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Testosterone Deficiency Accelerates Neuronal and
Vascular Aging of SAMP8 Mice: Protective Role of eNOS
and SIRT1
Hidetaka Ota
1
, Masahiro Akishita
1
*, Takuyu Akiyoshi
1
, Tomoaki Kahyo
2
, Mitsutoshi Setou
2
, Sumito
Ogawa
1
, Katsuya Iijima
1
, Masato Eto
1
, Yasuyoshi Ouchi
1
1Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan, 2Hamamatsu University School of Medicine, Department
of Molecular Anatomy, Hamamatsu, Shizuoka, Japan
Abstract
Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small
clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the
pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-
accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by
testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with
testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8.
Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD
+
-dependent
deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced
endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited
endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial
cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in
the prevention of vascular and neuronal aging.
Citation: Ota H, Akishita M, Akiyoshi T, Kahyo T, Setou M, et al. (2012) Testosterone Deficiency Accelerates Neuronal and Vascular Aging of SAMP8 Mice:
Protective Role of eNOS and SIRT1. PLoS ONE 7(1): e29598. doi:10.1371/journal.pone.0029598
Editor: Gian Paolo Fadini, University of Padova, Medical School, Italy
Received August 10, 2011; Accepted December 1, 2011; Published January 4, 2012
Copyright: ß2012 Ota et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (20249041,
21390220, 21790621). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: akishita-tky@umin.ac.jp
Introduction
Advancing age is the most significant risk factor for the
development of cognitive impairment [1,2]; however, what age-
related changes underlie this effect remains uncertain. With
advancing age, men experience a significant decrease in the
circulating level of testosterone. Although studies have shown
alterations in mood, libido, and cognition resulting from
testosterone deficiency [3], the full range of consequences of age-
related testosterone loss remains incompletely defined. In a small
clinical study of men recently diagnosed with cognitive impair-
ment, testosterone treatment improved performance on cognitive
tests [4]. In a prospective longitudinal study using subjects from
the Baltimore Longitudinal Study on Aging, men who developed
Alzheimer disease (AD) were observed to exhibit low testosterone
levels 5–10 years prior to the clinical diagnosis of AD [5]. With a
relationship between age-related testosterone decline in men and
increased risk for cognitive impairment reasonably well estab-
lished, a critical issue is how testosterone contributes to the
pathogenesis of cognitive decline with aging. The most likely
hypothesis is through the regulation of accumulation of amyloid ß
(Aß) peptides, which are widely believed to be the critical initiating
step in the pathogenesis of AD. However, it is becoming
increasingly clear that not all aspects of cognitive decline can be
explained by Aß [6,7]. Findings from such diverse lines of
investigations as neuroimaging and clinical trials suggest that non-
Aß factors also contribute to memory deficit in aged men.
In S. cerevisiae, the Sir2 (silent information regulator-2) family of
genes governs budding exhaustion and replicative life span [8,9].
Sir2 has been identified as an NAD
+
-dependent histone deacety-
lase and is responsible for maintenance of chromatin silencing and
genome stability. Mammalian sirtuin 1 (Sirt1), the closest homolog
of Sir2, regulates the cell cycle, senescence, apoptosis and
metabolism, by interacting with a number of molecules such as
p53. As recently reported, overexpression of SIRT1 in the brain
improved the memory deficit in a mouse model of AD via
activation of the transcription of a-secretase [10].
An increasing body of evidence suggests the presence of a link
between cognitive decline and vascular dysfunction, especially
atherosclerosis [11]. Senescence of endothelial cells is involved in
endothelial dysfunction and atherogenesis, and SIRT1 has been
recognized as a key regulator of vascular endothelial homeostasis,
controlling angiogenesis, endothelial senescence, and dysfunction
[12–14].
In the present study, we demonstrated that cognitive impair-
ment in senescence-accelerated mouse prone 8 (SAMP8), a model
of cognitive decline with aging, is associated with endothelial
senescence in the hippocampus and is ameliorated by testosterone
PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29598
replacement. SIRT1 plays an important role in prevention of
endothelial senescence induced by oxidative stress [13]. We
suggest that the protection against endothelial senescence in the
hippocampus through up-regulation of testosterone and SIRT1
could contribute to a novel therapeutic strategy against cognitive
decline with aging.
