. Protein carbonyl was measured with OxiSelect™ Protein Carbonyl ELISA Kit and data are
shown as nmol protein carbonyl per mg of protein (*P < 0.05, n=7, t-test). Lipid peroxidation was
measured using TBARS assay and data are presented as nmol malondialdehyde (MDA) equivalent per mg
of protein (*P < 0.05, n=7, t-test). Data are represented as mean±SEM.

The reduced oxidative damage in SIRT1 knockout mouse brain confirmed our findings in
culture that inhibition of SIRT1 increased stress resistance in neurons. However, other
groups have also shown that SIRT1 is beneficial in other organs, such as the heart
(Alcendor et al., 2007a). Considering the diverse roles SIRT1 play in different organs it is
important to assess its effect on overall stress resistance. Thus, we tested the effect of
SIRT1 inhibitor nicotinamide in response to exogenously induced oxidative stress. 2-
month old male C57BL/6J mice were intraperitoneally (i.p.) injected either nicotinamide
(500 mg per kg body mass) (n=12) or 0.09% saline (n=12) as control twice daily for 7
51

52

days. Then oxidative stress was induced by intraperitoneal paraquat injection (70 mg per
kg body mass). Nicotinamide or saline injection was continued until death of mice or end
of experiment. The mice were checked every hour and the test was censored at 100 h.
Kaplan −Meier analysis shows significantly fewer survivors among nicotinamide-treated
mice (Fig 21. A; P < 0.05, log-rank test, n =24). To overcome the non-specific effect of
nicotinamide we tested the oxidative stress resistance of SIRT1 +/+, +/-, -/- mice.
Oxidative stress was induced in 2-4 month old SIRT1 +/+ (n=14), SIRT1 +/- (n=15) or
SIRT1 -/- (n=15) mice by intraperitoneal paraquat injection (70 mg per kg body mass).
The mice were checked every 2 hr and the test was censored at 100 h. Kaplan −Meier
analysis shows significantly fewer survivors among SIRT1 -/- compared to SIRT1 +/+
mice (Fig 21. B; P < 0.001, log-rank test, n =44). These results confirmed the finding
using SIRT1 inhibitor nicotinamide and suggest that although SIRT1 inhibition is
beneficial in the brain it has a deleterious effect overall. As SIRT1 knockout mice had
been reported to have certain lung problems and paraquat is known to damage the lungs it
is also possible that the reduced survival of SIRT1 mice is due to specific effects of
paraquat on the lungs and that other oxidative species (like hydrogen peroxide) may not
induce the same effect. Nonetheless, the functions of SIRT1 appear more complex than
previously expected. Therefore, to further characterize the phenotype we decided to look
at the life span of SIRT1 knockout mice under different conditions.

Figure 21. SIRT1 inhibition decreases survival in paraquat induced oxidative stress
(A) 2-month old male C57BL/6J mice were intraperitoneally (i.p.) injected either nicotinamide (500 mg per
kg body mass) (n=12) or 0.09% saline (n=12) as control twice daily for 7 days, followed by intraperitoneal
paraquat injection (70 mg per kg body mass). Nicotinamide or saline injection was continued until death of
mice or end of experiment. The mice were checked every hour and the test was censored at 100 h. Survival
proportions were subjected to Kaplan −Meier analysis (P < 0.05, log-rank test, n =24). (B) Oxidative stress
was induced in 2-4 month old SIRT1 +/+ (n=14), SIRT1 +/- (n=15) or SIRT1 -/- (n=15) mice by
intraperitoneal paraquat injection (70 mg per kg body mass). The mice were checked every 2 hr and the test
was censored at 100 h. Survival proportions were subjected to Kaplan −Meier analysis (P < 0.001, log-rank
test, n =44).

Previously, we showed that lack of Sir2 in yeast extended further the lifespan of calorie
restricted (CR) cells, or long-lived Sch9 but did not extend the life span under standard
nutrients conditions (Fabrizio et al., 2005). Our results suggest that SIRT1 can also
sensitize mammalian neurons against oxidative damage. To determine the role of SIRT1
in vivo we monitored the life span of control mice and of mice homozygote or
heterozygote for disruption mutations in the SIRT1 gene (McBurney et al., 2003). SIRT1
+/+, +/-, -/- mice were fed ad-lib (Fig. 22 A) or a 40% reduced calorie diet for 2 years
(Fig. 22 B). Compared to wild type and heterozygous mice, SIRT1knockout mice
survived shorter on both the normal and calorie restricted diet. This is in agreement with
recent findings by others (Boily et al., 2008b). These results suggest that the important
53

role of SIRT1 in normal function under both ad lib and calorie restricted conditions
conceals the potential pro-oxidative damage role of SIRT1. The normal survival in the
SIRT1 +/- mice suggests that a reduction of SIRT1 expression does not reduce the life
span but also does not extend it.

Figure 22. Reduced life span of SIRT1 knockout mice under ad-lib and calorie restriction conditions
(A) Survival curves for SIRT1 +/+ (n=16), +/- (n=15), -/- mice (n=14) on ad-lib diet. P < 0.05, log-rank
test. (B) Survival curves for SIRT1 +/+ (n= 13), +/- (n=18), -/- (n=12) mice on 40% reduced calorie diet. P
< 0.001, log-rank test. The mice were 2-5 months old at the onset of calorie restriction.

2.5. Discussion
Sirtuins including Sir2 and SIRT1 have been described as mediators of the effect of
calorie restriction on longevity and are widely believed to protect against aging (Cohen et
al., 2004; Guarente, 2005a). However, others have proposed that sirtuins are not required
for the effects of CR on life span (Hansen et al., 2007; Kaeberlein et al., 2004; Kaeberlein
et al., 2006; Lee et al., 2006) and our previous results in yeast suggested that the lack of
Sir2 can also increase resistance to stress and extend further the chronological life span of
calorie restricted cells or yeast also lacking components of the Ras/cAMP/PKA or Sch9
pathway (Fabrizio et al., 2005; Longo and Kennedy, 2006). In agreement with our results
in S. cerevisiae, here we found that inhibition of SIRT1 reduces IGF-I signaling and
54

55

increases the resistance of mammalian cells to oxidative stress. Our data indicates that
SIRT1 increases insulin/IGF-I signaling in part by acetylating IRS-2, which promotes its
phosphorylation. These post-translation modifications in IRS-2 are followed by Ras/ERK
activation and sensitization of primary rat neurons to oxidative stress. Inhibition of
Ras/Mek1/ERK1/2 activity was sufficient to protect the neurons against oxidative stress.

Whereas the level of markers of oxidative damage was reduced in the brain of SIRT1-/-
mice compared to controls, these apparently protective effects were not sufficient to
counterbalance the positive effects of SIRT1 as evident from the reduced mean life span
of the homozygote knock out mice under both ad lib or calorie restricted diets. These
results are consistent with the existence of a pro-oxidative stress role for mammalian
SIRT1 and Ras similar to that described for Sir2 and Ras in S. cerevisiae but confirm that
sirtuins can play both positive and negative roles. SIRT1 was originally identified as a
histone deacetylase. Consistent with this function, it was found to be localized in the
nucleus in numerous studies (Michishita et al., 2005). Recently, however, Tanno et al
examined different tissues and found SIRT1 predominantly localized to the cytosol in
mouse brain tissue (Tanno et al., 2007). We checked SIRT1’s localization in the brain.
We performed sucrose-gradient centrifugation to separate the brain tissue into nuclear,
cytosolic and membrane fractions. Immunoblots with these fractions indicate that SIRT1
lies in both the nucleus and the cytosol with the preference of cytosol in postnatal day 3
brain; while SIRT1 is almost exclusively retained in the cytosol in the adult brain. This is
in line with increasing evidence pinpointing SIRT1 as exhibiting cytosolic localization
56

(Jin et al., 2007; Kim et al., 2007; Tanno et al., 2007; Zhang, 2007b). Besides histones,
various transcription factors have been identified as substrates for SIRT1. SIRT1-
mediated deacetylase activity on these substrates seems to occur in the nucleus.

It has been proposed that SIRT1 plays an anti-aging role in mammals by deacetylating
and activating FOXO (Brunet et al., 2004; Kobayashi et al., 2005). Similarly, SIRT1
exhibits antagonistic effects of insulin on PGC-1 in gluconeogenesis (Rodgers et al.,
2005). On the other hand, evidence is also accumulating in support of a synergic
relationship between Sir2 and insulin/IGF-1. SIRT1 was shown to increase the release of
insulin or improve insulin sensitivity (Bordone et al., 2006; Moynihan et al., 2005; Sun et
al., 2007). Moreover, SIRT1 lowered the expression of IGF-binding proteins (IGFBP), a
secreted inhibitory modulator of IGF function (Yang et al., 2005) and SIRT1 knockout
mice have increased expression of IGFBP1 (Lemieux et al., 2005). In agreement with
these results, knockdown of liver SIRT1 in mice reduced blood glucose concentration
(Rodgers and Puigserver, 2007). In our previous study in S. cerevisiae the deletion of
SIR2 increased further the resistance of mutants lacking sch9 (homologous to S6kinase
and Akt) to heat shock and oxidative stress but did not increase the resistance of mutants
with defects in the Ras/cAMP pathway raising the possibility that Sir2 and Ras function
in the same pathway. Although a Ras/cAMP/PKA pathway has not been described in
mammalian cells, we provide a mechanism linking SIRT1 activity, the IGF-I/IRS-
2/Ras/ERK pathway and stress resistance in mammalian cells. Our results suggest that
inhibition of SIRT1 downregulates insulin/IGF-I-dependent activation of ERK1/2 in part
57

through decreased IRS-2 phosphorylation and decreased Ras activation. These results are
consistent with findings that Ras induces premature replicative senescence in primary
mammalian cells and that SIRT1-deficient mouse embryonic fibroblasts (MEF) have a
dramatically extended replicative life span (Chua et al., 2005; Serrano et al., 1997). As
expected if SIRT1 acts upstream of Ras, SIRT1 was not required for Ras-dependent
accelerated replicative senescence (Chua et al., 2005). Thus, it is possible that inhibition
of IRS-2 upstream of Ras is responsible for this effect of SIRT1 deficiency on replicative
senescence. In agreement with this model our immunoblots indicated that SIRT1 is
almost exclusively retained in the cytosol of adult brain cells and SIRT1 co-precipitated
with IRS-2. Interestingly, the PI3K/Akt pathway, another major effector downstream of
IRS-2 was unaffected by SIRT1 inhibition, although over-expression of SIRT1 was
reported to increase Akt activation under insulin-resistant conditions but not under
normal conditions (Sun et al., 2007). It is interesting that only the ERK1/2 pathway but
not the PI3K/Akt pathway appears to be affected by IRS-2 in our system. Although IRS-1
and IRS-2 are distributed similarly in many tissues and their functions often overlap, the
relative contributions of IRS-1 and IRS-2 are different. In some organs, IRS-1 seems to
mediate the majority effects of insulin and transmit signal to PI3K and Ras/ERK
pathway. In other tissues like muscles, IRS-2 appears to selectively transduct to ERK
signaling, while IRS-1 preferentially activates Akt1; and Akt2 and p38MAPK lie
downstream of both IRS-1 and IRS-2 (Byron et al., 2006; Huang et al., 2005). These
imply that the coupling of IRS to the downstream signaling may depend on the cell type
58

or other environmental factors. The effect of SIRT1 on IRS-2 may have important
implications considering that brain irs2-/- mice live longer (Taguchi et al., 2007).
ERK is crucial for oxidative stress. ERK signaling cascade is known to be activated by
oxidative stress (Gaitanaki et al., 2003; Guyton et al., 1996; Zhang et al., 1998) and to
exhibit dual effects on cell death depending on its kinetics, duration, intensity and context
of its activation (Chu et al., 2004). It has been shown to promote cell survival under some
conditions (Cheung and Slack, 2004), but to increase the sensitivity to oxidative stress
under other conditions. Inhibition of ERK abrogates cell death induced by H
2
O
2
in
pancreatic cancer cells (Osada et al., 2007) and protects against glutamate-induced
neuronal death (Satoh et al., 2000). Furthermore, certain drugs protect neurons partly by
inhibiting ERK activation (Xu et al., 2006). Our results suggest that SIRT1 inhibition
protects neurons by decreasing Ras/ERK signaling and provide evidence for the pro-
aging role of the Ras/ERK pathway downstream of IGF-I, analogously to that of the Ras
pathways in S. cerevisiae. In agreement with our present results, a reduction in brain IRS-
2 increased the life span of mice and stabilized MnSOD activity after fasting (Taguchi et
al., 2007). These results in brain Irs2+/- mice are also consistent with our results in S.
cerevisiae showing that MnSOD is required for life span extension in both mutants with
defects in SCH9 or RAS/cAMP signaling (Fabrizio et al., 2003).

