博士論

文

Neuroprotective effects of food-derived compounds against
oxidative stress-induced cell death via mitochondrial enhancement
食品由来成分によるミトコンドリア増強作用を介した神経細胞保護効果

指導教員 佐々木 裕子

教授

甲子園大学大学院栄養学研究科
食品栄養学専攻
学籍番号・氏名

基礎栄養学部門

811851 伊藤章悟

Contents

Page
Abstract

2-3

1. Introduction

4-6

2. Materials and Methods

7-10

3. Results

11-13

4. Discussion

14-17

5. References

18-22

6. Figures

23-28

1

Abstract
Oxidative stress is involved in most neurodegenerative diseases. Although it has
been reported that the NAD+ precursor nicotinamide mononucleotide (NMN)
improves symptoms in some oxidative stress-associated disease models, the detailed
mechanisms have yet to be elucidated. In this study, the author hypothesized that
NMN attenuates oxidative stress-induced cell damage via sirtuin activation andincreased numbers of mitochondria. To investigate whether NMN can protect
neuronal cells from oxidative stress via the enhancement of mitochondrial function,
the author used PC12 cells treated with 6-hydroxydopamine (6-OHDA) or hydrogen
peroxide (H2O2) as oxidative stress models. With regard to cytoprotective effects,
pretreatment with NMN significantly reduced cell death induced by these stressors.
These protective effects were attenuated by the sirtuin inhibitor, sirtinol, and knockdown of the peroxisome proliferator-activated receptor gamma coactivator-1α(PGC1α), which activates mitochondrial biogenesis. To clarify if NMN increased
mitochondria, intracellular mitochondria were quantified using cytometric analysis.
NMN clearly increased the numbers of intracellular mitochondria, and this increase
was attenuated by sirtinol. Moreover, the author quantified the mitochondriaassociated proteins Sirt3 and superoxide dismutase 2 (SOD2) and found thattreatment with NMN increased these proteins. The author concludes that NMN
protects neuronal cells from oxidative stress via sirtuin activation followed by2

mitochondrial enhancement.

Keywords: NMN; oxidative stress; mitochondria; sirtuin; PGC1

3

1. Introduction

Nicotinamide mononucleotide (NMN) is known to be a precursor of nicotinamide
adenine dinucleotide (NAD+), and to activate sirtuins. NMN is biosynthesized in vivo,
but also occurs in foods such as broccoli, cabbage and avocado [1]. Administration of
NMN to cells activates intracellular sirtuins [2]. Sirtuins belong to the class III
histone deacetylase (Hdac) family, and deacetylate not only histones but also someother intracellular proteins, as well as regulating their activities [3,4,5,6]. Thus,
NMN affects intracellular signaling via sirtuin activation. Sirtuin activation has
been reported to show anti-neurodegenerative functions in both in vivo [7] and invitro [8] models. Therefore, the author hypothesized that NMN has a neuroprotectivefunction via sirtuin activation.
In most neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s
disease, oxidative stress is involved in neuronal cell death. Many neurodegenerative
diseases linked to oxidative stress have a long incubation period, in some cases
lasting more than 20 years. However, diagnosis before onset is rarely achieved.
Therefore, prevention many years prior to onset is important to reduce the risk ofthese diseases. One of the candidates for prevention is the ingestion of protective
ingredients from food, with dietary components such as polyphenols having beenreported to show neuroprotective effects against oxidative stress [9]. These
4