Results
Treatment with dihydrotestosterone ameliorated
cognitive function of SAMP8
In order to assess the effects of testosterone on cognitive function,
we used an in vivo model of aging, SAMP8, and a control
counterpart strain, SAMR1. SAMP8 was originally derived from
AKR/J strain, litters of which show the characteristic of cognitive
decline with aging. These mice exhibit age-related deficits in
learning and memory at an early age, and are considered a suitable
animal model to study aging and memory deficit. Body weight,
appearance, and plasma testosterone level of SAMR1 and SAMP8
at 12 weeks of age were determined. Body weight and appearance
did not differ between SAMR1 and SAMP8, but plasma
testosterone level in SAMP8 was lower than that in SAMR1
(Figure 1A). By determining the time required to find the platform
(escape latency) as a function of days of training in the Morris water
maze, we observed a marked decline in performance in SAMP8
compared with SAMR1 (Figure 1B). Because testosterone acts in
part through aromatase-dependent conversion to estradiol, non-
aromatizable dihydrotestosterone (DHT) was used to examine a
direct role of androgens through androgen receptor (AR). SAMP8
treated with DHT showed significantly reduced escape latency time
compared with untreated SAMP8. There was no difference in swim
speed between the groups; however, % time in the quadrant was
increased in DHT-treated SAMP8 (Figure 1B). These results
indicate that DHT treatment ameliorated cognitive dysfunction in
SAMP8. The water-maze is appropriate for hippocampal-depen-
dent paradigms. However, DHT administration may affect
behavior and how animals respond to different stimuli. Therefore,
we performed an open field test to examine locomotion, exploratory
behavior, and anxiety. No significant effect of DHT on locomotor
performance was observed in SAMR1 and SAMP8, whereas
SAMR1 moved significantly more compared with SAMP8
(Figure 1C). The ratio of the distance travelled in the central area
to that in the total area in the open- field, an indirect measure of
exploratory behavior and anxiety [15], was also observed. In
SAMP8, DHT increased this ratio (Figure 1C), suggesting that
DHT promoted exploratory behavior and diminished anxiety.
Figure 1. Testosterone deficiency causes senescence of hippocampus and cognitive impairment in SAMP8 mice. A. Body weight,
appearance, and plasma testosterone level of male SAMR1 and SAMP8 mice at 12 weeks of age. B. Escape latency of SAMR1 (N =10) and SAMP8 mice
(N = 10). Male mice were treated daily for 2 weeks with DHT (500 mg s.c) before trials. Swim speed during quadrant test on day 10. C. Total distance
and the ratio of central/total distance were measured in open field tests. D. Number of amyloid ß plaques, pyramidal cells, and SA-ßgal-positive cells
in CA1 and CA3 areas of hippocampus in SAMR1 and SAMP8. (*p,0.05, n.s: not significant).
doi:10.1371/journal.pone.0029598.g001
Testosterone Inhibits Hippocampal Senescence
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Next, we assessed the number of amyloid ß plaques, pyramidal
cells, and SA-ßgal-positive cells in CA1 and CA3 areas of the
hippocampus in these mice (Figure 1D). The number of plaques
was increased in SAMP8 compared with SAMR1, but was
unaltered by treatment with DHT. The number of SA-bgal-
stained cells was significantly increased in SAMP8 compared with
SAMR1, but treatment with DHT prevented this in SAMP8
despite no difference in pyramidal cell number (Figure 1D).
DHT treatment increased protein and mRNA expression
of SIRT1 in SAMP8
Furthermore, to estimate the role of testosterone deficiency in
SAMP8, we examined the effect of testosterone supplementation
on cognitive function in much older SAMR1 and SAMP8.
Similarly to young mice, we observed a marked decline in
performance in SAMP8 compared with SAMR1 at 18 months of
age. SAMP8 implanted with testosterone pellets showed signifi-
cantly reduced escape latency time compared with placebo-treated
SAMP8 (Figure 2A). Plasma testosterone level in SAMP8 at 18
months of age was lower than that in SAMR1, but implanted mice
showed recovery to the level in young mice (Figure 2A). These
results indicated that similar to DHT, testosterone also showed the
improvement of cognitive function in SAMP8. Next, we examined
the cause of low plasma testosterone in SAMP8. SAMP8 showed
no testicular atrophy (Figure S1A), but more senescent phenotypes
in Leydig cells, which produce testosterone in testes, than SAMR1
(Figure 2B). Moreover, we tried to allotransplant testes from
SAMR1 to SAMP8 (Figure S1B). Although performance gradually
responded to treatment up to 8–10 weeks, castrated SAMR1
showed a marked decline in performance whereas recipient
SAMP8 showed cognitive improvement (Figure 2C).
As recently reported, overexpression or activation of SIRT1
inhibits cellular senescence and protects cellular function in
various cell lines [13,16]. Therefore, we examined SIRT1
expression in the hippocampus of SAMP8 with or without DHT
treatment, at 12 weeks of age. DHT treatment increased the
protein and mRNA expression of SIRT1 in SAMP8 (Figure 2D).
To investigate further the involvement of AR, we examined the
expression of AR in SAMR1 and SAMP8 brains. The expression
of AR was more abundant in the hippocampus than in other brain
regions of SAMR1 and SAMP8 (Figure 2E).