Numerous studies point to SIRT1 as a key regulator of cell survival in response to stress.
It exhibits both pro- and anti- survival functions depending on the conditions. For
instance, SIRT1 countered p53-dependent apoptosis caused by etoposide in mouse
59

embryonic fibroblasts (MEF) (Luo et al., 2001). In an ALS mouse model SIRT1 rescued
neurons (Kim et al., 2007). But SIRT1 can also exacerbate cell death. As described
earlier SIRT1 knockout MEFs showed higher replicative life span under chronic
sublethal oxidative stress (Chua et al., 2005). SIRT1 also sensitized HEK293 cells to
TNF-induced apoptosis (Yeung et al., 2004). Many factors may contribute to the
seemingly contradictory effects of SIRT1. First different nutrient, growth or stress signals
may be sensed by SIRT1 and integrated into divergent outputs. Secondly, SIRT1’s
subcellular localization may also play a role in its regulation of cell death. Some studies
suggest that cytoplasm-localized SIRT1 may promote apoptosis (Jin et al., 2007; Zhang,
2007b) while the anti-apoptosis effect may come only from the nuclear-localized SIRT1
(Tanno et al., 2007). Thirdly, SIRT1 has a wide array of targets which may become
preferentially (de)activated in different contexts. Deacetylation of p53 and FOXO
contributes to the pro-survival effect of SIRT1 (Brunet et al., 2004; Langley et al., 2002;
Motta et al., 2004), while NFkB and p19
ARF
mediate the pro-death effect (Chua et al.,
2005; Yeung et al., 2004).

The detection of lower oxidative stress in the brain of SIRT1 knockout mice is consistent
with our cell culture data. The reduced production of hydrogen peroxide by mitochondria
and major changes in electron transport and leakage in SIRT1 mice recently shown by
McBurney and colleagues (Boily et al., 2008b) may explain part of the protective effect
observed after SIRT1 inhibition. Yet we cannot conclude that SIRT1 is pro-oxidative
damage in all organs in vivo as SIRT1 may play vastly different roles in various organs.
60

For example, Alcendor et al reported the beneficial effect of SIRT1 overexpression in the
heart against oxidative stress, although they showed that this anti-oxidant effect becomes
a pro-oxidant effect at a higher overexpression level (Alcendor RR et al., 2007).
Considering SIRT1’s subcellular localization and the many substrates (both inside and
outside the nucleus) this is not surprising. In fact, SIRT1 may also play additional
important roles in the brain, which could be beneficial. Using SIRT1 knockout mice
Boily et al. also showed that SIRT1 is involved in energy metabolism (Boily et al.,
2008b).

In agreement with the very different roles of SIRT1, here we show that the pro-oxidative
stress role of SIRT1 in neurons and in the mouse brain is not translated into a longer life
span. In fact, SIRT1 knockout mice live shorter than wild type controls under both
normal and calorie restricted diets. Thus, differently from our studies in yeast, we did not
find that calorie restriction extends further the life span of SIRT1 knockout mice, which
is consistent with a recent finding by others (Boily et al., 2008b). Considering that SIRT1
+/- mice display a normal mean life span and that SIRT1-/- mice have severe
developmental defects including a dwarf phenotype (McBurney et al., 2003), it is likely
that these defects are contributing to shortening the life span independently of the rate of
aging. This early death does not necessarily contradict our in vitro results especially
because the SIRT1+/- mice have a normal life span. Furthermore, our hypothesis is that
one of the roles of SIRT1 is to prevent entry into a phase with features of hibernation.
The recent results by McBurney and colleagues, showing that calorie restricted SIRT1
61

mice have a major reduction in metabolic rates (Boily et al., 2008b) support our
hypothesis. Thus, the absence of SIRT1 appears to protect neurons against oxidative
damage in vitro and in vivo. However, this deacetylase is so important for so many
functions under both ad lib and CR conditions that its absence reduced life span. A brain
specific SIRT1 knockout mouse model will be required to determine whether reduced
brain SIRT1 expression may result in increased life span extension. However, a pro-
oxidant role for SIRT1 in neurons does not necessarily translate into an overall negative
role for this deacetylase in the brain since it may play central roles in normal brain
function.

In summary, this study suggests that SIRT1 contributes to oxidative damage in mammals
by activating IRS-2/Ras/ERK signaling downstream of insulin/IGF-I receptors but also
plays a number of roles important for normal growth and life span (McBurney et al.,
2003; Moynihan et al., 2005; Picard et al., 2004). There has been a keen interest in
developing SIRT1 activators for human consumption. Our studies implicating SIRT1 in
both pro-aging and protective functions in yeast and mammalian cells suggest that
additional studies should be carried out before SIRT1 activators are considered for
chronic use.

62

Chapter 3. Role of SIRT1 in synaptic plasticity,
learning and memory
3.1. Summary
SIRT1, the mammalian NAD-dependent deacetylase ortholog to the aging regulator Sir2,
impacts a variety of processes involved in maintaining brain integrity including
neurogenesis, chromatin remodeling and gene expression, as well as redox status and
signal transduction. Here we report that SIRT1 is expressed in areas of the brain
important for learning and memory, and that the absence of this protein significantly
impaired cognitive abilities in mice. This deficit in learning and memory in SIRT1
knockout mice is accompanied by defects in synaptic plasticity as evidenced by reduced
capacity for long-term potentiation in hippocampus CA1 despite unaltered basal synaptic
transmission. In contrast, mice expressing increased brain levels of SIRT1 exhibited no
substantial change in associative or spatial memory tasks. Synaptic plasticity at CA1 area
was minimally affected. These data suggest that SIRT1 regulates brain molecular
processes underlying learning and memory and that SIRT1 is important for normal
cognitive function and synaptic plasticity.

63

3.2. Introduction
SIRT1, the mammalian ortholog of yeast sir2, was first identified as a histone deacetylase
and, in view of the role of sir2 in the regulation of yeast longevity, was proposed to be
involved in mammalian aging through NAD-dependent regulation of gene expression.
Over the last few years, several proteins have been identified as substrates of SIRT1-
mediated deacetylation, among which transcription factors and critical components of
signal transduction cascades. Consistent with this mechanism, SIRT1 was found to
modulate a diversity of biological functions in a variety of tissues. For example, SIRT1
is implicated in cell differentiation and developmental processes (Takata and Ishikawa,
2003; Zhao et al., 2005). It also regulates cell survival and cellular responses to stress and
DNA repair (Langley et al., 2002; Vaziri et al., 2001). In addition, it modulates glucose
homeostasis, insulin secretion and lipid metabolism (Bordone et al., 2006; Li et al., 2007;
Luo et al., 2001; Moynihan et al., 2005; Picard et al., 2004; Rodgers et al., 2005).

SIRT1 is expressed at a relatively high level in brain compared to other tissues
(Michishita et al., 2005). SIRT1 participates in brain development by by regulating
differentiation of neural progenitor cells into neurons or astrocytes (Prozorovski et al.,
2008). It is also involved in an array of neurodegenerative diseases. SIRT1 was found to
be neuroprotective against neuronal death in an animal model of amyotrophic lateral
sclerosis (ALS) (Kim et al., 2007) and of Alzheimer’s disease (Chen et al., 2005; Qin et
al., 2006). SIRT1 also plays a critical role in preconditioning-induced neuroprotection
against ischemia in field CA1 of hippocampus (Raval et al., 2008). On the other hand,
64

SIRT1 inhibition protects against oxidative stress in cultured cortical neurons (Li et al.,
2008, in press). Surprisingly, while numerous studies have focused on the role of SIRT1
in neuropathology and neuroprotection, very few studies have investigated its
involvement in normal brain function. Since SIRT1 is preferentially localized in
hippocampus (Wu et al., 2006), a brain structure critical for learning and memory, the
current study addressed the role of SIRT1 in synaptic function and plasticity and in
learning and memory.

SIRT1 deacetylates histones H1, H3 and H4 and regulates chromatin remodeling
(Vaquero et al., 2004). Increasing evidence indicates that regulation of gene expression
through histone acetylation is an essential component of learning and memory (Fischer et
al., 2007). Increasing histone acetylation levels by inhibition of histone deacetylase
facilitates induction of hippocampal long-term potentiation (LTP) (Levenson et al.,
2004b), a form of synaptic plasticity widely regarded as a cellular model of learning and
memory.

SIRT1 also deacetylates and regulates many transcription factors important for synaptic
function and learning and memory, such as NF κB (Yeung et al., 2004) and myocyte
enhancer factor 2 (MEF2) (Zhao et al., 2005). NF κB is activated during LTP and is
important for LTP maintenance (Kaltschmidt et al., 2006). It is also required for the
initial formation, reconsolidation and retrieval of long-term memory (Lubin and Sweatt,
2007; Meffert et al., 2003; Merlo et al., 2005). MEF2 regulates activity-dependent
65

synaptic formation, an effect regulated by MEF2-mediated post-translational
modifications including acetylation (Mao et al., 1999; Shalizi et al., 2006). Furthermore,
SIRT1 directly modulates many signal transduction pathways involved in synaptic
plasticity and memory. In particular, SIRT1 modulates insulin/IGF-1 signaling through
multiple mechanisms. It upregulates IGF-1 level by depressing IGFBP-1 [define!]
(Lemieux et al., 2005) and augments insulin/IGF-1 signaling by deacetylating insulin
receptor substrate-2 (IRS-2), which increases downstream signaling kinases, mitogen-
activated protein kinase (MAPK) and PI3 kinase (Huang et al., 2008; Zhang, 2007a); Li
et al., 2008 in press). Insulin/IGF-1 and its downstream signaling are important for
synaptic plasticity and learning and memory. Mice with low IGF-1 levels exhibited
impaired spatial learning, which was partially reversed by IGF-1 injection (Trejo et al.,
2007). MAPK activation is also required for LTP (English and Sweatt, 1997) and for
spatial learning and fear conditioning (Selcher et al., 1999).