ingredients are considered to protect neuronal cells mainly via the reduction ofreactive oxygen species (ROS). A few polyphenols, such as resveratrol and quercetin,
have been reported to protect neuronal cells against oxidative stress [10] and are
known to activate sirtuin [11]. This suggests that sirtuin activation could contributeto protection against oxidative stress. However, no neuroprotective function against
oxidative stress by NMN has yet been reported. In this study, the author usedhydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA) as oxidative stressmodels. 6-OHDA is also used as a stressor in Parkinson’s disease models [12,13].
Mitochondria are where oxidative metabolism takes place. Mitochondria
themselves produce ROS as by-product of their metabolic activity, and excess
intracellular ROS are known to damage mitochondria. It was reported thatmitochondrial numbers were reduced in neurodegenerative-diseased brain tissue[14]. Studies using a neuronal cell culture model have shown that oxidative stressinduces mitochondrial damage [15]. However, mitochondria contain the antioxidant
superoxide dismutase 2 (SOD2), which works to reduce ROS. This suggest that if
there are sufficient mitochondria for eliminating ROS then this can reduce the riskof neurodegeneration. Therefore, natural products which increase mitochondria and
SOD2 could attenuate oxidative stress and reduce the neurodegeneration.
Seven sirtuins (SIRT1–7) have been identified in mammals. Sirtuins belong to theclass III Hdac family, which function to deacetylate intracellular proteins and5regulate their activity. PGC1 is known to be a crucial factor for mitochondrial
biogenesis, and its activity is regulated by the acetylation of lysine residues [16]. The
cytosolic sirtuin Sirt1 deacetylates PGC1 and stimulates mitochondrial biogenesis
activity [6]. This suggests that Sirt1 activation by NMN induces mitochondrial
biogenesis via PGC1 deacetylation. Another sirtuin, Sirt3, is localized within
mitochondria and regulates the activities of mitochondrial proteins by deacetylation.
The mitochondrial antioxidative protein SOD2 has been proposed as one of thetargets of Sirt3 [17]. Deacetylation of SOD2 by Sirt3 was reported to stimulate
antioxidant activity [17], which suggests that Sirt3 activation by NMN increases
mitochondrial antioxidant activity via the activation of SOD2.
In this study, the author focused on NMN as an ingredient in food for preventing
neurodegenerative diseases caused by oxidative stress, and explored the mechanism
for neuroprotection via sirtuin activation.

6

2. Materials and Methods

2.1. Cell Culture

PC12 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St
Louis, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS),
10 U/ml penicillin, and 100 g/ml streptomycin in a 5% CO2 humidified atmosphereat 37ºC. During treatment the cells were cultured in medium without FBS.

2.2. Cell treatment

PC12 cells were seeded in 96-well plates at a density of 3,000 cells/well. After 24 hculture, the cells were treated with 1 mM NMN (-Nicotinamide mononucleotide)(Oriental Yeast, Tokyo, Japan) with or without 20 M sirtinol(Wako, Osaka,

Japan), a sirtuin inhibitor, for 2 h at 37ºC. Then, an oxidative stressor was added,
either 6-OHDA (SIGMA) (final concentration 100 μM) or H2O2 (Wako, Osaka, Japan)(final concentration 50 μM). After incubation for 24 h at 37ºC, treated cells were
subjected to cell viability assays.

2.3. Cell Viability Assay
7

Cell viability was determined using the MTT assay and live/dead cell-staining withcalcein AM and propidium iodide (PI, Dojindo, Kumamoto, Japan). Regarding the
MTT assay, treated cell-medium was replaced with serum-free medium containing
0.25 mg/ml 3-(4,5-dimethyl-2-thyazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT,
Dojindo, Kumamoto, Japan), and incubated at 37ºC for 2 h, and then terminated byadding the same volume of 15% (w/v) sodium dodecyl sulfate (SDS) and 50% (v/v)
dimethylformamide in water. After termination, absorbance was measured at 570
nm using a microplate reader (Molecular Devices, San Jose, USA). Data are shownas means ± SD. Differences were analyzed using one-way ANOVA followed by aTukey–Kramer post hoc test, with p-values <0.05 considered to be significant.
For live/dead cell staining, treated cells were stained with 1 g/ml calcein AM and1 g/ml PI in medium without FBS at 37ºC for 20 min. After staining, cells wereobserved under a fluorescent microscope (Bio-Rad, Hercules, USA).

2.4. siRNA transfection

PC12 cells were transfected with 3 pmol PGC-1α siRNA (SC-72151, Santa Cruz
Biotechnology, Dallas, USA) using 10 µl/ml Lipofectamine RNAiMAX reagent(Thermo Fisher Scientific K. K., Tokyo, Japan). After culture for 24 h, the cells were8

subjected to treatment.

2.5. Mitochondrial analysis

PC12 cells were seeded in 6-well plates at a density of 100,000 cells/well. After
culture for 24 h, the cells were subjected to treatment. Treated cells were harvested
andstained

with

2

M

5,5’,6,6’-Tetrachloro-1,1’,3,3’-Tetraethyl-

benzimidazolocarbocyanine Iodide (JC-1) (SIGMA) at 37ºC for 15 min. Afterincubation, cells were collected and resuspended in serum-free medium andanalyzed using a Tali image-based cytometer (Life Technologies Japan Ltd., TokyoJapan).