Figure 2. Supplementation of testosterone improves cognitive function in SAMP8 mice. A. Escape latency and plasma testosterone level
of male SAMR1 (N = 10) and SAMP8 mice (N = 10) at 18 months of age. These mice were implanted subcutaneously with a placebo or a 21-day-release
2.5 mg testosterone pellet in the dorsal neck. B. Number of SA-bgal-stained Leydig cells in testes in SAMR1 and SAMP8. Arrows indicate Leydig cells.
Representative SA-bgal-stained testes from SAMR1 and SAMP8. C. Escape latency of castrated SAMR1 (upper, N = 5) and recipient SAMP8 (lower,
N = 5). Observation (0–10 weeks) was started from 3 weeks after operation. D. SIRT1 expression in hippocampus of SAMP8 with or without DHT
treatment. Immunofluorescent staining for SIRT1 (green) and DAPI (blue). E. Expression of AR in SAMR1 and SAMP8 brains. (*p,0.05).
doi:10.1371/journal.pone.0029598.g002
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Oxidative stress was increased in hippocampal cells of
SAMP8
Oxidative stress may be closely related to senescence and age-
related diseases. Also, an increase in oxidative stress has been
suggested to be one of the earliest pathological changes in the
brain in conditions with cognitive impairment such as AD [17].
Then, we examined the level of oxidative stress, using the SAMR1
and SAMP8 hippocampus at 12 weeks of age. SAMP8
hippocampus showed an increase in the level of oxidative stress
compared with SAMR1 as judged by detection of carbonylated
proteins. DHT treatment decreased carbonylated proteins in the
SAMP8 hippocampus (Figure 3A). In parallel, the concentration of
the neurotransmitter acetylcholine in hippocampal lysates was
decreased in SAMP8 compared with that in SAMR1, and DHT
treatment prevented this (Figure 3B).
Testosterone and DHT acts on vascular endothelial cells and
stimulates the PI3K/Akt pathway, leading to eNOS activation
through direct interaction of AR [18,19]. The eNOS/SIRT1 axis
is recognized as one of the fundamental determinants of
endothelial senescence, and SIRT1 acts as a driver of cellular
stress resistance [20]. To examine the influence of DHT treatment
on endothelial cells, we determined the degree of senescence and
the expression of SIRT1 in endothelial cells around the CA3 area
of the hippocampus. DHT-treated SAMP8 showed a reduction of
SA-bgal-stained endothelial cells and increased SIRT1 expression
compared to untreated SAMP8 (Figure 3C and D). To confirm
that these cells were endothelial cells, not neuronal cells, cerebral
microvessels were isolated from SAMR1 and SAMP8. In parallel
with immunohistological staining, SAMP8 showed a reduction of
SIRT1 expression compared to SAMR1, and DTH treatment
increased SIRT1 expression compared to that in untreated
SAMP8 (Figure 3E). These results suggest that vascular endothelial
senescence in the hippocampus may be related to the memory
deficit in SAMP8. Since testosterone and DHT activates eNOS, a
NOS inhibitor, N
G
-nitro-L-arginine methyl ester hydrochloride (L-
NAME), and N
5
-(1-lmino-3-butenyl)-L-ornithine (L-VNIO), a
Figure 3. Senescent endothelial cells of hippocampus are decreased by treatment with DHT. A. Oxidative stress level was measured by
detection of carbonyl groups introduced into proteins. B. Acetyl-choline concentration was measured by a colorimetric method. C. SA-bgal-stained
endothelial cells and SIRT1 expression in CA3 area of hippocampus in SAMR1 and SAMP8 with or without DHT treatment. Immunofluorescent
staining for SIRT1 (green), PECAM-1 (red), and DAPI (blue). D. Number of SA-bgal-stained endothelial cells in CA3 area of hippocampus in SAMR1 and
SAMP8 with or without DHT treatment. E. Expression of SIRT1, PECAM-1, and b-actin was analyzed using cerebral micro vascular cells. F. Escape
latency of SAMR1 (N = 10) and SAMP8 mice (N = 10). Male mice were treated daily for 2 weeks with DHT (500 mg s.c) and L-NAME (20 mg/kg gavage)
before trials. G. Escape latency of SAMR1 (N = 5) and SAMP8 mice (N = 5). Male mice were treated daily for 2 weeks with DHT (500 mg s.c) and L-VNIO
(5 mg/kg IP) before trials. (*p,0.05, n.s: not significant).
doi:10.1371/journal.pone.0029598.g003
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selective neuronal NOS (nNOS) inhibitor, were applied to
examine the involvement of NOS in this process. L-NAME
abrogated the effects of DHT on cognitive function (Figure 3F). In
contrast, L-VNIO did not change the effect of DHT (Figure 3G).