All these studies suggest that SIRT1 may play an important role in synaptic plasticity and
learning and memory. In the present study, we tested this hypothesis by evaluating
synaptic plasticity and learning and memory in SIRT1 transgenic and knockout mice. Our
results indicate that synaptic plasticity and several forms of learning and memory are
impaired in SIRT1 knockout mice. On the other hand, spatial memory appears to be
altered to a certain degree in SIRT1 over-expressing mice.

66

3.3. Methods and Materials

Animals SIRT1+/+ and SIRT1-/- genotypes have been described previously (McBurney
et al., 2003). NeSTO mice overexpressing SIRT1 specifically in the brain were generated
by breeding the floxed-SIRT1 transgenic mice (Firestein et al., 2008) with Nestin-Cre
mice (Tronche et al., 1999). NeSTO mice from 6-8 months were use for
immunohistochemistry and immunoblotting. For behavioral tests, 18 male and 20 female
naïve Cre+ mice served as test subjects in this study. Of these, six males and 11 females
over-expressed SIRT1.

Behavioral tests
Y Maze Test The Y maze is a three-arm maze, made of black Plexiglas with equal angles
between all arms, which is used to evaluate immediate memory in rodents. 2-5 months
old mice were initially placed within one arm and allowed to move in the maze freely.
The sequence and number of arm entries were recorded for each mouse over an 8-min
period. The percentage of triads in which all three arms were represented (e.g. ABC,
CAB, or BCA but not BAB) was recorded as an alternation to estimate short-term
memory of the last arms entered. The total number of possible alternations is the number
of arm entries minus two. Additionally, the number of arm entries serves as an indicator
of spontaneous activity. Results were analyzed by student's t-test.

Fear conditioning 5-10 months old mice were trained on day 1 and tested on day 2 and
day 3. On day 1 each mouse was placed in the conditioning chamber and after a 3 min
67

baseline period it was exposed to 3 tone-footshock pairings (tone, 20 sec, 80 dB, 2kHz;
footshock, 1 sec, 0.8mA) separated by 1 min intervals. Twenty-four hrs later (day 2),
contextual fear conditioning was assessed by returning the mice to the conditioning
chamber and measuring freezing behavior (i.e. motionless position) for 5 min. Whether
or not the mouse was freezing was scored once every 10 sec and % freezing is calculated
by dividing the number of times the mouse froze by the total possible freezings in 5 min
* 100%. Extinction data show % freezing to context over an 8 min period. 48 hrs after
training (day 3), each mouse was placed into a novel chamber and freezing behavior was
scored for 3 min as altered context test. Then it was tested for auditory fear conditioning
for 5 min. Freezings in the next 3 min were also used for 8-min extinction graph. Data are
expressed as means ± SEM and analyzed by t-test.

Vision test The reaction of mice to light was examined by shining a bright light in the
eye to look for the pupillary reflex. To test visual perception, a black bar was inserted
quickly into the home cage, and the escape behavior of mice was observed. SIRT1-/-
mice showed comparable vision to SIRT1+/+ mice.

Barnes maze The Barnes maze is a white circular platform (91 cm in diameter, 91 cm
height) with 20 holes (5 cm in diameter) located 2.5 cm from the perimeter. A black
escape box (EB) was placed under one of the holes and 19 false target boxes under all
other holes. All mice were 2 months old at the onset of the experiment. Each mouse was
randomly assigned a unique position for the EB. Distinct spatial cues were located all
68

around the maze and kept constant throughout the study. All mice were brought into the
testing room 1 hour before the experiment. Each mouse was trained once daily from day
0 till day 7 and tested twice daily from day 1 through day 7. During the training session
the mouse was placed in the middle of the maze in a start chamber for 30 sec and then
allowed to freely explore the maze until it entered the EB or after 2 min elapsed. When
the mouse entered the EB it was allowed to remain there for 30 sec. When the mouse did
not enter the EB by itself it was gently guided there and allowed to stay there for 30 sec.
The EB was always located underneath the same hole for a particular mouse. On days 1
to 7, following one training session the mice were tested twice. Testing was performed
similarly to training, except that if after 2 min the mouse still did not enter the escape box
it would not be guided to the EB but returned to the cage. Several measures were
recorded for each testing including, the latency (second, s) (the time it took the mouse to
enter the EB), the number of errors before entering the EB (errors were defined as nose
pokes and head deflections over any false target box), deviation of the first error (how far
away the first error was from the EB) and strategy employed by the mouse to locate the
EB (random search strategy-localized hole searches separated by crossings through the
maze center), serial search strategy-systematic hole searches in a clockwise or
counterclockwise direction, or spatial search strategy-navigating directly to the EB with
both error and deviation scores of no more than 3. Success rate for each test was either
100% (finding the EB within 2 min) or 0 (not finding the EB within 2 min). For mice that
did not find the EB within 2 min the latency was represented as 120 (seconds). All
measures were averaged from 2 tests to get the daily value for each mouse. Data were
69

analyzed with two-way ANOVA with genotype and days as sources of variation. On day
14 (7 days after the last training and tests) retention was assessed by testing each mouse
once.

Open field exploratory behavior Mice were placed in a level, square, open field box
(55cm
2
wire mesh grid [0.6 cm
2
], surface area bordered by a 15 cm high, black plexiglass
surface). Movement was tracked and recorded for 300 s using Field 2020 tracking
software from HVS Image. Both total distances traveled and time spent in the 40 cm
2

square area at the center of the field was averaged for each group.

Inclined screen balance and coordination The same wire mesh surface box used for
open field testing was used for inclined screen. Mice were placed in the center of the
tilted (60 degree incline), open field box. Movement was recorded for 300s using Field
2020 tracking software from HVS Image (Buckingham, UK). Total distance traveled was
averaged for each group.

Rota-rod motor performance Locomotor performance was tested using an automated,
motorized Rota-Rod Treadmill for mice (Med Associate Inc., St. Albans, VT). The
rotating drum (3 cm in diameter) is divided into test zones, by round plates, allowing for
5 mice to be tested at one time. Mice were habituated to the Rota-rod one day prior to
testing by first being placed on the non-rotating drum for 10s immediately followed by
120s period with the drum rotating at a constant 4.0 rpm. During testing, mice were
70

placed on the rotating drum which was set to gradually accelerate from 4 to 40 rpm over
a 300s interval. Mice were forced to move at increasing speeds to avoid the 16.5 cm fall
to the platform. Each mouse received 3 trials, with a 15-min inter-trial interval. Latencies
before falling were measured and averaged for each group.

Water Maze
The water maze apparatus consisted of a white circular plastic tank (100 cm diameter and
70 cm high) which was filled with water (24±1 ◦C) made opaque by the addition of white
DryTemp® paint powder (Palmer Paint Products Inc.,Troy, MI, USA). Visual cues of
objects varying in geometric shape and color and were affixed to a clear, plastic cylinder
inserted adjacent to the interior wall of the tank and extending approximately 30 cm
above the water surface. A clear, circular (10 cm diameter) escape platform was
submerged a few millimeters below the water surface.

Mice were trained to locate the hidden platform with 4 trials per day for 6 days. Each
trial was started by placing the mouse in the water, adjacent to and facing the wall of the
tank. The location of entry of the mouse changed for every trial such that mice entered
the maze from each of 4 different start positions each day, with the order of start position
randomly set each day. During trials, mice were given 60s to find the submerged
platform. If a mouse did not find the platform, it was gently led to the platform. After
either finding or being led to the platform, mice were left on the platform for 30s
allowing it time to familiarize itself of the platform’s location with respect to the visual
71

cues. On day 7 mice performed a probe trial where the escape platform was removed and
the amount of time spent searching in each quadrant of the pool is recorded for 60
seconds to assess recall of the platform location. Data were collected using a
computerized animal tracking system (HVS Image with Water 2020 Software,
Buckingham, UK) which recorded path length, swim speed, time spent in each quadrant
of the pool, and time taken to reach the platform (latency). Animals were tested in squads
of 8–12 mice, and all treatment groups were present within each testing squad. Time (%)
spent in each quadrant was recorded and averaged for each group.

14-Unit T-Maze
An integrated straight runway and 14-unit T-maze was constructed from black Plexiglas
and over-laid with a single sheet of transparent Plexiglas. The maze was situated
diagonally on a stainless steel grid floor which was wired, in series, to receive a constant-
current scrambled shock (Model El 3-08, Coulbourn Instruments, Inc., Lehigh Valley,
PA). A timer, with hand-held switch, was used to initiate shock contingencies. Two
black Plexiglas boxes with a movable rear walls served as both a goal- and start-boxes
which could be inserted into either end of either the straight runway or maze. The layout
of the 14-unit T-maze is a mirror image of the one described in Spangler, Rigby and
Ingram (1986) and built at 21% scale. As in Spangler et al, the maze was separated into
five distinct segments by guillotine gates, which were used to prevent reentry into
previous segments of the maze. Additionally, the maze incorporated non-functional
sham gates to ensure that gates would not serve as cues. Maze trials were recorded using
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a digital video camera, located above the maze, on a fixed boom. Analysis was
conducted from the resulting video files.

Maze pretraining. Before the start of preliminary one-way avoidance training in the
straight runway, mice were removed from the colony room and transported, in their home
cages, to the testing room where they were acclimated for at least 30 minutes (m). During
the first trial, each mouse was transferred from its home cage to the start-box. The start-
box was then inserted into the runway entrance and the mouse was gently pushed from
the box into the runway. The mouse then had 10 seconds (s) to avoid a 0.8-mA foot-
shock by running to the goal box at the opposite end of the runway. Once the mouse
entered the goal box, a gate was lowered, and the entire box was transferred back to the
runway entrance. All animals received 15 massed practice trials with a 1m intertrial
interval (ITI) between 1000 and 1200 hours.

Maze acquisition. Maze training began 1 hr after the final pretraining session. During this
interval, mice remained in the maze room (in their home cages). Prior to maze training, a
shallow tray of ethanol was placed below the shock grid to discourage navigation via
scent trails. During maze training, each mouse was removed from its home cage and
placed into the start-box. The start box was then placed into the maze entrance and the
mouse was gently pushed from the box. To avoid a continuous mild foot-shock (0.8 mA),
the mouse had to navigate through the first gate in the maze within 10s. Once the mouse
passed through the gate, it was lowered and the avoidance contingency was reset. This
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sequence was repeated in each of the five segments of the maze. Once the mouse entered
the goal-box at the end of the final maze segment, the goal-box was exchanged with the
start-box such that the mouse was again at the maze entrance. All animals received 15
massed training trials with a 1m intertrial interval (ITI) between 1300 and 1800 hours.
All trials lasted a maximum of 360s. Testing was immediately halted for animals which
failed to complete the maze in less than 360s on more than two maze trials.