2.6. Western blot analysis

Treated cells were collected and lysed in 1% (v/v) Triton X-100 (MP Biomedicals,
Santa Ana, USA) and 1% (v/v) protease inhibitor cocktail (Nacalai Tesque, Kyoto,
Japan) in PBS. The protein concentration was then measured using a BCA Protein
Assay Reagent Kit (Thermo Fisher Scientific K. K., Tokyo, Japan). Samples were
subjected to 10% or 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gelelectrophoresis. After separation, the proteins were transferred electrophoretically
9onto polyvinylidene fluoride (PVDF) blotting membrane. The membrane was blockedwith 5% defatted milk in Tris-buffer containing 0.05% Tween 20 for 30 min at room
temperature. Samples were then incubated at 4ºC overnight with anti-SIRT1 (H300) (1:500 rabbit polyclonal sc-15404, Santa Cruz Biotechnology, Dallas, USA), antiSIRT3 (1:500

rabbit polyclonalab75434, Abcam,

Cambridge,UK), anti-

SOD2/MnSOD (1:1,000 rabbit polyclonal ab13534, Abcam, Cambridge, UK), antiPGC1α (1:500 rabbit polyclonal GTX37356, GeneTex Inc., USA), or anti-GAPDH(1:1,000 rabbit polyclonal NB100-56875, Novus Biologicals, Centennial, USA)
primary antibodies. Afterwards, the membrane was incubated at room temperaturefor 1 h with horseradish peroxidase-conjugated secondary antibody anti-rabbit
immunoglobulins from swine (1:1,000 polyclonal P0399, Dako, Santa Clara, USA).
The bands were developed using ECL reagent (GE Healthcare Japan Ltd., TokyoJapan) and analyzed using a C-DiGit Blot Scanner (LI-COR, Lincoln, USA). Data
are shown as means ± SD (n=3).

2.7. Statistical analysis
Data are shown as means ± SD. Differences were analyzed using one-way ANOVA
followed by a Tukey–Kramer post hoc test, with p-values <0.05 considered to besignificant.

103. Results

3.1. NMN protects PC12 cells against H2O2- and 6-OHDA-induced oxidative stress

The author investigated whether NMN can protect PC12 cells from oxidative stress.
PC12 cells were pretreated with 1 mM NMN for 2 h, and then an oxidative stressor wasadded (50 M H2O2 or 100 M 6-OHDA) for 24 h. After treatment, the number of live
cells was quantified using an MTT assay. Both stressors were found to induce cell death,but NMN-pretreatment significantly reduced cell death (Fig. 1A). A live/dead cellstaining assay showed that oxidative stressors increased PI-positive dead cells, and that
NMN-pretreatment clearly decreased the ratio of dead cells (Fig. 1B). These data
indicate that NMN reduced both H2O2- and 6-OHDA-induced cell death.

3.2. PC12 cells express Sirt1 and PGC1a, and their expression is increased by NMN

Before studying the contribution of Sirt1 and the mitochondrial biogenesis inducer
PGC1, the author elucidated the expression of Sirt1 and PGC1 in PC12 cells. As
shown in Fig. 2, the PC12 cells used in this study expressed both. The author found that
NMN treatment increased the expression of both Sirt1 and PGC1. This indicates thatNMN not only activates Sirt1 followed by PGC1 but also increases the expression of11both Sirt1 and PGC1, suggesting that NMN has a strong enhancing effect on Sirt1 and
PGC1 signaling.

3.3. Protective effects of NMN against oxidative stress were sirtuin activation-dependent

To determine whether the protective effects of NMN were sirtuin activation-dependent,
the author performed a PC12 cell-protection assay in the presence of the sirtuin inhibitor
sirtinol. PC12 cells were pretreated with 1 mM NMN for 2 h in the presence or absenceof 20 µM sirtinol, then oxidative stressors (50 M H2O2 or 100 M 6-OHDA) were added
and incubated for 24 h. After treatment, the number of live cells was quantified using an
MTT assay. In the presence of sirtinol, the cell-protective effects of NMN against both
H2O2 and 6-OHDA were abolished (Fig. 3), indicating that the protective effect of NMN
was sirtuin activation-dependent.