These results suggest that eNOS/SIRT1 in endothelial cells may
play an important role in the protective effect of testosterone
against senescence of the hippocampus.
SIRT1 plays an important role in the protective effect of
testosterone against endothelial senescence
Following the animal experiments, we examined whether
testosterone inhibited endothelial senescence in vitro using cultured
cells. We induced premature endothelial senescence by addition of
H
2
O
2
100 mmol/L for 1 hour. DHT or testosterone treatment
inhibited SA-bgal activity and the morphological appearance of
senescence (Figure 4A). We observed that oxidative stress
decreased eNOS and SIRT1 and increased PAI-1 expression,
and DHT or testosterone treatment prevented these changes and
increased the phosphorylation of eNOS at Ser1177 (Figure 4B).
Overexpression of SIRT1 significantly inhibited oxidative stress-
induced senescence, and DHT accelerated the effect of SIRT1
through phosphorylation of eNOS at Ser1177 (Figure 4C). To
determine the role of endogenous SIRT1, DHT-treated endothe-
lial cells were transfected with SIRT1 siRNA or treated with
sirtinol, a chemical inhibitor of SIRT1. SIRT1 siRNA or sirtinol
abrogated the effect of DHT on SA-bgal activity (Figure 4D). We
previously reported that testosterone activated eNOS [18], and
eNOS activation promoted SIRT1 expression [21]. Accordingly,
we examined the role of eNOS in the protective effect of
testosterone. We observed that DHT or testosterone treatment
increased NOS activity that was reduced by oxidative stress
(Figure 4E). Treatment with eNOS siRNA or L-NAME decreased
the inhibitory effect of DHT on a senescent phenotype in parallel
with SIRT1 expression (Figure 4F and G). These results indicate
that eNOS/SIRT1 play an important role in the protective effect
of testosterone and DHT against a senescent phenotype.
Figure 4. Testosterone inhibits oxidative stress-induced endothelial senescence through eNOS/SIRT1. A. Testosterone inhibited SA-
bgal activity and senescent morphological appearance induced by hydrogen peroxide (100 mmol/L). B. Expression of eNOS, SIRT1, and PAI-1 in
hydrogen peroxide (100 mmol/L)-treated HUVEC under treatment with DHT or testosterone. C. Overexpression of SIRT1 and DHT reduced SA-bgal
activity. eNOS expression was increased by overexpression of SIRT1, and DHT increased phosphorylation of eNOS (Ser1177). D. SIRT1 inhibition by
siRNA or sirtinol (100 mmol/L) abrogated the effect of testosterone on SA-bgal activity. E. Treatment with testosterone or DHT increased eNOS
activity. F. eNOS inhibition by siRNA or L-NAME (10 mM) abrogated the effect of testosterone on SA-bgal activity. G. Treatment with L-NAME
decreased SIRT1 expression in DHT-treated HUVEC. (*p,0.05, N = 3).
doi:10.1371/journal.pone.0029598.g004
Testosterone Inhibits Hippocampal Senescence
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Senescent endothelial cells induced by oxidative stress
promoted neuronal senescence
Finally, we hypothesized that endothelial senescence promotes
senescence of adjacent neuronal cells. To test this hypothesis, we
used a co-culture system of endothelial cells (HUVEC) with
neuronal cells (mouse hippocampal neuronal cells; MHC)
(Figure 5A). Both cells were co-cultured, but were separated by
a microporous polycarbonate membrane, for 10 days after
endothelial cells were treated with hydrogen peroxide, and the
senescent phenotype of MHC was analyzed. We found that the
number of SA-bgal-positive cells and the senescent appearance of
MHC were increased, and the concentration of acetylcholine in
cells was decreased by co-culture with senescent endothelial cells
(Figure 5B). In parallel with this, MHC showed increased PAI-1
and p53, and decreased SIRT1 expression (Figure 5C). We also
found that senescent endothelial cells showed increased expression
of inflammatory cytokines such as IL-6, IL-8, MCP-1, and TNF-a
(Figure 5D). Both MHC and HUVEC, or HUVEC alone were
treated with testosterone at 3 days before HUVEC were treated
with hydrogen peroxide, and both cells were co-cultured for 10
days, and the senescent phenotype of MHC was analyzed. We
found that the number of SA-bgal-positive MHC was decreased
by treatment of HUVEC with testosterone irrespective of the
treatment of MHC with testosterone (Figure 5E). In addition, we
found that a SIRT1 activator, resveratrol treatment rescued the
senescent phenotype of MHC (Figure 5F). These results suggest
that senescent endothelial cells exhibit a senescence-associated
secretory phenotype [22], induce neuronal senescence, and
testosterone rescues it through up-regulation of SIRT1 (Figure 5G).