Nissl Staining, Immunohistochemistry and Western blotting

SIRT1 wild type and KO mice were deeply anesthetized with isoflurane and
intracardially perfused with cold PBS followed by 4% formaldehyde. Brains were
removed, fixed for 2 hr and serially incubated in 15% and 30% sucrose for 15 hr. Then
brains were snap frozen and cut into 30 μm thick coronal sections on a cryostat. Brain
sections were stained with cresyl violet for Nissl staining or immunostained with
corresponding antibodies. Antigen retrieval was performed with 0.1 N HCl. Half brains
from the NeSTO and Nestin-Cre mice were fixed for 24-48 h in 4% paraformadehyde,
paraffin embed and 5 um thick coronal sectioned. Slices were deparaffinized, rehydrated
and antigen retrieval was performed by microwave irradiation. Both frozen and paraffin
sections were incubated with Sirt2a 1:1000 (UPSTATE), NeuN 1:500 (Chemicon
International) or GFAP 1:500 (Sigma) as primary antibodies overnight at 4°C. Alexa
Fluor 488 and 568 (1:500) were used as secondary antibodies. Photographs were taken
with an Olympus confocal microscope using the Kalman filter and sequential scanning
mode. For immunoblotting hippocampus was finally dissected from the brain and protein
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lysates run in a PAGE-SDS gel. Protein was electro-transfer to a PVDF membrane and
blot with Sirt2-alpha antibody (UPSTATE). Immunoblots were quantified using the
ImageQuant software.

Electrophysiology
Hippocampal slices preparation The animals were deeply anesthetized with halothane
(Sigma) and then decapitated. Brains were quickly removed and transferred to
oxygenated, ice-cold, high magnesium ACSF cutting medium containing (in mM): 124
NaCl, 26 NaHCO
3
, 10 glucose, 3 KCl, 1.25 KH
2
PO
4
, 5 MgSO
4
, and 3.4 CaCl
2
. 400-µm-
thick transverse hippocampal slices were made by using a McIlwain-type tissue chopper.
Slices were then maintained in a recovery chamber with oxygenated normal ACSF (in
mM: 124 NaCl, 26 NaHCO
3
, 10 glucose, 3 KCl, 1.25 KH
2
PO
4
, 2.5 MgSO
4
, and 3.4
CaCl
2
) at room temperature for 40 minutes and then transferred to an interface recording
chamber in which slices were exposed to a warm, humidified atmosphere of 95% O
2
/ 5%
CO
2
with preheated (33 ±0.5 °C) oxygenated normal ACSF perfused at 1 mL/min.

Field recording After a minimum of one-hour incubation in the recording chamber, field
excitatory postsynaptic potential (fEPSP) were recorded from stratum radiatum of field
CA1 using a single glass pipette filled with normal ACSF (yielding a resistance of 3-5
M Ω) in response to the stimulation on Schaffer collateral-commissural projection in field
CA1 with twisted nichrome wires (single bare wire diameter: 50 μm). Before each
experiment, the input-output relation was examined and the stimulation intensity was
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adjusted so as to obtain 40-50% of the maximum fEPSP without spike component in the
response. The adjusted stimuli were then delivered at 0.05Hz and the responses were
recorded as the baseline. After establishing a 10 minutes stable baseline, LTP was
induced by 2 trains of theta burst stimulation (TBS) serially. During induction, the
duration of the stimulation pulses was increased two-fold. The first train (5x3) was 5
bursts separated by 200 ms, with each burst consisting of 3 pulses at 100 Hz. The second
train (10x10) was 10 bursts separated by 200 ms, 10 pluses at 100 Hz within each burst.
After each train of stimulation, the responses were followed for 40 minutes. Paired-pulse
facilitation (PPF) responses were also examined but on different slices from those to be
experimented for LTP. Five different paired-pulse intervals were tested from 30, 50, 100,
200, to 300 ms on the same slice. Data were collected and digitized by NAC 2.0
Neurodata Acquisition System (Theta Burst, Irvine, CA).

fEPSP responses were analyzed for both amplitude and fall slope. The input-output (I/O)
curve data were presented as a percentage of maximal fEPSP without spike component
while LTP data were presented as a percentage of baseline. Besides I/O cuves and LTP,
we also looked at the PPF, burst responses, and short-term potentiation (STP). PPF were
based on the fEPSP amplitude, calculated with the following formula: PPF = [(2
nd
fEPSP
– 1
st
fEPSP) / 1
st
fEPSP] x 100%. Responses to individual bursts during single train of
TBS were expressed as the area of each burst responses, which was further normalized
with the first burst area for analysis. Lastly, STP was measured as the mean fEPSP within
2 minutes after each train of TBS.
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Statistics
The t-test was used to compare 2 groups. One-way ANOVA was used to compare
multiple groups, followed by Bonferroni's Multiple Comparison Test. For the Barnes
maze, Morris water maze, inclined screen, rotarod and open field data, a two-way
analysis of variance (ANOVA) was used. For the data, two-way ANOVA followed by a
Fisher’s PLSD tests employed. An alpha level of 0.05 was used as the criterion for
significance for all analyses.

3.4. Results
3.4.1. SIRT1 deficiency does not alter gross brain anatomy
To investigate the possible role of SIRT1 in learning and memory, we first examined
brain morphology in SIRT1 wild type (WT) and knockout (KO) mice. Brains of SIRT1
KO mice were generally smaller in size compared with WT. Nissl staining did not reveal
any discernable abnormality in brains from SIRT1-/- mice compared to those from WT
mice (Fig. 23 A). Similar results were obtained with immunostaining for NeuN (a marker
for neuronal nuclei) and DAPI (labeling nucleus in general) (Fig. 23 B).
Immunohistochemistry with NeuN and MAP2 (a marker of neuronal dendrites) did not
show obvious differences in neurons or dendrites between KO and WT mice, suggesting
that SIRT1 knockout mice does not affect gross brain anatomy(Fig. 23 C).
A

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Figure 23, Continued
B

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Figure 23, Continued
C

Figure 23. SIRT1 deficiency does not change gross brain anatomy
A, Representative images of Nissl stain of coronal brain sections of WT and SIRT1-/- mice. B,
Representative images of immunoreactivity for NeuN and DAPI of coronal brain sections of WT and
SIRT1-/- mice. C, Representative images of immunoreactivity for NeuN (a marker for neuronal nuclei) and
MAP2 (a marker of neuronal dendrites) of WT and SIRT1-/- mice in CA1 area of hippocampus.

3.4.2. SIRT1 is essential for normal cognitive capabilities in mice
Next we assessed the effect of lack of SIRT1 on learning and memory by testing SIRT1
KO mice in three different behavioral paradigms: Y-maze for immediate memory, fear
conditioning for classical conditioning and Barnes maze for spatial learning. We first
subjected 2-5 months old SIRT1+/+ (n=16) and SIRT1-/- (n=16) mice to a Y maze task
for assessment of immediate memory (Fig. 24 A). When allowed to move freely, rodents
tend to sequentially visit different arms of the maze, and the percentage of triads in which
all three arms are represented (e.g. ABC, CAB, or BCA but not BAB) is scored as
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spontaneous alternation (SA) to estimate immediate memory of the last arms entered.
Compared to wild type controls (WT), knockout (KO) mice exhibited lower SA during
the 8 min period (P < 0.05, t-test), suggesting that KO mice have decreased immediate
memory compared to WT. Since previous studies reported the regulation of SIRT1 on
androgen receptor, we extended the analysis to evaluate possible differences between
sexes. We found significantly lower SA in male KO mice (Fig. 24 C; male WT vs. male
KO, P < 0.05 one-way ANOVA, Bonferroni's test) whereas female KO did not exhibit
significant decrease in SA (female WT vs. female KO, P >0.05, one-way ANOVA,
Bonferroni's test). In addition, we recorded the total number of arm entries as a partial
indicator of locomotor activity level and found that KO mice moved more than controls
(Fig. 24 B, P < 0.05, t-test, n=16 for SIRT1+/+ and n=16 for SIRT1-/-). Further gender
analysis revealed significantly more arm entries by female KO mice compared to WT
mice, while male KO mice did not differ significantly from male WT mice (Fig. 24 D,
male WT vs. male KO, P >0.05 one-way ANOVA, Bonferroni's test; female WT vs.
female KO, P < 0.01, one-way ANOVA, Bonferroni's test). We also tested vision for
both WT and KO mice and did not find any difference in pupillary reflex or perception.

Figure 24. SIRT1KO mice exhibit impaired immediate memory in Y maze test
(A) 2-5 months old SIRT1+/+ (n=16) and SIRT1-/- (n=16) mice were allowed to move freely in a Y maze
for 8 minutes. The percentage of triads in which all three arms were represented was recorded as
spontaneous alternation. SIRT1-/- mice show a lower spontaneous alternation of arm entries (P < 0.05, t-
test). (B) The total number of arm entries was recorded as a partial indicator of spontaneous activity.
SIRT1-/- mice show a lower spontaneous alternation of arm entries (P < 0.05, t-test). (C) Male SIRT1 KO
mice show decreased spontaneous alternation that male WT controls (male WT vs. male KO, P < 0.05,
one-way ANOVA, Bonferroni's test), while no significant difference is found between female WT and
female SIRT1 KO mice (female WT vs. female KO, P >0.05, one-way ANOVA, Bonferroni's test). (D) No
significant difference is found between male WT and male SIRT1 KO mice in the total number of arm
entries (male WT vs. male KO, P >0.05, one-way ANOVA, Bonferroni's test); however, female SIRT1 KO
mice entered more arms than female WT controls (female WT vs. female KO, P < 0.01, one-way ANOVA,
Bonferroni's test). Data represent means ± SEM.

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We then employed fear conditioning to assess associative memory. In this paradigm,
mice learn to associate a neutral context and a neutral tone with noxious foot shocks
during training. When they are later exposed to the same context or tone in the absence of
shock, they exhibit fear, which is reflected by a stereotyped behavior - freezing or an
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absence of movement (except for respiration). During the training session, when mice
were exploring the chamber, the number of crossings (how many times a mouse crosses
the midline of the chamber) in the first 3 min was recorded to provide a partial indicator
of locomotor activity level. SIRT1-/- mice exhibited increased activity compared to
SIRT1+/+ mice (Fig. 25 A, P < 0.01, t-test, SIRT1+/+ [n=13] vs. SIRT1-/- [n=10]). Then
the mouse was subjected to 3 pairs of tone-foot shocks. During training both groups of
mice displayed similar responses (jumping, rushing, and freezing) following foot shocks,
suggesting that the sensing, perception and response to the electric shock is not affected
by SIRT1 deletion. Mice were tested after 24 hr in the same chamber for conditioned fear
to context. During a 5 min period SIRT1 KO mice showed much less freezing than WT
controls (Fig. 25 B, P <0.001, t-test, SIRT1+/+ [n=13] vs. SIRT1-/- [n=10]), indicating
that they had decreased learning and memory in an associative type of task. The
extinction curves over 8 min period were plotted in Fig. 25 C. In the gender analysis,
both males and females showed diminished freezing response to context (Fig. 25 D, male
WT vs. male KO, P < 0.05; female WT vs. female KO, P < 0.001, one-way ANOVA,
Bonferroni's test). These findings implicate that SIRT1 plays a role in contextual fear
conditioning.

Figure 25. SIRT1KO mice exhibit impaired contextual fear
(A) In fear conditioning the number of times a mouse crossed the midline of the chamber in the first 3 min
during the training session serves as a partial indicator of spontaneous activity. Compared with WT, SIRT1
KO mice cross the midline of the chamber for more times in the first 3 min during training, P < 0.01, t-test,
SIRT1+/+ (n=13) vs. SIRT1-/- (n=10). (B) 24 hr after training mice were tested for conditioned fear to the
same context for 5 min. SIRT1 KO mice show diminished conditioned fear to the context compared with
WT controls, P <0.001, t-test, SIRT1+/+ (n=13) vs. SIRT1-/- (n=10). (C) Extinction curves over 8 min
period. (D) Both male and female SIRT1 KO mice exhibit diminished contextual fear compared to their
respective WT controls, male WT vs. male KO, P < 0.05; female WT vs. female KO, P < 0.001, one-way
ANOVA, Bonferroni's test. Data represent means ± SEM.