3.4. Protective effects of NMN against oxidative stress were PGC1 activation-

dependent

To determine whether the protective effects of NMN were PGC1 activationdependent, the author performed a PC12 cell-protection assay using PGC1 knock-down
PC12 cells. PC12 cells were transfected with PGC1 siRNA, incubated for 24 h, and then12

subjected to a cell-protection assay. In siRNA-transfected cells, the protective effect of
NMN against H2O2 and 6-OHDA toxicity was abolished (Fig. 4), indicating that theprotective effect is PGC1 activation-dependent. This also suggests that mitochondrial
biogenesis is involved in the protective effect.

3.5. NMN increased mitochondria in the presence or absence of oxidative stress via

sirtuin activation

PC12 cells were treated with NMN and oxidative stressors as a cell-protection assay
in the presence or absence of sirtinol. Treated cells were harvested, stained, andsubjected to cytometric analysis. As shown in Fig. 5, NMN increased the amount of
mitochondria, but this effect was abolished by sirtinol, indicating that NMN increases
mitochondrial numbers via sirtuin activation. This increase was observed even in thepresence of both oxidative stressors (Fig. 5), suggesting that the increase in mitochondria
is involved in the protective effect against oxidative stress.
The increase in mitochondria was also demonstrated by the expression levels of
mitochondrial sirtuin Sirt3 and the mitochondrial antioxidative protein SOD2. As shownin Fig. 6, NMN treatment increased the expression of both proteins. Since Sirt3 canactivate SOD2 [17], it is likely that NMN treatment not only increases the number ofmitochondria but also enhances mitochondrial antioxidant activity.
13

4. Discussion

NMN, a precursor of NAD+, was reported to have a protective role against amyloid toxicity and cognitive impairment [18]. Oxidative stress has been linked toneurodegeneration in many neurodegenerative diseases, such as Alzheimer’s disease
and Parkinson’s disease. However, few studies have investigated whether NMN canprotect neuronal cells from oxidative stress. Since NMN was reported to activate
Nrf2 signaling, which is involved in cell protection, in an in vivo brain injury model[19], it seems likely that NMN would also protect neuronal cells against oxidative
stress. In this study, the author focused on the protective function of NMN against
oxidative stress-induced neurotoxicity. The author used H2O2- or 6-OHDA-induced
cell death in PC12 cells as an oxidative stress-induced neurodegeneration model; 6OHDA is also used as a stressor in Parkinson’s disease models [12, 13].
First, the author analyzed the protective effects of NMN against H2O2 and 6OHDA toxicity. NMN significantly suppressed cell death induced by these oxidative
stressors (Fig. 1). Although NMN can be converted to NADPH, which is known to bea cofactor of ROS-reducing proteins, such as thioredoxin and glutathione reductase,the author focused on the effects of NMN via sirtuin activation because the cytosolic
14

sirtuin Sirt1 was reported to suppress oxidative stress-induced cell damage [11].
Thus, the author analyzed whether this protective effect was sirtuin activationdependent. In the presence of the sirtuin inhibitor sirtinol, the protective effect of
NMN against both H2O2 and 6-OHDA was abolished (Fig. 3). Since NMN is aprecursor of NAD+, the data suggest that NAD+ converted from NMN directly
activates sirtuins followed by cell-protective signals. In addition, NMN treatment
also increased the expression of Sirt1 (Fig. 2). This indicates that NMN strongly
activates cellular Sirt1 activity not only via direct activation but also by increasing
its expression.
Regarding the antioxidant function of Sirt1, Sirt1 activity was reported to be
associated with mitochondrial biogenesis [20]. In some oxidative stress-induced cell
death, mitochondrial damage was reported [21]. Since mitochondrial damage isknown to cause cell death [22], any treatment that protects or enhances
mitochondrial function should protect cells from oxidative stress. Sirt1 was reported
to activate the mitochondrial biogenesis factor PGC1 via its deacetylating activity[6]. Thus, the author analyzed the contribution of PGC1 activation to the protective
effects of NMN. In PC12 cells administered with PGC1 siRNA, the protective effectof NMN against both H2O2 and 6-OHDA was abolished (Fig. 4). In addition, NMN
increased PGC1 expression in PC12 cells. These data indicate that NMN stimulates
mitochondrial biogenesis via the upregulation of PGC1, followed by the activation
15