Discussion
Testosterone level and cognitive function show a decline with
age in men. A series of evidence suggests that this association is not
just age related [23]. Results from cell culture and animal studies
provide evidence that testosterone could have protective effects on
brain function, especially in the hippocampus [24]. Here, we
demonstrated that administration of testosterone restored cogni-
tive function in male SAMP8 in association with improvement of
the senescent phenotype in the hippocampus and cerebral vessels.
Figure 5. Oxidative stressed-induced endothelial cell senescence promotes adjacent neuronal cell senescence. A. Co-culture cell
culture dish. B. Number of SA-bgal-stained MHC and senescent appearance of MHC were increased, and acetyl-choline concentration was decreased
by co-culture with senescent endothelial cells. Senescent MHC are indicated by arrows. C. Expression of SIRT1, PAI-1, p53, and b-actin in MHC co-
cultured with senescent endothelial cells. D. Expression of IL-6, IL-8, MCP-1, and TNF-ain endothelial cells were analyzed by RT-PCR. E. The number of
SA-bgal-stained MHC was decreased by treatment with testosterone in both MHC and HUVEC (MHC, testosterone (+)), or HUVEC (MHC, testosterone
(2)) alone. F. Resveratrol decreased the number of SA-bgal-stained MHC co-cultured with senescent endothelial cells. (*p,0.05, N = 3). G.
Hypothetical signal transduction pathways of testosterone in endothelial cells.
doi:10.1371/journal.pone.0029598.g005
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We also showed that testosterone ameliorated endothelial
senescence through eNOS/SIRT1-dependent mechanisms in vitro.
The present study demonstrated that testosterone and SIRT1
interacts with each other and inhibited the senescence of
hippocampal vascular and neuronal cells, suggesting that testos-
terone replacement therapy is a treatment option for cognitive
decline with aging.
Testosterone may act in part through aromatase-dependent
conversion to estradiol. To estimate a direct effect of androgens
through AR, testosterone and DHT were used in this study. Both
compounds showed significant protective effects on cognitive
function.
In the present study, we used SAMP8 mice. SAMP is comprised
of 14 strains derived from selective inbreeding of the AKR/J
strain. SAMP8 exhibits age-related learning and memory deficits,
as well as amyloid-like deposits in the brain [25]. Increased
expression of hyperphosphorylated tau has also been detected in
SAMP8 [26]. Given such features, SAMP8 has been proposed as a
plausible age-associated AD animal model, and a suitable rodent
model for studying the molecular mechanism underlying cognitive
impairment [27]. A previous study has shown an age-related
decrease in serum testosterone in SAMP8, and suggesting that
impaired cognitive function in SAMP8 is due to reduced
testosterone [28]. We observed that AR expression was abundant
in the hippocampus of SAMR1 and SAMP8. Several studies have
demonstrated that testosterone has a neuroprotective effect
through AR in the hippocampus [29,30], and testosterone induced
NO productions via AR-dependent activation of eNOS in
endothelial cells [18,19].
Accumulating evidence suggests that NAD
+
-dependent deace-
tylase SIRT1 play an essential role for cellular senescence and
cognitive function. SIRT1 modulates endothelial cellular senes-
cence [13], and overexpression of SIRT1 exhibits neuroprotective
effects in hippocampus, and cognitive function of Sirt1-KO mice is
markedly impaired [10,31,32].
The precise etiologic mechanism of the cognitive decline with
aging is unclear, but it has been identified that cardiovascular risk
factors are associated with a higher incidence of cognitive
impairment [33]. In addition, age-associated vascular inflamma-
tion is an early manifestation of chronic stress responses, i.e.
overloading of ROS on endothelial cells [34]. Indeed, SAMP8
showed enhancement of oxidative stress and a senescent
phenotype in the hippocampus. Notably, senescent endothelial
cells were increased in the hippocampus of SAMP8 accompanied
by a reduction of SIRT1, and L-NAME abrogated the effect of
DHT on cognitive function. Therefore, we hypothesized that
testosterone influenced cerebral endothelial senescence via eNOS/
SIRT1, and that pro-inflammatory cytokines, which were derived
from senescent endothelial cells, promoted senescence in adjacent
neuronal cells. Indeed, we observed that testosterone induced
eNOS activity, and subsequently increased SIRT1 expression in
endothelial cells. Inhibition of eNOS/SIRT1 abrogated the effect
of testosterone on endothelial senescence. In a co-culture system,
we found that senescent endothelial cells promoted senescence of
adjacent neuronal cells, and treatment of endothelial cells with
testosterone inhibited senescence of adjacent neuronal cells. It can
reasonably be speculated, therefore, that SIRT1 may exert
salutary actions against cognitive decline with aging by preventing
a senescence-associated secretory phenotype of endothelial cells.