Two days after training, mice were placed in a novel chamber and tested for fear
conditioning in response to the auditory cue. The levels of freezing shown by both mice
in the altered context test were not significantly different (Fig. 26 A, P >0.05, t-test,
SIRT1+/+ [n=13] vs. SIRT1-/- [n=10]). When tested for conditioned fear to tone, SIRT1-
/- mice exhibited less freezing compared to WT controls (Fig. 26 B, P <0.05, t-test,
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SIRT1+/+ [n=13] vs. SIRT1-/- [n=10]). The extinction curves over 8 min period were
plotted in Fig. 26 C.

Figure 26. SIRT1KO mice exhibit decreased conditioned fear to auditory cue
(A) Two days after training, mice were placed in a novel chamber for 3 min and tested for fear response in
the altered context test. SIRT1 KO mice show similar levels of freezing response to WT controls in the
altered context, P >0.05, t-test, SIRT1+/+ (n=13) vs. SIRT1-/- (n=10). (B) Conditioned fear to auditory cue
was recorded for 5 min. SIRT1 KO mice show impaired conditioned fear to the tone, P <0.05, t-test,
SIRT1+/+ (n=13) vs. SIRT1-/- (n=10). (C) Extinction curves over 8 min period. (D) Gender analysis of
freezing response to tone. No significant difference is found between male WT and male SIRT1 KO mice
in conditioned fear to tone (male WT vs. male KO, P > 0.05, one-way ANOVA, Bonferroni's test);
similarly, no significant difference is uncovered between female WT and female SIRT1 KO mice in
conditioned fear to tone (female WT vs. female KO, P > 0.05, one-way ANOVA, Bonferroni's test). Data
represent means ± SEM.

Further analysis reveals that despite the trend for KO to underperform WT controls,
neither male nor female SIRT1 KO mice exhibited significantly decreased freezing
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response to tone compared to WT (Fig. 26 D, male WT vs. male KO, P > 0.05; female
WT vs. female KO, P > 0.05, one-way ANOVA, Bonferroni's test). These findings
implicate that SIRT1 may play a role in auditory fear conditioning. Taken together, our
results suggest that SIRT1-/- mice have impairment in a type of associative memory.

The Barnes maze test was also used to assess spatial learning and retention of memory. In
this test a mouse is placed in the middle of an open circular dry-land platform in a start
chamber for 30 sec and then allowed to move freely until it locates among 20 holes the
one unique hole that leads to a black target escape box or after 2 min elapses. To
accurately find the escape hole, the mice need to learn, memorize and use the
relationships among the visual cues in the room were the maze is located. 2 month old
SIRT1+/+ and SIRT1-/- mice were trained once daily for 8 consecutive days (day 0 till
day 7) and tested twice daily on day 1 through day 7. Although the success rate to find
the target hole in a two-minute period over the course of the acquisition phase steadily
improved over the course of 7 days in both groups, the SIRT1 null mice exhibited
significantly lower percentage of success and slower rate of improvement than the WT
controls (Fig. 27 A, SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], P < 0.001, two-way ANOVA).
In accordance, the latency to locate the escape box declined steadily for both groups of
mice, but it was markedly slower in the SIRT1 null mice than in the WT ones (Fig. 27 B,
SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], P < 0.001, two-way ANOVA). Over the course of
one week, both groups of mice showed a gradual decrease in the number of errors
(visiting a false box) before entering the escape box, and although WT showed a
tendency of fewer total number of errors in comparison with the SIRT1 KO mice, no
significant difference was found (Fig. 27 C, SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], P >
0.05, two-way ANOVA). However, when we analyzed the deviation of the first error
(how far away the first error was from the escape box), we found that by day four the
control mice started to exhibit less deviation to the target and by the end of one week the
deviation was significantly lower than that observed in the SIRT1 mutant mice, who, in
contrast, maintained their high level of deviation as the days progressed (Fig. 27 D,
SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], P < 0.001, two-way ANOVA).

Figure 27. SIRT1KO mice show impaired spatial learning in Barnes maze test
2 month old SIRT1+/+ (n=6) and SIRT1-/- (n=5) mice were subjected to the Barnes maze test over a 2-
week period. (A) SIRT1 KO mice show a slower increase with training in success in finding the escape box
within 2 min compared to WT controls, P < 0.001, two-way ANOVA, SIRT1+/+ vs. SIRT1-/-. (B) SIRT1
KO mice show a slower decline in latency for finding the escape box compared to WT controls, P < 0.001,
two-way ANOVA, SIRT1+/+ vs. SIRT1-/-. (C) The number of errors (visiting a false box before entering
the escape box) declined for both WT and SIRT1 KO mice and no significant difference is found, P > 0.05,
two-way ANOVA, SIRT1+/+ vs. SIRT1-/-. (D) Deviation of the first error decreased for WT, but not for
SIRT1 KO mice, P < 0.001, two-way ANOVA, SIRT1+/+ vs. SIRT1-/-.
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Furthermore, we classified the search strategies employed by mice to locate the escape
box as random (localized hole searches separated by crossings through the maze center),
serial (systematic hole searches in a clockwise or counterclockwise direction), or spatial
(navigating directly to the escape box with both error and deviation scores of no more
than 3). Both WT and KO mice started out using primarily random and serial strategies-
83.3% for WT and 100% for KO; but striking differences emerged over the course of one
week. By day 3 the WT mice dropped the random strategy to negligible levels (8.3%) and
steadily gained the spatial or direct search strategy, which became equally used with the
serial strategy and later by day seven spatial strategy represented 83.3% of the search
patterns used to find the target (Fig. 28 A, C,D). In contrast, although the SIRT1 null
mice decreased the use of the random strategy by half over the course of 6 days (from
60.0% to 30.0%) and acquired a fair level of the spatial search (20.0%), the spatial
strategy failed to dominate as the days went on and serial strategy (by day 7 serial
strategy accounted for 50% of all strategies used) was preferentially employed (Fig. 28
B,C,D). A comparison for the percentage of random strategy (P < 0.05, SIRT1+/+ [n=6]
vs. SIRT1-/- [n=5], two-way ANOVA) as well as the percentage of spatial strategy (P <
0.01, SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], two-way ANOVA) illustrates the difference
between WT and KO mice. These results are consistent with the impairment in spatial
learning in SIRT1 KO mice. During the retention test on day 14, both WT and KO mice
displayed some degree of forgetting and did not perform as well as (but there was no
significant change) that on day 7 of the acquisition phase in terms of latency and number
of errors (Fig. 27, 28). The similar tendency of inferior performance was evident in the
SIRT1 KO mice as revealed by lower success rate yet higher latency, number of errors
and deviation. Albeit the disappearance of the random strategy from the SIRT1 KO mice
and the marginal gain of random strategy by the WT mice, the previous preferred serial
and spatial search patterns, respectively, were still preserved.

Figure 28. Differential search strategies employed by WT and SIRT1KO mice in Barnes maze test
(A) Proportions of search strategies employed by SIRT1+/+ mice to locate the escape box. WT mice
gradually acquire spatial strategy as the dominant search strategy while eliminating random strategy with
training. (B) Proportions of search strategies employed by SIRT1-/- mice to locate the escape box.
Although random strategy declined with training and spatial strategy was gained SIRT1 KO mice failed to
employ spatial strategy as the dominant one. (C) A comparison of the two genotypes on the percentage of
random strategy used. The use of random strategy declined in SIRT1 KO mice with training, but not as fast
as in WT controls. (P < 0.05, SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], two-way ANOVA). (D) A comparison
of the two genotypes on the percentage of spatial strategy used. Both groups of mice gained the use of
spatial strategy with training, but WT mice came to use it predominantly while spatial strategy failed to
dominate in SIRT1 KO mice. (P < 0.01, SIRT1+/+ [n=6] vs. SIRT1-/- [n=5], two-way ANOVA).

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89

Together all these data demonstrate that SIRT1 null mice exhibit a deficit in spatial
memory and learning acquisition but no impairment in cognitive retention. Our results
prompted us to further analyze the impact that increased SIRT1 levels in the brain might
have in cognitive functions.

3.4.3. Effect of SIRT1 overexpression on learning and memory
In order to increase brain SIRT1 levels, we bred the floxed SIRT1 transgenic mice
previously described (Firestein et al., 2008) with the brain specific Nestin-Cre strain
(Tronche et al., 1999). The double transgenic animals obtained from these breedings were
referred to as NeSTO—mice overexpressing SIRT1 specifically in the brain, and matched
Nestin-Cre mice which served as controls. By Western blot quantification we determined
that SIRT1 protein levels in hippocampus were increased about 15 fold in NeSTO mice
as compared to Nestin-Cre mice (Fig. 29). Immunofluorescence revealed that similar to
the endogenous protein, exogenous SIRT1 in NeSTO mice was restricted to hippocampal
granule cells in dentate gyrus and pyramidal neurons in CA1 and CA3 (Fig. 30). In
addition, SIRT1 also colocalized with NeuN and DAPI but not with the glial protein
GFAP.

Figure 29. SIRT1 protein levels in the hippocampus of NeSTO mice
A. Western blot of hippocampus lysates from NeSTO and Nestin-Cre mice using a polyclonal anti-SIRT1
antibody. B, Quantification of Western blot signal shows substantially increased level of SIRT1 in the
hippocampus of NeSTO mice compared to Nestin-Cre mice. C, Average level of expression reveals that
NeSTO mice contain ~15-fold increased of SIRT1 in hippocampus analyzed either in males and females.
90

Figure 30. SIRT1 overexpression in hippocampal neurons of NeSTO mice
91

Immunofluorescence of paraffin-embedded coronal brain sections (4 μm) of Nestin-Cre and NeSTO mice
using antibodies against SIRT1 and GFAP and counterstained with DAPI. Pictures were taken with an
Olympus Confocal Microscope. SIRT1 signal colocalizes with DAPI in different regions of hippocampus
including the dentate gyrus, CA1 and CA3 area. The increased SIRT1 levels in NeSTO mice hippocampus
show similar localization pattern as in Nestin-Cre mice.
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Both strains of mice, NeSTO and Nestin-Cre, were first tested for exploratory behavior
and general locomotor activity. Performance in neither open field nor inclined screen test
indicated any significant difference in path length or in percent of time spent in
movement between the genotypes (Fig. 31 A-D). Likewise, the rotarod test which
evaluates locomotor performance and fatigue showed no significant effect of SIRT1
overexpression on latency to fall as assessed by two-way ANOVA (Fig. 31 E). These
data demonstrate that both genotypes have similar spontaneous activity and locomotion.