of PGC1 via Sirt1 activation. This has the effect of preventing a reduction in
mitochondria caused by oxidative stress and helps to protect cells. Some polyphenols,such as resveratrol and quercetin, were reported to protect neuronal cells against
oxidative stress [10] and to activate sirtuin [11]. Another, epigallocatechin-3-gallate(a green tea catechin), has been reported to activate Sirt1 [23], suppress oxidative
stress, and increase the amount of PGC1α mRNA in PC12 cells [24]. Thus, it issuggested that NMN could protect cells via similar mechanisms to food ingredients,
such as epigallocatechin-3-gallate.
Next, the author investigated whether NMN treatment increased the number ofmitochondria, using cytometric analysis. We found that NMN treatment clearly
increased the number of cells containing high numbers of mitochondria, and that
this effect was sirtuin activation-dependent (Fig. 5). In mitochondrial biogenesis,
PGC1 is known to be crucial [16]. PGC1 is activated via Sirt1 activation [6], andthe data suggest that Sirt1 activation following NMN treatment activates PGC1,
which in turn induces mitochondrial biogenesis in PC12 cells. In addition, theexpression of the mitochondrial proteins Sirt3 and SOD2 was also increased by NMN
treatment (Fig. 6), supporting the cytometric analysis data. Mitochondrial Sirt3 canalso be activated by NAD+, and SOD2 is activated by Sirt3 via deacetylation [16].
SOD2 is known to reduce intracellular ROS. This indicates that NMN-induced
activation of Sirt3, followed by SOD2 activation, enhances mitochondrial antioxidant
16

activity. The increase and activation of SOD2 contributes to the protective effects of
NMN against oxidative stress.
This study demonstrated the antioxidant effects of NMN via sirtuin activation andmitochondrial enhancement. The data for this study suggest that NMN or othermolecules that activate sirtuins can, via mitochondrial enhancement, reduce the riskof neurodegenerative diseases. Furthermore, it is anticipated that the consumptionof sirtuin-activating food ingredients, including NMN, could be a novel means ofpreventing neurodegenerative diseases caused by oxidative stress.

17

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6. Figures

Fig. 1. Protective effects of NMN against 6-OHDA or H2O2 toxicity. PC12 cells werepre-treated with NMN for 2 h, then 6-OHDA or H2O2 was added and cells wereincubated for 24 h. Cell viability was analyzed using an MTT assay (A). Data arepresented as means ± SD (n=6). *P<0.05, **P<0.01. For live/dead cell-staining,
treated cells were stained with calcein AM and PI, and observed under a fluorescent
microscope (B).
23

Fig. 2. Effect of NMN on SIRT1 and PGC1α expression. PC12 cells were treated with
1 mM NMN. After incubation for 24 h, cells were subjected to Western blot analysis.
Expression levels were quantified by the intensity of ECL luminescence. Data arepresented as means ± SD (n=3).

24

Fig. 3. Effect of sirtuin inhibition on the protective effects of NMN against 6-OHDAor H2O2 toxicity. PC12 cells were pre-treated with NMN for 2 h in the presence orabsence of sirtinol, then 6-OHDA (A) or H2O2 (B) was added and cells were incubatedfor 24 h. Cell viability was analyzed using an MTT assay. Data are presented asmeans ± SD (n=6). *P<0.05, **P<0.01. For live/dead cell-staining, treated cells were
stained with calcein AM and PI, and observed under a fluorescent microscope (C).

25

Fig. 4. Effect of PGC1α knock-down on the protective effects of NMN against 6OHDA or H2O2. PC12 cells were transfected with PGC-1α siRNA and incubated for
24 h. Then, the cells were treated with NMN for 2 h, 6-OHDA (A) or H2O2 (B) was
added, then cells were incubated for 24 h. Cell viability was analyzed using an MTT
assay. Data are presented as means ± SD (n=6). *P<0.05, **P<0.01

26

Fig. 5. Effect of NMN on the number of intracellular mitochondria in the presenceor absence of 6-OHDA or H2O2, and their sirtuin dependence. PC12 cells were treatedwith NMN in the presence or absence of sirtinol for 2 h, 6-OHDA (A) or H2O2 (B) was
added, then cells were incubated for 24 h. Mitochondria were stained with JC-1 andanalyzed using image-based cytometry.
27

Fig. 6. Effect of NMN on mitochondrial sirtuin and SOD2 expression. PC12 cells
were treated with NMN and subjected to Western blotting analysis. Expression
levels were quantified by the intensity of ECL luminescence. Data are presented asmeans ± SD (n=3).

28