Because L-NAME is a non-selective inhibitor of NOS, it is possible
that the effect of L-NAME might be in part a result of inhibition of
nNOS in concert with eNOS. However, a specific nNOS
inhibitor, L-VNIO did not change the effect of DHT in SAMP8.
In co-culture experiments, we found that treatment with
resveratrol or testosterone did not change the expression or
activation of nNOS in MHC (Figure S1C and D). Further studies
are needed to address the differential role of eNOS and nNOS,
and the exact role of SIRT1 in vivo.
In conclusion, supplementation of testosterone prevented
cognitive impairment of SAMP8, in which testosterone secretion
was decreased in association with the senescence of testis Leydig
cells, through an eNOS/SIRT1-dependent mechanism. Unprec-
edented reversal of the senescent hippocampal changes and
vascular protection may justify exploration of a neuronal
rejuvenation strategy by utilizing testosterone for the prevention
of cognitive decline with aging, particularly through up-regulation
of eNOS/SIRT1.
Methods
Materials
Dihydrotestosterone (DHT), testosterone, and N
G
-nitro-L-argi-
nine methyl ester hydrochloride (L-NAME) were purchased from
Sigma (St. Louis, MO). Hydrogen peroxide (H
2
O
2
) and resveratrol
were purchased from Wako Pure Chemical Industries (Osaka,
Japan). Testosterone and placebo pellets were purchased from
Innovative Research of America (Sarasota, FL). N
5
-(1-lmino-3-
butenyl)-L-ornithine (L-VNIO) was purchased from Enzo Life
Sciences (Plymouth Meeting, PA).
Cell culture
Human umbilical vein endothelial cells (HUVEC) were
purchased from CAMBREX (Walkersville, MD). Population
doubling levels (PDL) were calculated as described previously
[35], and all experiments were performed at PDL of 10–11. In our
preliminary experiments, HUVEC were cultured in EBM without
phenol red (Clonetics, Walkersville,MD) with 10% dextran-
charcoal-stripped serum to remove steroids from the culture
medium. This condition, however, induced marked growth arrest
and an increase in senescent cells. Consequently, we performed all
experiments in EBM-2 (Clonetics) with 10% complete serum-
supplemented medium.
Animal experiments
The animal experiments were approved by our institutional
review board (animal experiments ethics board, Graduate School
of Medicine and Faculty of medicine, The university of Tokyo
(approval ID: M-P-09-056)). Senescence-accelerated mice prone
(SAMP) 8 and control senescence-accelerated mice resistant
(SAMR) 1 male mice were all housed and maintained in a room
at 2262uC with automatic light cycles (12 h light/dark) and
relative humidity of 40–60%. Mice were purchased from Japan
SLC, Inc. (Shizuoka, Japan). Food and tap water were provided
ad libitum throughout the study. In the water maze test of this
study, a grou p of male SAMR1 (N = 10) and SAMP8 (N = 10)
was first tested. Male mice of 12 weeks of age were treated daily
for 2 weeks with DHT (500 mg in 0.05 ml/mouse) by subcuta-
neous injection (s.c.) in the neck before the water maze test. Male
mice of 18 months of age underwent subcutaneously implantation
of a placebo (N = 5) or a 21-day-release 2.5 mg testosterone
(N = 5) pellet into the dorsal neck region. L-NAME was given by
gavage once a day (20 mg/kg) [36]. L-VNIO was given by
intraperitoneal injection (0.5 mg/kg) [37]. Small fragments of
testis tissue fragments from SAMR1 were grafted under the back
skin of castrated male SAMP8 as previously described [38].
Briefly, after removal of the capsule and obvious connective
tissue, donor testes were cut into small fragments. Testis
fragments were kept in Dulbecco’s modified Eagle’s medium
Testosterone Inhibits Hippocampal Senescence
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(Gibco Lab Inc., Grand Island, NY, USA) on ice until grafting.
SAMR1 were anesthetized and castrated, and testicular tissue
fragments were grafted under the back skin of SAMP8. Mice
were anesthetized with enflurane, killed by cervical dislocation,
and trunk blood collected within 1 min. The blood was
centrifuged and plasma testosterone was measured by radioim-
munoassay method. The brain was removed for histological
examination, after systemic perfusion with phosphate-buffered
saline (PBS). For immunohistochemical studies, mouse brains
were processed and labeled with anti-amyloid-bantibody
(Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) to
visualize extracellular amyloid plaques, anti-NeuN antibody
(Millipore, Billerica, MA) to assess pyramidal cell number, or
DAPI (Dojindo Molecular Technologies, Inc., Tokyo, Japan) for
nuclear staining. The primary antibody was purified rat anti-
mouse CD31 (platelet endothelial cell adhesion molecule;
PECAM-1) monoclonal antibody from Pharmingen (San Jose,
CA, USA). Secondary antibodies (Alexa Fluor 488 donkey anti-
rat IgG and Alexa Fluor 594 donkey anti-rat IgG) and antifade
reagent were from Molecular Probes (Invitrogen). Fluorescent
images were analyzed using a fluorescence microscope (BZ-9000,
KEYENCE, Osaka, Japan).