Figure 31. Overexpression of SIRT1 does not alter spontaneous exploratory and locomotor activities
Male and female Nestin-Cre and NeSTO mice were examined in open field exploratory test (A-B), inclined
screen balance and coordination test (C-D) as well as rotarod performance test (E). A, Total pathlength
traveled during open field test. B, Percent of time spent on moving during the open field test. C, Total
pathlength traveled during inclined screen test. D, % of time spent on moving during the inclined screen
test. E, Latency to fall from an accelerating rotarod. Males Nestin-Cre n = 8 and NeSTO n = 9, females
Nestin-Cre n = 10 and NeSTO n = 12.

93

Figure 32. SIRT1 overexpression in the brain does not alter associative memory
A, Fear conditioning to the context. Nestin-Cre wild type controls: n=14, NeSTO SIRT1 overexpressing
mice: n = 18. B, Fear conditioning to the tone. Data represent mean ± SEM.

To study the effect of increased SIRT1 brain levels on learning and memory, we first
assessed associative memory using the fear conditioning paradigm but no significant
difference between the two genotypes was detected in the freezing behavior to either the
context or the tone (Fig. 32). Next we employed two hippocampus-dependent spatial
tasks, the 14-unit T-maze and the water maze. Although different from the Barnes maze,
these two tests are widely used to study acquisition and retention of spatial memories. In
the 14-unit T-maze test (stone maze), mice were motivated with electric foot shocks to
make serial position discriminations in order to complete the maze. All mice passed pre-
training trials and exhibited no difference in performance between groups (data not
shown). For maze performance two-way ANOVA analysis was used to determine the
effect of SIRT1 overexpression on the number of shocks received, latency to complete
the maze and errors committed during trials. There was no significant difference in the
number of shocks received between the two genotypes (Fig. 33 A). However, males and
females exhibited marked difference in performance. Whereas NeSTO males committed
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significantly fewer errors and completed the maze in shorter time than Nestin-Cre males
(Fig. 33 C; errors p < 0.05), NeSTO females exhibited a marked increase in latency
compared to Nestin-Cre controls (Fig. 33 B; p < 0.05) which was accompanied by
negligible difference in the number of errors. These data suggest that SIRT1
overexpression in the brain may differentially modulate performance in male versus
female mice in the 14-unit T-maze test.

In the Morris water maze test both genotypes of mice learned to locate the platform, since
they all reached an average minimum latency of 23 sec on the last training day, which
represents a 61% improvement in efficiency to complete the task as compared to the first
day (Fig. 34 A). No significant difference was found between NeSTO and Nestin-Cre
mice in the latency to find the escape platform during training days (Fig.34 A).While
Nestin-Cre mice did not appear to show an apparent preference for the correct quadrant
(where the platform used to be) in the probe trial, NeSTO mice spent moderate but
significantly more time in the correct quadrant compared to Nestin-Cre controls (Fig. 34
B, p < 0.05, t-test). Taken together, these results suggest that increased levels of SIRT1 in
the brain appear to alter spatial learning and memory to a certain degree without
substantially affecting associative memory.

Figure 33. Effect of SIRT1 overexpression on performance in 14-unit T-maze test
A, Number of shocks received. B, Latency to complete the maze. C, Number of errors made. Male Nestin-
Cre n = 10, male NeSTO n = 3, females Nestin-Cre n = 7, females NeSTO n = 8.
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Figure 34. Effect of SIRT1 overexpression on performance in Morris water maze test
A, Latency to find the platform during the five-day acquisition phase. B, Time spent in each quadrant
during platform searching. Nestin-Cre (n = 17) and NeSTO mice (n = 19), p < 0.05, t-test. Data represent
mean ± SEM.

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Figure 35. SIRT1 deficiency has no effect on input/output properties at Schaffer collateral CA1
synapses
Input-output (I/O) curves were generated from field responses to single pulse stimulation in stratum
radiatum of CA1. Results were expressed as percent of maximal fEPSP amplitude (A) or slope (B). I/O
curves were not statistically different between WT and SIRT1 KO mice (WT: n = 7; KO: n = 8; two-way
ANOVA).

3.4.4. Impaired LTP in SIRT1 knockout hippocampus
The impairment in cognitive functions as a result of SIRT1 loss led us to investigate
whether this phenomenon was associated with defects in hippocampal synaptic plasticity,
particularly in long-term potentiation (LTP), a cellular mechanism underlying certain
forms of learning and memory. In order to address this question, hippocampal slices from
four WT and three SIRT1 KO male mice, aged from three to four months old, were used
to determine the electrophysiological characteristics of synaptic responses recorded in
stratum radiatum of field CA1 elicited by stimulation of the Schaffer collateral pathway.
We first measured input/output (I/O) responses produced by stimulation at different
intensities of the Schaffer collateral pathway. As shown in Fig. 35, there was no
significant difference between the two genotypes in I/O function (amplitude: p =0.6058,
slope: p= 0.3612; two-way ANOVA, n =12 for WT, n=11 for KO), which demonstrated
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that loss of SIRT1 did not cause a deleterious effect on basal synaptic transmission at
Schaffer collateral CA1 synapses.

Figure 36. Absence of SIRT1 impairs long-term potentiation
Five and ten theta bursts (five trains of 3 pulses at 100 Hz or 10 trains of 10 pulses at 100 Hz delivered at
200 ms intertrain intervals) were applied at 10 min and 50 min (arrows) to the Schaffer collateral pathway
in field CA1. fEPSP responses in stratum radiatum of CA1 to single pulses were collected for 40 min after
each TBS. Results (means ± SEM) were expressed as percent of the average fEPSP amplitude (A) or slope
(B) recorded during the baseline (pre-TBS) period.

We next determined whether there were changes in synaptic plasticity by examining
long-term and short-term potentiation (LTP and STP). Two protocols were used to induce
LTP, the first one using 5 trains of 3 pulses (5x3) and followed by 10 trains of 10 pulses
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(10x10), both delivered at theta frequency (Fig. 36; Fig. 37 A, B ). The average fEPSPs
measured 30-40 min after 5x3 TBS were not different in slices from KO and WT mice
whether fEPSP amplitude (5x3 amplitude: WT, 184 ± 13%, n =5; KO, 163 ± 11%, n=7;
p =0.241, t test) or initial slope (5x3 slope: WT, 213 ± 14%; KO, 183 ± 16%; p =0.2186,
t test) were analyzed. However, the maximal degree of LTP was significantly smaller in
SIRT1 KO group (10x10 amplitude: WT, 244 ± 9%, n =5; KO, 204 ± 13%, n =7;
p=0.0435. 10x10 slope: WT, 310 ± 17%; KO, 238 ± 16%; p =0.0140; t-test).

Figure 37. Absence of SIRT1 impairs long-term potentiation but not short-term potentiation
A-B, Summary of LTP data. LTP was determined at 30-40 min after each TBS in slices from WT and
SIRT1 KO mice. A, Amplitude. B, Slope. LTP obtained from “10x10” TBS showed significant difference
between WT and KO hippocampal slices. C-D, Summary of the short-term potentiation (STP) results. STP
was determined as the average of fEPSP slopes for the first two minutes post TBS. C, Amplitude. D, Slope.
There were no statistical differences in STP in slices from WT and KO mice after either train of TBS (t-
test).

STP was determined by averaging the responses obtained in the first 2 min after TBS and
there were no differences between STP in SIRT1 KO or WT mice (Fig. 37 C, D; 5x3
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amplitude: p =0.6815; 10x10 amplitude: p =0.3530; 5x3 slope: p =0.7276; 10x10 slope: p
=0.8144; t test).

Figure 38. SIRT1 loss does not affect burst responses or paired pulse facilitation in field CA1 of
hippocampal slices
A-B, WT and SIRT1 KO displayed similar burst responses in both the 5x3 and 10x10 TBS protocol,
respectively (two-way ANOVA). C, Paired pulses were delivered at various interpulse intervals (30, 50,
100, 200, 300 ms). fEPSP amplitudes were measured. Paired-pulse facilitation (PPF) was calculated as the
difference between the second and the first response normalized to the first response. There was no
significant difference in PPF between WT and KO mice (n = 3 for each genotype, two-way ANOVA).

Analysis of synaptic responses elicited during TBS is often used to determine potential
changes in NMDA receptor-mediated synaptic responses. Our results indicated that there
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was no difference between KO and WT mice (Fig. 38 A, B; 5x3: p =0.8883; 10x10: p
=0.0544; two-way ANOVA). We also evaluated potential changes in paired-pulse
facilitation (PPF), which is generally considered to represent presynaptic events; there
was no difference in PPF between the two genotypes (Fig. 38 C; p =0.2966; two-way
ANOVA, n=3 for each genotype).

Together, these results indicate that the lack of SIRT1 in hippocampal neurons does not
modify the basic features of synaptic transmission but results in a substantial deficit in
LTP at CA3-CA1 synapses. And the absence of SIRT1 does not appear to affect the
normal function of NMDA receptors.

3.4.5. Overexpression of SIRT1 in brain has no effect on long-term
potentiation
We proceeded to examine the potential changes in synaptic plasticity by SIRT1
overexpression. Hippocampal slices were prepared from three pairs of age-matched
NeSTO and Nestin-Cre male mice (9~12 months old). In contrast to what was observed
in the KO mice, I/O curves from the NeSTO mice were shifted to the left (Fig. 39,
p<0.0001 for both amplitude and slope data; two-way ANOVA, n=10 for WT, n=14 for
SIRT1 O/E).

Figure 39. SIRT1 overexpression in the brain alters input/output properties at Schaffer collateral
CA1 synapses
Input/output (I/O) curves were generated by increasing the stimulation intensity in stratum radiatum of
CA1. Results were expressed as percent of maximal fEPSP amplitude (A) or slope (B). I/O curves of
NeSTO (SIRT1 O/E) mice were shifted to the left compared to those in Nestin-Cre wild type (WT) controls
(n = 10 slices for WT, n = 14 for O/E; p <0.0001, two-way ANOVA).

However, no significant differences were found for LTP between these two genotypes
(Fig. 40; Fig. 41 A, B; 5x3 amplitude, p=0.5058, 5x3 slope, p=0.3255; 10x10 amplitude,
p=0.7624, 10x10 slope, p=0.7480; t-test). Likewise, there were no differences in STP
(Fig. 41 C, D; post-5x3 TBS: amplitude, p=0.3565, slope, p=0.0833; post-10x10 TBS:
amplitude, p=0.9169, slope, p=0.9439; t-test), burst responses elicited by 5x3 TBS (Fig.
42 A, p=0.7875, two-way ANOVA) or PPF (Fig. 42 C, p=0.9470; two-way ANOVA,
n=5 for each genotype). Only burst responses elicited by 10x10 TBS exhibited a
significant facilitation in NeSTO mice compared to WT (Fig. 42 B, p=0.0016, two-way
ANOVA). These results led us to conclude that SIRT1 overexpression in the
hippocampus did not alter synaptic plasticity although it resulted in increased excitability,
possibly due to changes in certain properties of AMPA receptors.
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Figure 40. Effect of SIRT1 overexpression on long term potentiation recordings in field CA1 of
hippocampal slices
LTP induction protocols (5x3 and 10x10 TBS) were applied to field CA1 in hippocampal slices of Nestin-
Cre wild type (WT) controls and NeSTO (SIRT1 O/E) mice. fEPSP amplitude (A) or slopes (B) were
continuously measured for 40 min after each TBS.