Plasmids and siRNA transfection
Proliferating cells were washed three times with growth medium
and exposed to the indicated concentrations of testosterone or
DHT diluted in medium. pIRES-SIRT1 plasmid was provided by
Dr. M. Takata [39], and Dr. R.A. Weinberg [40]. Each plasmid
was overexpressed by transfection using Lipofectamine LTX and
PLUS reagents (Invitrogen) for HUVEC according to the
manufacturer’s instructions. Proliferating cells were transfected
with each siRNA using silMPORTER (Upstate Cell Signaling
Solutions). siRNAs for SIRT1 (GAT GAA GTT GAC CTC CTC
A [41] and TGA AGT GCC TCA GAT ATT A), and eNOS were
purchased from Santa Cruz Biotechnology, Inc.
Immunoblotting and immunoprecipitation
Cells were lysed on ice for 1 hour in buffer (50 mmol/L
Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1% NP-40, 0.1% SDS,
1 mmol/L dithiothreitol, 1 mmol/L sodium vanadate, 1 mmol/
L phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 10 mg/
mL leupeptin and 10 mmol/L sodium fluoride). Equal amounts
of protein were separated by SDS/PAGE gel electrophoresis and
transferred to nitrocellulose membranes. After blocking, the
filters were incubated with the following antibodies; anti-SIRT1,
anti-nNOS, anti-AR (Cell Signaling, Danvers, MA), anti-eNOS
(BD Transduction Laboratories, San Jose, CA), anti-PAI-1
(Molecular Innovations, Southfield, MI), anti-PECAM-1 (Santa-
Cruz Biotechnology, CA), and anti-ß-actin (Sigma). After
washing and incubation with horseradish peroxidase-conjugated
anti-rabbit or anti-mouse IgG (Amersham, Piscataway, NJ) for
1 hour, antigen-antibody complexes were visualized by using an
enhanced chemiluminescence system (Amersham).
Senescence-associated ß-galactosidase (SA-ßgal) staining
HUVEC were pretreated with diluted EGM-2 medium for 3
day. HUVEC were then washed three times with EGM-2 and
treated for 1 hour with 100 mmol/l H
2
O
2
diluted in EGM-2.
After treatment, HUVEC were trypsinized, re-seeded at a
density of 1610
5
in 60-mm dishes, and cultured with EGM-2
containing DHT or testosterone for 10 days. The proportion of
SA-ßgal-positive cells was determined as described by Dimri
et al [42].
NOS activity assay
NOS activity was determined using an NOS assay kit
(Calbiochem) according to the manufacturer’s instructions.
Measurement of acetylcholine
The concentration of acetylcholine was measured with a
choline/acetylcholine quantification kit (BioVision, CA, USA)
according to the manufacturer’s instructions.
Real-time quantitative reverse transcription PCR
Total RNA was isolated with ISOGEN (Nippon Gene Inc.,
Toyama, Japan). After treatment with Rnase-free Dnase for
30 min, total RNA (50 ng/ml) was reverse transcribed with
random hexamers and oligo d(T) primers. The expression levels
of SIRT1, IL-6, IL-8, MCP-1, and TNF-arelative to ß-actin
were determined by means of staining with SYBR green dye and
a LineGene fluorescent quantitative detection system (Bioflux
Co., Tokyo, Japan). The following primers were used: SIRT1 F
59-CCTGACTTCAGGTCAAGGGATGGTA-39,R59-CTGA-
TTAAAAATATCTCCTCGTACAG-39;ß-actinF59-TGGGC-
ATGGGTCAGAAGGAT-39,R59-AAGCATTTGCGGTGGA-
CCAT-39;IL-6F59-GGGAAGGTGAAGGTCGG-39,R59-T-
GGACTCCACGACGTACTCAG-39,IL-8F59-CTGGCCGT-
GGCTCTCTTG-39,R59-CCTTGGCAAAACTGCACCTTT-
39;TNF-aF59-GTAGCCCACGTCGTAGCAAAC-39,R59-
CTGGCACCACTAGTTGGTTGTC-39; MCP-1 F 59-CATT-
GTGGCCAAGGAGATCTG-39,R59-CTTCGGAGTTTGG-
GTTTGCTT-39.