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Figure 41. Overexpression of SIRT1 does not affect long or short term potentiation in field CA1 of
hippocampal slices
A-B, Mean fEPSP amplitude (A) or slopes (B) at 30-40 min post-TBS were expressed as percent of the last
10 min of baseline. No significant difference was found in LTP between Nestin-Cre wild type (WT)
controls and NeSTO (SIRT1 O/E) groups. C-D, STP was calculated as the average increase in responses
(amplitude [A] or slopes [B]) obtained during the first two minutes post TBS; there was no significant
difference between the two genotypes.
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Figure 42. Effect of SIRT1 overexpression on burst responses and paired-pulse facilitation
A, Burst responses elicited by 5x3 TBS were not different between NeSTO (SIRT1 overexpressing) and
Nestin-Cre wild type (WT) mice. B, The burst responses of SIRT1 O/E mice in the 10X10 protocol showed
a significant increase compared to the WT (p = 0.0016, two-way ANOVA). C, Paired pulses were delivered
at various interpulse intervals (30, 50, 100, 200, 300 ms). Paired-pulse facilitation (PPF) was calculated as
the difference between the second and the first response normalized to the first response. There was no
significant difference in PPF between WT and SIRT1 O/E mice (n = 5 for each genotype, two-way
ANOVA).

3.5. Discussion
The results in the current study demonstrate for the first time that the NAD-dependent
deacetylase, SIRT1, plays an important role in learning and memory in the mammalian
brain. We showed that in the hippocampus, a brain region crucial to learning and
memory, SIRT1 is localized in pyramidal neurons of CA1-CA3 and granule cells of the
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dentate gyrus. SIRT1 deficiency resulted in significant cognitive impairment in different
forms of learning and memory: immediate, associative and spatial memories. Consistent
with this cognitive decline we observed in the SIRT1 KO mouse hippocampus a
reduction in LTP, which is believed to be a cellular mechanism underlying certain forms
of learning and memory. On the other hand, brain specific overexpression of SIRT1 did
not impact associative memory, but it appeared to affect certain spatial learning to some
degree. At the same time LTP was not significantly changed by SIRT1 overexpression.
These results suggest that SIRT1 is required for synaptic plasticity, learning and memory,
although overexpression of SIRT1 may not be sufficient to enhance synaptic plasticity or
learning and memory.

Our results demonstrate that SIRT1 deficiency leads to certain behavioral and learning
deficits, especially hippocampus-dependent contextual fear and spatial memories. In Y-
maze test that assess immediate (working or short-term) memory these mice exhibited a
decrease in spontaneous alternation, however, it is much less striking than their impaired
performance in tasks for evaluating associative and long-term spatial memories.
Hippocampus is required for performance in both Y-maze and Barnes maze and is also
critical for contextual fear conditioning. Since we also verified that the vision in the
tested SIRT1 KO mice were similar to those in WT controls, and that the spontaneous
locomotor activity, partially represented by the total number of entries in the Y-maze or
the number of crossings in the fear conditioning, were not lower but higher than WT
controls, the poor performance of SIRT1 KO mice in these tasks was unlikely due to
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either vision problems or activity levels. These data, therefore, implicate the essential role
of SIRT1 in hippocampus-dependent learning and memory. In the Barnes maze test
although we could not record the amount of time each mouse spent in different quadrants
of the maze, a reduced success rate, increased latency, the non-declining deviation as well
as the limited acquirement of spatial search strategy exhibited by SIRT1 KO mice all
clearly manifest the role of SIRT1 in this type of spatial learning. In contrast, auditory
fear conditioning relies on the amygdala (Huang et al., 2008). In SIRT1 KO mice the
decrease in freezing response to auditory cue, although to a lesser degree, may imply that
SIRT1 in other brain regions such as the amygdala also played a part. Consistent with our
data, preliminary results from Dr. Michael Mcburney's lab also suggest that SIRT1 KO
mice show a deficit in certain forms of learning (data not shown). Although SIRT1 KO
mice have brains which are 20% smaller than in WT controls (Boily et al., 2008a), we
did not detect visible alterations in the neuroanatomical architecture of the brain nor in
neuronal morphology. In the current study we focused on the hippocampus, however,
other brain regions may also be assessed in future studies. Specifically, better antibodies
or other probes of SIRT1 need to be developed to reveal the location of SIRT1 in
different brain regions since SIRT1 may exhibit a differential distribution in different
brain regions. Moreover, the subcellular distribution of SIRT1 (for instance, nuclear
versus cytosolic) may also be different in different brain regions and a detailed
characterization will be desirable. The exact localization of SIRT1 in neurons, for
example nuclear, pre-synaptic or post-synaptic locations, will provide us with different
clues on its functional mechanisms. In addition, considering the ubiquitous knocking out
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of SIRT1 in our model, future research should confirm these results with brain specific
SIRT1 knockout mice.

In contrast to SIRT1 KO mice, increased levels of SIRT1 in the brain did not appear to
significantly alter the performance in fear conditioning or spatial learning. Several
possibilities may explain why we did not observe improved learning and memory in
SIRT1 overexpressing mice. Firstly, the beneficial effect of SIRT1 on synaptic plasticity,
learning and memory may be already saturated in wild type mice. Secondly, it is still
possible that SIRT1 may alter some other forms of learning and memory not yet tested.
Thirdly, it remains to be tested whether the enzymatic activity of SIRT1 is elevated
although protein expression levels of SIRT1 are augmented. In addition, as SIRT1 plays
diverse fundamental roles massively increased SIRT1 levels could potentially exert
different effects from moderately increased levels, as has been shown by a previous
report on the effect of overexpressing SIRT1 on the heart (Alcendor et al., 2007b).

To try to understand how SIRT1 modulates learning and memory we examined LTP in
the hippocampus. LTP in CA1 pyramidal neurons is generally considered to underlie
certain types of memories like contextual fear conditioning and spatial learning (McHugh
et al., 1996; Chen and Tonegawa, 1997). We found that LTP was impaired in SIRT1 KO
mice, yet unchanged in NeSTO mice. More research needs to be conducted to reveal the
mechanisms. For example, apart from a more thorough mapping of SIRT1 localization at
the subcellular and brain region levels, it is possible to test if pre-synaptic release of
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glutamate, the major excitatory transmitter, is altered, or if the number and/or activity of
the post-synaptic glutamate receptors are changed. The fact that short-term potentiation
as well as the basic electrophysiological properties of synaptic transmission including
presynaptic mechanisms were unaltered by the absence of SIRT1, may suggest that
SIRT1 acts through a mechanism working post-synaptically downstream of glutamate
receptors. We can also try to identify through which signaling pathway(s) SIRT1
regulates synaptic plasticity. A few signaling pathways which are implicated in synaptic
plasticity have been shown to be regulated by SIRT1, such as insulin/IGF-I signaling
(both insulin and ERK1/2) and endothelial nitric oxide synthase (eNOS) signaling.
Applying the inhibitors or activators of these signaling pathways may uncover the effects
of these signal transductions on SIRT1-mediated regulation of synaptic plasticity.
Chromatin remodeling via changes in histone acetylation has been shown to be involved
in synaptic plasticity and learning (Levenson et al., 2004a). We expect increased brain
chromatin acetylation in SIRT1 KO mice and this effect may participate in the
modulation of learning and memory. Another possibility is that SIRT1 regulates the
expression of certain genes that are involved in learning and memory. In this case,
analysis of microarray data from wild type and SIRT1-null, SIRT1-overexpressing mice
can potentially offer some insights. Furthermore, SIRT1 may also contribute to the
process by directing the redox-dependent fate of neural progenitors (Prozorovski et al.,
2008), since adult neurogenesis may be involved in learning and memory (Aimone et al.,
2006). SIRT1 may also participate in cognitive functions by modulating transciptional
factors like NF κB and MEF2 (Yeung et al., 2004; Zhao et al., 2005) Activated during
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LTP, NFκB is required for several phases of long-term memory. (Kaltschmidt et al.,
2006). MEF2 regulates activity-dependent synapse formation and function (Mao et al.,
1999; Shalizi et al., 2006). We have to keep in mind that several different mechanisms
may together account for the effect of SIRT1 on learning and memory and if that is the
case it is not surprising to see differential performance in different memory tasks and it
would be consistent with the diverse substrates of SIRT1. Whatever the case, gaining a
clearer understanding of SIRT1 will likely benefit us in solving the mystery of learning
and memory, in the treatment of neurodegenerative disease and in the application of its
activators or inhibitors in human use.

In summary, our results obtained from SIRT1 KO and brain specific SIRT1
overexpressing mice indicate that SIRT1 is important for LTP as well as different forms
of hippocampus-dependent memories. Future research should uncover in detail the
cellular and molecular mechanisms underlying the roles of SIRT1 in mammalian
cognitive function.
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Summary and Conclusions
In this dissertation I studied the mechanism of aging, with special attention paid to the
central nervous system (CNS). CNS is one of the organs which are most vulnerable to
age-related damages and functional decline and is also critical to the control of aging. In
this study, I focused on a protein SIRT1, the ortholog of yeast sir2, which was proposed
to be an anti-aging factor by some researchers and attracted extensive attention in the last
few years. My experiments on the mammalian system, both in vitro and in vivo, indicate
multifaceted roles of SIRT1 in neuronal oxidative damage and normal brain functions.

The study of the roles of SIRT1 in neuronal oxidative damage is built on the knowledge
about aging accumulated in the last few decades. Some milestone findings revealed (1)
oxidative stress as an important mechanism for aging, (2) calorie restriction (CR) as an
effective approach for extending life span, and (3) insulin/IGF-I signaling as a key
regulatory system for determining longevity. Aging is a complex process and no single
theory can yet explain all the causes. Yet several types of evidence supports oxidative
stress as a crucial mechanism for aging: oxidative damage increases with age; decreasing
oxidative damage delays aging; manipulations that increase life span also reduce the age-
related increase in oxidative damage; and oxidative stress contributes to many age-related
diseases. CR extends life span in a wide range of organisms, including yeast, worms, fruit
flies and mice. However, what mediates the effects of CR is still not clear and more
research is needed. Apart from CR, genetic mutations in the insulin/IGF-I signaling
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pathway elongate life span in worms, flies and mice, and this life span extension is often
accompanied by an increase in stress resistance.

Sir2 was first brought to broad attention by a study showing that it was required for the
elongation of CR-induced elongation of replicative life span in yeast. Some follow up
studies supported this notion and revealed its function as a NAD-dependent histone
deacetylase, which raised hopes for delaying aging with its activators. However, when
Sir2 was tested in our laboratory with a yeast model of chronological aging, which more
closely resembles mammalian aging, it was shown to have detrimental roles in some
conditions partly through regulation of stress resistance to oxidative damage. This called
for a more careful analysis of Sir2 functions and in my study I extended the observation
to the mammalian system and found that SIRT1 indeed holds a detrimental role in
neuronal oxidative stress. In addition, my finding provides further evidence that SIRT1 is
functionally related to insulin/IGF-1 signaling pathway, the key regulatory signaling
system that determines the longevity.