Co-culture system
For these experiments, co-culture dishes were used as outlined
in Figure 5A. They were obtained from BD Biosciences
(Erembodegem, Belgium) with a 6-well format. HUVEC were
treated with H
2
O
2
(100 mM) for 1 h and cultured on the
permeable microporous (0.4 mm) membrane in the insert, and
mouse hippocampus neuronal cells on the base of the culture dish,
kept physically separated but allowing the passage of micromol-
ecules through the porous membrane for 10 days. Mouse
hippocampus neuronal cells were purchased from DS Pharma
Biomedical Inc. (Osaka, Japan).
Quantitative analysis of amyloid b
Measurement of amyloid bwas performed using an amyloid b
(1–40) (FL) assay kit (Immuno-Biological Laboratories Co., Ltd.,
Gunma, Japan) according to the manufacturer’s instructions.
Morris water maze test
The procedure of the Morris water maze test was described
previously [43]. SAMR1 and SAMP8 mice were trained to find a
visible platform with three trials on the first day, and then tested to
find the hidden platform for 10 consecutive days. In each trial, the
mice were allowed to swim until they found the hidden platform,
or until 2 min had passed, and the mouse was then guided to the
platform. On the test days, the platform was hidden 1 cm beneath
the water. The escape latency was recorded by a video camera.
The swim speed of each mouse was calculated by means of a video
tracking system. Probe tests were performed on the 10
th
day.
During percent time quadrant test, the platform was removed
from the pool. Mice were started in a position opposite the
location of the platform position and allowed to swim for
60 seconds.
Testosterone Inhibits Hippocampal Senescence
PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29598
Open field test
The open field test fear response to novel stimuli was used to
assess locomotion, exploratory behavior, and anxiety. Open field
test protocols were modified from that of Lukacs et al [44]. The
open field test consisted of a wooden box (60660660 cm) and was
indirectly illuminated by two fluorescent lights. A 10 cm area near
the surrounding wall was delimitated and considered the
periphery. The rest of the open field was considered the central
area. The distance travelled, the ratio of the distance travelled in
the central area/total distance travelled, and the time in the center
of the open- field were analyzed as a measure of anxiety-like
behavior. During the test, mice were allowed to move freely
around the open field and to explore the environment for 15 min.
Isolation of cerebral microvessels
Cerebral microvessels were isolated from the remaining brain
tissue as previously described by Zhang et al [45] with minor
modifications. Brain tissue, devoid of large vessels, was homoge-
nized in ice cold PBS with Dounce homogenizer and centrifuged
twice at 2000 g at 4uC. The supernatant, containing the
parenchymal tissue, was discarded. The pellet was resuspended
in PBS and centrifuged as described above. The resulting pellet
was resuspended and layered over 15% Dextran (in PBS) (Sigma,
St.Louis, MO) and centrifuged at 4500 g for 30 minutes at 4uC.
The top layer was aspirated and discarded and the remaining
pellet resuspended in 15% Dextran and centrifuged. The final
pellet was resuspended in 1% bovine serum albumin (BSA), the
suspension was then passed though a 40-mm nylon mesh (BD
Falcon). Microvessels retained on the mesh were washed with
BSA/PBS and collected by centrifugation at 900 g for 10 minutes
at 4uC.
Data analysis
Values are shown as mean 6S.E.M in the text and figures.
Differences between the groups were analyzed using one-way
analysis of variance, followed by Bonferroni test. Probability values
less than 0.05 were considered significant.
Supporting Information
Figure S1 Testes of SAMP8 and SAMR1 mice and role of
nNOS in neuronal senescence. A. Testis weight of SAMR1
and SAMP8 with or without testosterone. B. Photographs of
SAMR1 donor and SAMP8 recipient mice. White arrows indicate
operation scar. C. Expression of nNOS in MHC treated with
resveratrol or testosterone under the oxidative stress. D. Activity of
nNOS in MHC treated with resveratrol or testosterone under the
oxidative stress. (
*
p,0.05, N = 3, n.s: not significant).
(TIF)
Acknowledgments
We are grateful to Dr. L. Guarente (Paul F. Glenn Laboratory and
Department of Biology, Massachusetts Institute of Technology, Boston,
USA) for advice and discussion. We thank Dr. M. Takata (Department of
Immunology and Molecular Genetics, Kawasaki Medical School,
Okayama, Japan) and R.A. Weinberg (Professor of Biology, Whitehead
Institute for Biomedical Research, MIT, Boston, USA) for providing the
pIRES-Sirt1 plasmid.
Author Contributions
Conceived and designed the experiments: HO MA YO. Performed the
experiments: HO TA. Analyzed the data: HO SO KI ME MA.
Contributed reagents/materials/analysis tools: TK MS. Wrote the paper:
HO MA.
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