We first tested the role of SIRT1 in the regulation of resistance to oxidative stress in
neurons. Neurons are a suitable system since they have a relatively high metabolic rate
and are subject to high levels of oxidative stress. And in addition, as generally non-
renewable post-mitotic cells, the oxidative damages will continuously accumulate in
these cells. We found that inhibition of SIRT1 using inhibitors or specifically knock
down SIRT1 with siRNA increased resistance to hydrogen peroxide or menadione
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induced oxidative stress in neurons. These data were consistent with those findings in
yeast mentioned above. But SIRT1 inhibition did not rescue the cells against some other
forms of insults, like UV irradiation or a DNA alkylating agent, suggesting that the
protective effect could be specific for oxidative stress. To figure out the mechanisms
underlying the protection rendered by SIRT1 inhibition we checked the levels of the
endogenous antioxidant system including SOD and catalase but could not find any
significant change that could explain the protection effect. Then we drew upon the
insights from yeast studies showing that both Ras and Sch9 (homologs of mammalian
Ras and Akt) are functionally related to Sir2 and exhibit pro-aging activity and sensitize
cells to oxidative stress. We first checked if SIRT1 regulates mammalian Ras and Akt
signaling. We found that when SIRT1 was inhibited, the activation level of Akt remained
unaltered but Ras activity and its downstream ERK1/2 activation were significantly
down-regulated. Using inhibitors of MEK1/ERK1/2, we found that inhibition of ERK1/2
could protect neurons against oxidative stress, further verifying that Ras/ERK1/2 at least
in part mediate the effects of SIRT1. Further analysis indicated inhibition of SIRT1 or
ERK1/2 protected the neurons primarily against necrosis induced by oxidative stress. To
see if a similar mechanism occurs in vivo, we measured the oxidative damage in the
brains of old mice and found that mice lacking SIRT1 had reduced levels of oxidized
proteins as well as oxidized lipids, confirming the results obtained with cultured neurons.

The characterization of the molecular mechanisms underlying SIRT1-mediated effects on
Ras/ERK1/2 signaling identified a protein insulin receptor substrate 2 (IRS-2) as a
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substrate for the deacetylation activity of SIRT1. This finding further linked SIRT1
function with the key pro-aging system, the insulin/IGF-I pathway. We found that SIRT1
can physically interact with IRS-2, an adaptor protein upstream of Ras in the insulin/IGF-
I pathway. SIRT1 inhibition increases acetylation and decreased phosporylation of IRS-2,
which in turn leads to reduced Ras/ERK1/2 activation. These data indicate a synergistic
relationship between SIRT1 and insulin/IGF-I pathway, in line with yeast findings. Some
other studies have also shown that overexpression of SIRT1 in pancreatic beta cells
enhances insulin secretion. SIRT1 also represses the expression of PTP1B, a negative
regulator of insulin pathway. In addition, SIRT1 depresses the expression of IGF-binding
protein 1 (IGFBP1), one of secreted modulators of IGF-I, which may in turn enhance
IGF-I function. Hence, our result provides one more link between SIRT1 and the pro-
aging insulin/IGF-I signaling pathway.

Intriguingly, although SIRT1 inhibition in neuronal culture and in the brain showed
beneficial effects in reducing oxidative damage, deletion of SIRT1 did not result in
longer life span. SIRT1 deficient mice displayed severe developmental defects and were
short lived under both ad libitum and calorie restricted conditions. These results suggest
that the role of SIRT1 deficiency in protecting against oxidative damage may be
overridden by the vital importance of this deacetylase in many normal functions. This is
consistent with findings in yeast in that SIRT1 can be both anti- and pro-aging depending
on the circumstances. Isolating scenarios where SIRT1 exert “harmful” effect from those
beneficial ones would be important for understanding aging, the insulin/IGF-I signal
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transduction and oxidative stress in the neurons. Future research may help to solve
unanswered questions. For example, a brain specific SIRT1 knockout mouse can be used
to test if the shortened life span persists or if the shortened life span is a mere result of
severe developmental defects specific in our model. It will also be interesting to test
whether or not overexpression of SIRT1 extends mouse life span. As more and more
substrates are identified for SIRT1 it is clear that SIRT1 is a master regulator of many
different processes and any subtle increase or decrease in SIRT1 activity may have severe
system wide consequences.

The finding that SIRT1 has significant roles in regulating Ras/ERK1/2 activity turned our
attention from the health and aging of the brain to normal brain functions, especially
learning and memory. ERK1/2 is an important signaling factor in synaptic plasticity and
learning and memory. Some other studies on SIRT1 functions also support the possible
roles in learning and memory.

To test if SIRT1 plays a role in learning and memory, we first subjected SIRT1 deficient
mice to different behavioral tasks. We found that these mice did not perform as well as
wild type controls in tasks testing immediate memory, associative memory and spatial
memory, respectively. These mice maintained normal gross brain anatomy despite a
smaller brain size (their body is smaller than wild type too). Electrophysiological analysis
on hippocampal slices revealed that the basic synaptic transmission in SIRT1-null mice
was not significantly altered. But LTP, a well studied model of synaptic plasticity, was
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impaired to some degree. LTP is believed to be one of the mechanisms underlying
memory and this finding is in line with the results from behavioral tests. We also checked
if learning and memory is augmented in brain specific SIRT1 overexpressing mice.
SIRT1 overexpression did not change the performance in fear conditioning, nor did it
appear to substantially alter the performance in two different spatial tasks. Consistent
with that, the synaptic plasticity in these mice did not change significantly. These results
suggest that SIRT1 is important for synaptic plasticity and learning and memory,
although enhancement of its activity may not be sufficient to improve these functions.
Some research may be done in the future to enable us to further understand how exactly
SIRT1 regulates learning and memory. For example, a brain specific or conditional
SIRT1 knockout mouse may be used to confirm the deficit in cognitive functions. The
SIRT1 overexpressing mice may be tested more carefully and comprehensively for
behavior in learning and memory tasks. More importantly, the exact mechanism by which
SIRT1 regulates synaptic plasticity needs further analysis. To start with, better SIRT1
antibodies should be utilized to map the distribution of SIRT1 in different brain regions.
Here we examined the hippocampus but cortex, amygdala and other regions should also
be checked and it would not be surprising to see different patterns in different areas.
Next, the subcellular distribution of SIRT1 may not be the same in different brain regions
and localizing SIRT1 to the nucleus, pre-synaptic or post-synaptic sites will have
different functional implications. To figure out how SIRT1 may be involved in LTP, we
can test in SIRT1 null hippocampus slices for any change in the pre-synaptic release of
glutamate, the major excitatory transmitter, or in the activity or/and number of the post-
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synaptic glutamate receptors. Along the same lines, the signaling pathways through
which SIRT1 may regulate synaptic plasticity remains to be identified. SIRT1 has been
shown to modulate insulin/IGF-I signaling as well as endothelial nitric oxide synthase
(eNOS) signaling, both of which are involved in synaptic plasticity. Inhibiting or
activating these signaling pathways may uncover the roles of these transductions on
SIRT1-mediated modulation of synaptic plasticity. In addition, analysis of microarray
data from wild type, SIRT1 overexpressing and SIRT1 deficient mice may yield certain
genes involved in learning and memory whose expression is regulated by SIRT1. It is
very likely that several different mechanisms may together explain the effect of SIRT1 on
learning and memory and that SIRT1 modulates synaptic plasticity with different
mechanisms in different brain regions. Therefore, it is not unlikely to observe differential
performance in different memory tasks.

Together, my parts of research indicate that SIRT1 can be both pro- and anti- aging and it
plays multifaceted functions in aging and normal brain function.

Apart from the above important scientific implications, our results on the connection
between SIRT1 and neuronal oxidative damage and the relationship between SIRT1 and
learning and memory are of practical significance too, especially due to the fervid pursuit
of sirtuins as potential anti-aging drug targets. The initial studies showed that Sir2
extended longevity in several lower organisms and suggested Sir2 as a key mediator of
the life span extension from calorie restriction. Adding to the excitement is the
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identification a small molecule found in red wine called resveratrol, which was reported
to activate Sir2 and to increase life span in yeast, worms and flies as well as to deliver
health benefits to diabetic mice. All these studies led to the ever more popular
development of drugs targeting SIRT1 which could be the “magic bullet” for aging.
However, the above mentioned findings in our laboratory first shed light on the other side
of Sir2: it can under certain conditions be pro-aging and block life span extension.
Consistent with this result, it was also found that Sir2 increases the sensitivity of yeast
cells to oxidative stress. These findings suggest that the functions of SIRT1 can be more
complicated than formerly expected. Indeed in this study we found that SIRT1 can play
an “evil” role in neuronal oxidative stress both in vivo and in vitro. But on the other hand,
SIRT1 deletion shortened the life span in mice. At the same time, we also showed that
SIRT1 is required for normal brain functions. These contrasting results appropriately
highlight the complexity of SIRT1 and its vital roles in diverse biological processes and
that SIRT1 is not simply “good” or “bad”. Instead, it exerts its effects depending on the
environment, location, context and developmental stages. According to previous studies
SIRT1 may deter apoptosis in some cancer cell lines through the transcription factor p53
but it may also aggravate cell death in other cells through NF κB. Some reported that
inhibiting SIRT1 may curb cancer while some others found that activating SIRT1 may
ameliorate diabetes. Our current results indicate that inhibition of SIRT1 can protect
neurons against oxidative stress; but another group also reported activating SIRT1 offers
protection against neurodegeneration in an amyotrophic lateral sclerosis model. As more
and more evidence emerges for both “good” and “bad” sides of SIRT1, it is becoming
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clearer that no simple answer fits all cases: activating SIRT1 may be beneficial in some
cells under some conditions while inhibiting SIRT1 may be advantageous in other cells
under other conditions. Overlooking the multiple effects of SIRT1 would be to mislead
the consumers. The general public may not have the resources or time to sort through the
intricacy of research findings in order to reach a sound decision and public policies have
limitations preventing the fully safeguarding of consumers’ welfare in a timely manner. It
is therefore the responsibility of us researchers to spread the understanding, spur interest
and invite discussions for the sake of advancing closer to the truth. Capitalizing on the
knowledge pharmaceutical companies may need to rely on targeted activation or
inhibition of SIRT1 or even other sirtuins when designing therapeutically useful drugs for
certain diseases. The work in this dissertation and other studies call for an ever more
cautious attitude by the companies and media and elevated consumer vigilance so that
they may stay safe and well.
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Abstract (if available)

Abstract Aging is a common phenomenon of multiple organisms. In humans aging is frequently accompanied by cognitive decline and occurrence of neurodegenerative diseases which reduce the quality of life and impose financial stress on society. Delaying the aging process, extending life span and decreasing the occurrence of age-related brain function deficit have always been aspirations of human kind. Extensive research has advanced our understanding of the mechanisms underlying aging, among which is the ability of calorie restriction to increase longevity, and the pivotal regulatory roles of insulin/IGF-1 signaling pathway. Some recent studies identified silent information regulator 2 (Sir2

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Asset Metadata

Creator Li, Ying (author)

Core Title Roles of SIRT1 in neuronal oxidative damage and brain function

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 10/30/2008

Defense Date 09/12/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag brain,learning and memory,neurons,OAI-PMH Harvest,oxidative damage,SIRT1

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Longo, Valter D. (committee chair), Baudry, Michel (committee member), Madigan, Stephen A. (committee member), Pike, Christian J. (committee member)

Creator Email lying@usc.edu,yingraceli@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1723

Unique identifier UC1424895

Identifier etd-LI-2405 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-118411 (legacy record id),usctheses-m1723 (legacy record id)

Legacy Identifier etd-LI-2405.pdf

Dmrecord 118411

Document Type Dissertation

Rights Li, Ying

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu