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Year: 2009
Common effects of lithium and valproate on mitochondrialfunctions: protection against methamphetamine-induced
mitochondrial damage
Bachmann, R F; Wang, Y; Yuan, P; Zhou, R; Li, X; Alesci, S; Du, J; Manji, H K
Bachmann, R F; Wang, Y; Yuan, P; Zhou, R; Li, X; Alesci, S; Du, J; Manji, H K (2009). Common effects oflithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrialdamage. International Journal of Neuropsychopharmacology, 12(6):805-822.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:International Journal of Neuropsychopharmacology 2009, 12(6):805-822.
Bachmann, R F; Wang, Y; Yuan, P; Zhou, R; Li, X; Alesci, S; Du, J; Manji, H K (2009). Common effects oflithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrialdamage. International Journal of Neuropsychopharmacology, 12(6):805-822.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:International Journal of Neuropsychopharmacology 2009, 12(6):805-822.
Common effects of lithium and valproate onmitochondrial functions: protection againstmethamphetamine-induced mitochondrialdamage
Rosilla F. Bachmann*, Yun Wang*, Peixiong Yuan, Rulun Zhou, Xiaoxia Li,
Salvatore Alesci, Jing Du and Husseini K. Manji
Mood and Anxiety Disorders Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA
Abstract
Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of
a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could
potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a
variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood
stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that
long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore,
chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial
membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term
treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the
mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial
cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase
(COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to
the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were
attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and
VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in
mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function
and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in
the treatment of other diseases associated with impaired mitochondrial function, such as neurodegen-
erative diseases and schizophrenia.
Received 14 May 2008 ; Reviewed 25 June 2008 ; Revised 5 November 2008 ; Accepted 25 November 2008 ;
First published online 19 January 2009
Key words : Bcl-2, lithium, methamphetamine, mitochondria, valproate.
Introduction
In recent years, research has linked mood disorders
with structural and functional impairments related to
neuroplasticity in various regions of the central ner-
vous system (CNS). Research on the biological under-
pinnings of mood disorders has therefore moved away
from focusing on absolute changes in neurochemicals
such as monoamines and neuropeptides, and instead
has begun highlighting the role of neural circuits and
synapses, and the plastic processes controlling their
function. Indeed, accumulating evidence from micro-
array, biochemical, neuroimaging, and post-mortem
brain studies all support the role of mitochondrial
dysfunction in the pathophysiology of bipolar dis-
order (BPD) (Quiroz et al. 2008). Kato and colleagues
recently found that mitochondrial DNA (mtDNA)
polymorphisms and mutations in BPD affect mito-
chondrial calcium regulation (Kato, 2006, 2008). In
addition, Konradi and colleagues found that among
Address for correspondence : H. K. Manji, M.D., Laboratory of
Molecular Pathophysiology, National Institute of Mental Health,
Building 35, 1C912, Bethesda, MD 20892, USA.
Tel. : (301) 451-8441 Fax : (301) 480-0123
Email : [email protected]
* These authors contributed equally to this work.
International Journal of Neuropsychopharmacology (2009), 12, 805–822. Copyright f 2009 CINPdoi:10.1017/S1461145708009802
ARTICLE
CINP
the 43 genes that were decreased in BPD compared
with schizophrenia in post-mortem brain microarray
studies, 42% coded for mitochondrial proteins, and
were involved in regulating oxidative phosphoryla-
tion in the mitochondrial inner membrane (Konradi
et al. 2004).
Key to the present discussion is that it is now clear
that mitochondria regulate not only long-term cell
survival and cell death, but also immediate synaptic
function – both of which are clearly very relevant for
BPD. It is important to emphasize at the outset that it is
not our contention that BPD is necessarily a classical
mitochondrial disorder. Indeed, the vast majority of BPD
patients do not show the symptoms of classical mito-
chondrial disorders [e.g. optic and retinal atrophy,
seizures, dementia, ataxia, myopathy, exercise intol-
erance, cardiac conduction defects, diabetes, lactic
acidosis (Fadic & Johns, 1996)]. Instead, emerging data
suggest that many of the upstream abnormalities
(probably nuclear genome coded) converge to regulate
mitochondrial function implicated both in acute ab-
normalities of neurotransmitter synaptic plasticity as
well as in long-term cellular resilience (Quiroz et al.
2008).
The possible involvement of Bcl-2 in the pathophy-
siology and treatment of BPD initially arose from
mRNA differential display studies suggesting that
Bcl-2 might be a common target for the actions of both
chronic lithium and valproate (VPA), mood stabilizers
commonly used to treat BPD (Chen et al. 1999).
Chronic treatment of rats with therapeutic doses of
lithium and VPA doubled Bcl-2 levels in the frontal
cortex, an effect due primarily to markedly increased
numbers of Bcl-2 immunoreactive cells in layers II and
III of the anterior cingulated cortex (Chen et al. 1999;
Manji et al. 1999, 2000). Furthermore, at least in
cultured cell systems, lithium reduces levels of the
pro-apoptotic protein p53. As demonstrated recently,
repeated electroconvulsive shock also significantly
increases precursor cell proliferation in the dentate
gyrus of the adult monkey, an effect that appears to
be due to increased expression of Bcl-2 (Perera et al.
2007).
We therefore undertook this study to determine
whether one of the major downstream actions of the
mood stabilizers lithium and VPA is the enhancement
of mitochondrial function. We used methampheta-
mine (Meth) treatment to investigate this issue be-
cause Meth induces mitochondrially mediated toxicity
in the brain (Burrows et al. 2000 ; Cadet et al. 2005;
Deng et al. 2002). The molecular mechanisms via
which lithium and VPA protect against Meth-induced
mitochondrial toxicity were investigated in vivo.
A mitochondria-related microarray study was con-
ducted to explore the multiple functional groups of
genes altered by Meth and protected by lithium and
VPA. Finally, among these molecules, the role of Bcl-2
in lithium- and VPA-induced mitochondrial function
was further investigated.
Method
SH-SY5Y cell culture
Cell cultures were prepared as previously described
(Yuan et al. 1999, 2001). [See Supplementary Material
(available online) for a detailed explanation of the
procedure.]
Preparation of mitochondrial fraction from SH-SY5Y
cells
Mitochondrial fractions were prepared as previously
described with slight modifications (Almeida &
Medina, 1998). (See Supplementary Material for a de-
tailed explanation of the procedure.)
Cellular respiration rate measurement
Dulbecco’s Modified Eagle’s Medium (DMEM) was
used for oxygen consumption measurements with
the Clark electrode fitted to a small (200 ml) airtight
chamber Oxygraph System (Hansatech, UK). The O2
electrode was calibrated by immersion in electrode
buffer and DMEM equilibrated with room air. The cell
suspension in DMEM (107 cells/ml) was slowly in-
troduced to the bottom of the chamber to drive out
micro-bubbles through a corresponding opening at the
top. Cellular oxygen consumption was recorded with
Oxygraph (Biaglow et al. 1998 ; Mamchaoui & Saumon,
2000) and replicated on three separate occasions. The
respiration rate was normalized as percent of control
for comparison.
JC-1, MitoTracker Red (MTR) and MitoTracker
Green (MTG) staining
For JC-1 staining, SH-SY5Y cells were cultured in four-
well chamber glass slides and were treated (or trans-
fected with siRNA-Bcl-2 and then treated) with
various concentrations of lithium or VPA for 3–7 d.
A total of 10 nM JC-1 (Molecular Probes, USA) was
applied to the SH-SY5Y cells and incubated for 20 min
at 37 xC. The cells were washed twice with Phenol
Red-free DMEM plus 10 mM Hepes, then viewed un-
der a Zeiss 510 confocal microscope (Zeiss, USA) in
Phenol Red-free DMEM with the excitation and emis-
sion wavelength for the red fluorescence (excitation
806 R. F. Bachmann et al.
543 nM, emission 560 nM) and for the green fluor-
escence (excitation 488 nM, emission 530 nM).
The use of MTR and MTG to determine mitochon-
drial oxidation has been well-established (Buckman
et al. 2001 ; Shanker et al. 2005). MTR CM-H2XRos
(M7513) is a derivative of dihydro-X-rosamine com-
pound. The reduced probe does not fluoresce until
it enters an actively respiring cell, where it is oxidized
to the corresponding fluorescent mitochondrion-
selective probe and then sequestered in the mito-
chondria. We confirmed that it is a mitochondrial
dye by co-localization of MTR dye with MTG. The co-
localization efficiency of these two dyes was >80%.
For MTR (Molecular Probes) and MTG (Molecular
Probes) staining, 300 nM of MTR and 60 nM of MTG
(dissolved in 1r PBS) were added to the cells and in-
cubated at 37 xC for 20 min. The cells were washed
twice with Phenol Red-free DMEM plus 10 mM Hepes.
The live cell images were randomly taken under ex-
actly the same conditions for each group using LSM
510 software under a Zeiss 510 confocal microscope.
For each well, only four images were taken to ensure
the fluorescent signal was not quenched.
Qualitative analysis was performed using LSM 510
software (Zeiss). To avoid bias, all cells touching the
line of a four-line ‘#’-shaped grid on the image were
quantified for fluorescent intensity. For quantification
of both JC-1 and MTR/MTG signals, red and green
fluorescent intensities on the whole cell region were
determined by LSM 510 software (Zeiss). For each ex-
perimental condition, 34–59 cells were quantified. The
experiment was performed at least 2–3 times.
Animals and drug treatments
Adult male Wistar Kyoto rats (Charles River
Laboratories, USA) weighing 200y250 g were housed
2–3 per cage with free access to food and water (12 h
light/dark cycle ; lights on 06:00 hours). All animal
procedures were conducted according to the Guide for
the Care and Use of Laboratory Animals and were
approved by the NIMH Animal Care Committee. The
Supplementary Material provides a detailed expla-
nation of the treatment procedure, which was similar
to that used in a previous study (Du et al. 2004).
Preparation of mitochondrial fractions from brain
tissue
See the Supplementary Material for a detailed expla-
nation of the procedure for preparing the mitochon-
drial fractions from brain tissue, which was similar to
a previously published method (Zini et al. 1998).
Western blot analysis
Tissue or cell homogenates and mitochondrial fraction
samples were prepared and analysed as previously
described (Du et al. 2004). (See Supplementary
Material for a detailed explanation of the procedure.)
Cytochrome c oxidase activity measurements
A Cytochrome c Oxidase Activity Assay kit (Cytocox
I ; Sigma, USA) was used to measure the activity of
electron transport chain (ETC) complex IV according
to the manufacturer’s instructions (see Supplementary
Material for a detailed explanation of the procedure).
RNA extraction and oligo-microarray analysis
Total RNA of the rat brain cortex was isolated using
Trizol according to the manufacturer’s protocol
(Invitrogen, USA), and was further purified using the
RNeasy Mini kit according to the manufacturer’s re-
commendations (Qiagen, USA), with the addition
of DNase digestion with a RNase-Free DNase set
(Qiagen). The concentration of RNA was determined
spectrophotometrically at a ratio of 260/280 nm.
The oligo-microarrays used for this study contained
603 custom-designed rat mitochondria-related gene
70-mer oligos spotted onto poly-L-lysine-coated slides
at the National Human Genome Research Institute
(NHGRI) using an OmniGrid arrayer (GeneMachines,
USA). Methods for probe-labelling reaction andmicro-
array hybridization have been described elsewhere
(Wang et al. 2003). Microarrays were scanned at 10 mm
resolution on a Scanarray-5000 scanner (PerkinElmer,
USA) and images were organized in IPLab software
(BD Biosciences, USA). The Cy5- and Cy3-labelled
cDNA samples were scanned at 635 nm and 532 nm,
respectively. The resulting TIFF images were analysed
by IPLab software (BD Biosciences).
Each sample was tested in duplicate by alternating
the dyes. Thus, a total of eight microarrays for each
group was completed, which provided enough data-
points for statistical significance. The ratios of the
sample intensity to the reference intensity (green
[Cy3]/red [Cy5]) for all targets were determined.
Because a normal distribution could not be applied to
all components of the dataset, a Mann–Whitney test
was used to ascertain statistical significance among
microarray replicates (Wang et al. 2003). Well fluor-
escence was corrected for background fluorescence,
and ratios of intensity were established relative to ap-
propriate controls. We selected a 1.3-fold threshold
difference because the multiple repeats in our exper-
imental scheme increased the likelihood of statistical
reliability.
Lithium and valproate increase mitochondrial function 807
Bcl-2 siRNA transfection
siRNA expression vectors against Bcl-2 were con-
structed. Bcl-2 gene 423–440 and x6 to +12 were sel-
ected as the siRNA targeting sequences. A scrambled
sequence for Bcl-2 was used as the control sequence.
Oligonucleotides were designed using the software
provided by Ambion (Austin, USA) and were synthe-
sized by Sigma Genosys (USA). siRNA oligos were
then inserted into linearized P-Silencer 3.0 (Ambion,
USA) according to the manufacturer’s instructions.
SH-SY5Y cells were cultured in six-well plates or four-
well glass slides for 24–48 h. When SH-SY5Y cells
reached>90% confluence, they were transfected with
1 mg Bcl-2 siRNA pSilencer or scrambled siRNA
in pSilencer using LipofectamineTM 2000 (Invitrogen).
A cyan fluorescent protein (CFP) plasmid (Invitrogen)
was co-transfected as a marker for confocal image
analysis. The transfection procedures followed the
manufacturer’s instructions (Invitrogen). After trans-
fection, the cells were treated with 1.2 mM lithium
or 1.0 mM VPA the next day for 3 or 7 d. The down-
regulation of Bcl-2 protein expression was determined
by Western blot, and mitochondrial functions were
analysed by mitochondrial dye staining.
Results
Long-term lithium or VPA increases cell respiration
rate in SH-SY5Y cells
Oxygen consumption of cells is closely related to mi-
tochondrial function for ATP production (McCully
et al. 2007), thus we first sought to determine whether
lithium and VPA had a common effect on oxygen
consumption in human neuroblastoma SH-SY5Y cell
lines, using Oxygraph. We chose this cell line because
of its human origin, neuronal phenotype, and its
demonstrated usefulness as an experimental model
of mitochondria-mediated toxicity (Bar-Am et al. 2005;
Kluck et al. 1997). We used therapeutically relevant
concentrations of lithium and VPA in human plasma
for both in-vitro and in-vivo studies. The clinical
therapeutic ranges are 0.7¡0.3 mM for lithium
(Lauritsen et al. 1981 ; Vestergaard & Schou, 1984) and
39¡3 mg/ml for VPA (Bowden et al. 1996). We found
that treatment with either lithium or VPA increased
oxygen consumption time- and dose-dependently in
SH-SY5Y cells (Fig. 1).
When SH-SY5Y cells were exposed to ther-
apeutically relevant concentrations of lithium (1.2 mM)
and VPA (1.0 mM), cell oxygen consumption increased
significantly after 1 d treatment, and these effects were
sustained for 6 d (Fig. 1a). We also found that oxygen
consumption increased after 0.5 mM of lithium treat-
ment, and peaked at 1 mM of lithium treatment
(Fig. 1b). The increase in oxygen consumption fell
slightly after 2.0 mM of lithium treatment, progressing
1 d 3 d 6 d
Res
pira
tion
rate
, % o
f con
trol
(m
mol
O2/
min
per
107
cells
)Con Li/0.5 mM Li/1.0 mM Li/2.0 mM
Con VPA/0.4 mM VPA/0.8 mM VPA/1.2 mM
*
*
*
**
**
** *
(a)
250
200
150
100
50
0
250
200
150
100
50
0
200
150
100
50
0
(b)
(c)
Fig. 1. Effects of lithium (Li) and valproate (VPA) on cellular
respiration in human SH-SY5Y cells. (a) Time-course of
lithium and VPA effects on cellular respiration in SH-SY5Y
cells. %, Control (Con) ; , lithium; , VPA. SH-SY5Y cells
were exposed to lithium (1.2 mM) or VPA (1.0 mM) for 1, 3, or
6 d in serum-free, inositol-free Dulbecco’s Modified Eagle’s
Medium. Cellular respiration was determined as described in
the Methods section. (b) Dose–response curve for the effect of
lithium on cellular respiration. (c) Dose–response curve for
the effect of VPA on cellular respiration. Both lithium
and VPA increased SH-SY5Y cell respiration rate at
therapeutically relevant concentrations. Data are expressed
as mean¡S.E.M. (one-way ANOVA, N=3, n=5 for each
group; Tukey’s multiple comparison test : * p<0.05).
808 R. F. Bachmann et al.
in an inverted U-shaped pattern (Fig. 1b). Similarly,
VPA also regulated respiration rate in an inverted
U-shaped manner, showing a narrow concentration
window for this biological effect (Fig. 1c).
Long-term lithium or VPA enhances mitochondrial
membrane potential in SH-SY5Y cells
Next, we investigated the effects of lithium and VPA
on additional mitochondrial functions. Because mito-
chondrial membrane potential is a key indicator of
mitochondrial function, we assessed the effects of
long-term lithium or VPA on this important par-
ameter. We found that long-term treatment with
lithium significantly increased the green and red flu-
orescence ratio in JC-1-stained SH-SY5Y cells, indi-
cating dose-dependently increased mitochondrial
membrane potential (p<0.01) (Fig. 2). VPA treatment
also enhanced the red/green ratio ; this effect reached
a plateau after 0.8 mM treatment, which is a thera-
peutically relevant concentration.
Common effect of lithium and VPA on mitochondrial
oxidation in SH-SY5Y cells
Mitochondrial oxidation is one of the key mito-
chondrial functions involved in ATP synthesis. We
therefore investigated mitochondrial oxidation after
treatment with either lithium or VPA using MTR and
MTG staining. MTR dye will become a red fluorescent
colour only when oxidized. We measured both the
fluorescent intensity of MTR and theMTR/MTG ratio ;
this ratio filtered the bias caused by quenching of the
fluorescence signals and the alteration in MTR and
MTG dye uptake. We found that after 7 d treatment
with VPA (1.0 mM), MTR staining was significantly
enhanced, indicating increased mitochondrial oxi-
dation (Fig. 3c, d). After VPA treatment, the MTR/
MTG ratio also showed significant enhancement
(Fig. 3e). Seven days of lithium treatment similarly
enhanced MTR intensity (Fig. 3a, b) and MTR/MTG
ratio (Fig. 3e).
Lithium and VPA attenuate Meth-induced decreases
in mitochondrial cytochrome c and mitochondrial
Bcl-2/Bax ratio in vivo
We further investigated the protective effects of lithium
and VPA on mitochondrial function against Meth-
induced neurotoxicity. The doses of lithium and VPA
in rodent chow were established by previous studies
from our laboratory that found that, after chronic
treatment with lithium- and VPA-containing chow,
plasma concentrations of lithium and VPA in rats
were within the range of human therapeutically rel-
evant concentrations (Du et al. 2004). Rat serum drug
concentrations were in the range of 0.74¡0.25 mM for
lithium and 0.55¡0.17 mM for VPA. Because the pre-
frontal cortex is highly implicated in the pathophys-
iology of mood disorders (Adler et al. 2006; Nitschke
& Mackiewicz, 2005), we investigated the protective
effects of lithium and VPA in this brain region.
Cell survival is thought to be critically dependent
on a molecular balancing act regulating the ratio of
anti-apoptotic protein Bcl-2 to pro-apoptotic protein
Bax; imbalance of Bcl-2 family members results in the
consequent release of cytochrome c and activation of
effector caspases that cause apoptosis (Kluck et al.
1997). In this study, Meth treatment indeed caused a
marked decrease of the anti-apoptotic protein Bcl-2
and induction of the pro-apoptotic protein Bax, re-
sulting in a significant decrease in Bcl-2/Bax ratio
in the mitochondrial fractions of rat frontal cortex
(Fig. 4). After Meth administration, cytochrome c lev-
els also decreased in the mitochondrial fraction of rat
frontal cortex, indicating that some of the cytochrome
c was released into cytoplasm as an apoptotic signal.
Four weeks of pretreatment with lithium or VPA pre-
vented Meth’s effects on mitochondrial Bcl-2/Bax and
cytochrome c (Fig. 4), suggesting that these agents play
a protective role in mitochondrial homeostasis. Porin
was used as a loading control for Western blot analysis
of mitochondrial fractions.
150
125
100
75
50
25
0
Red
/Gre
en (
% o
f co
ntr
ol) *
**
* *
0.0 mM 0.6 mM 1.2 mM 2.0 mM 0.0 mM 0.6 mM 1.0 mM 2.0 mM
Lithium Valproate
Fig. 2. Effects of lithium and valproate on mitochondrial
membrane potential. SH-SY5Y cells were treatedwith various
concentrations of lithium or valproate for 6 d and
mitochondrial membrane potential was determined by
JC-1 staining (one-way ANOVA, N=2, n=34 ; * p<0.05).
Lithium and valproate increase mitochondrial function 809
Lithium and VPA preserve rat frontal cortex
mitochondrial function by inhibiting the
Meth-induced reduction of cytochrome oxidase
(COX) activity
COX is one of the key mitochondrial enzymes
and a reliable marker of mitochondrial efficiency.
Cyclosporin A (CsA) was selected as a positive control
because it exerts its effect by inhibiting permission
transition pore (PTP), thereby preventing cytochrome
c release from the mitochondria. In the present
study, we found that Meth profoundly decreased
COX activity in the mitochondrial fraction of rat
frontal cortex. CsA treatment prevented Meth’s effect
on COX activity (Fig. 5), and chronic pretreatment
with lithium or VPA prevented the reduction of
COX activity induced by Meth (Fig. 5). COX activity
was measured as an arbitrary optic density reading/
mg protein per minute. Treatment with lithium or
VPA alone did not significantly affect COX activity
(Fig. 5).
Lithium and VPA prevent Meth-induced reduction
of tyrosine hydroxylase (TH), a functional enzyme
in dopaminergic neurons
Previous studies found that Meth decreases TH
staining as a marker for dopaminergic neurons
(Deng et al. 2007). We investigated TH levels in
the prefrontal cortex after Meth treatment with or
without chronic pretreatment with lithium or VPA.
We found that TH levels were significantly lower
in prefrontal cortex after 1 d of Meth treatment,
an effect that was prevented by pretreatment with
lithium or VPA; this suggests that these agents
protect against Meth-induced reductions in TH
levels (Fig. 6). b-actin was used as a loading con-
trol. Under similar conditions, we also investi-
gated the general neuronal marker neuron-specific
enolase (NSE). Levels of NSE and actin remained
unchanged, suggesting that the effects of lithium
and VPA on TH are relatively specific (data not
shown).
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
MT
R/M
TG
rat
io
Con Con VPALi
p<0.01 p<0.01
200180160140120100806040200
180160140120100806040200
MT
R (
% o
f co
ntr
ol)
MT
R (
% o
f co
ntr
ol)
ConLi (1.2 mM)
3 d 7 d 3 d 7 d
* *ConVPA (1.0 mM)
MTG MTR MTG MTR
Con Con
VPALi
(a) (c) (e)
(b) (d )
Fig. 3. Lithium (Li) and valproate (VPA) enhance mitochondrial oxidation in SH-SY5Y cells. SH-SY5Y cells were treated with
lithium (1.2 mM) or VPA (1.0 mM) for 3–7 d. The mitochondrial oxidation of VPA- or lithium-treated SH-SY5Y cells was
determined by MitoTracker Red (MTR) staining. After 6–8 d of treatment, lithium (a, b) and VPA (c, d ) significantly enhanced
mitochondrial oxidation (N=2–4, n=39–53, Student’s t test : * p<0.05). The MTR/MTG ratio showed similar changes (e).
Data are expressed as mean¡S.E.M.
810 R. F. Bachmann et al.
Mitochondria-relevant microarray analysis for
lithium and the protective effects of VPA against
Meth-induced neurotoxicity
We used customized ‘mitochondria-relevant ’ oligo
cDNA expression for protein profiling to investigate
the protective effects conferred by lithium and VPA
against Meth-induced neurotoxicity. The microarrays
used for this study included 603 custom-designed rat
mitochondria-related genes, including ETC proteins,
enzymes for tricarboxylic acid (TCA) cycle and sub-
strate metabolism, the Bcl-2 family proteins, and cha-
perone proteins. Cluster analysis using GeneSpring
revealed different patterns of gene expression in rats
1 d after Meth administration with or without pre-
treatment with lithium or VPA. The positive targets
were identified based on eight independent tests (n=8
animals per group). In addition, fold-change levels
were set at >30%, and stringent statistical tests were
performed in accordance with previously established
methods (Jin et al. 2001).
Specifically, Meth up-regulated (>1.3-fold) 39
genes (Table 1) and down-regulated 20 genes (Table
2). Among the 39 genes up-regulated by Meth, Meth’s
effects on 11 genes were also significantly prevented
by lithium and VPA. These 11 genes belonged to three
major gene groups : (1) apoptotic genes, including
caspase-3, caspase 11, and cytochrome c ; (2) ETC
genes, including NADH dehydrogenase, NADH de-
hydrogenase 1 alpha subcomplex 5, cytochrome b5,
cytochrome P450, and COX10 homolog cytochrome c
assembly protein fernesyltransferase ; and (3) chaper-
one interacting protein genes, including FK506 bind-
ing protein 4, suppression of tumorigenicity 13 (Hsp70
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
ConM
eth
Met
h+CsA
Met
h+Li
Met
h+VPA Li
VPACsA
#
##
*
CO
X1
acti
vity
(Arb
itra
ry u
nit
/µg
pro
tein
per
min
)
Fig. 5. Protective effects of lithium (Li) and valproate (VPA)
on rat frontal cortex mitochondrial COX activity after Meth
administration. Lithium and VPA preserved rat frontal cortex
mitochondrial function by inhibiting the Meth-induced
reduction of COX activity in frontal cortex homogenate.
Cyclosporin A (CsA) was used as a positive control (N=2,
n=6–28 animals per group; one-way ANOVA, Tukey’s
multiple comparison test : * p<0.05 ; Student’s t test :# p<0.05).
150
125
100
75
50
25
0
Bcl
-2 le
vels
(%
co
ntr
ol)
Bax
leve
ls (
% c
on
tro
l)
Rat
io B
cl-2
/Bax
Cyt
ochr
ome
c le
vels
(% c
ontr
ol)
Control Meth Li+Meth VPA+Meth
Control Meth Li+Meth VPA+Meth Control Meth Li+Meth VPA+Meth Control Meth Li+Meth VPA+Meth
Mitochondrial fraction
Control Meth Li+Meth VPA+Meth
Mitochondrial fraction
Control Meth Li+Meth VPA+Meth
Mitochondrial fraction
Control Meth Li+Meth VPA+Meth
Mitochondrial fraction
**
#
Bcl-2
Porin
Bax
Porin Porin
Cyt c
200
150
100
50
0
2.5
2.0
1.5
1.0
0.5
0.0
150
100
50
0
#
#
##* *
*
(a) (b) (c) (d )
Fig. 4. Protective effect of lithium (Li) and valproate (VPA) on mitochondrial cytochrome c (Cyt c) and anti-apoptotic Bcl-2/Bax
ratio in mitochondrial fractions after Meth treatment. Chronic treatment of rats with lithium or VPA attenuated Meth-induced
decreases in anti-apoptotic Bcl-2 and increases in pro-apoptotic Bax in mitochondrial fraction of rat frontal cortex. Mitochondrial
fraction was isolated by differential centrifugation (N=3, n=4–10, one-way ANOVA, Tukey’s multiple comparison test :
** p<0.01, * p<0.05 ; Student’s t test : # p<0.05). Data are expressed as mean¡S.E.M. Porin was used as a loading control. (a)
Representative blot and quantification of Bcl-2 changes in the mitochondrial fraction of rat frontal cortex. (b) Bax changes in
the mitochondrial fraction of rat frontal cortex. (c) Ratio of Bcl-2/Bax in the mitochondrial fraction of rat frontal cortex.
(d ) Decrease of mitochondrial cytochrome c levels in rat frontal cortex after Meth administration, indicating that some of the
cytochrome c was released into cytoplasm. Chronic treatment with lithium prevented Meth’s effects on cytochrome c levels.
Quantified results are presented relative to controls.
Lithium and valproate increase mitochondrial function 811
interacting protein), and stress-induced phosphopro-
tein 1 (Hsp70/Hsp90 organizing protein) (Table 1). For
the 20 genes down-regulated by Meth, Meth’s effects
on seven genes were prevented by lithium or VPA.
These seven genes belonged to two major groups:
(1) anti-apoptotic genes, including cyclin D1 and
cyclin A1; and (2) enzymes involved in substrate
metabolism, such as glutamic-pyruvate transaminase,
glutamate oxaloacetate transaminase 2, glutaminyl-
tRNA synthase, arylalkylamine N-acetyl-transferase,
and vitamin D(3)-25 hydroxylase (Table 2).
Western blot analysis was performed to assess
whether the changes in mRNA levels were reflected in
protein expression. For confirmation, five targets were
selected from the pro-apoptotic gene group (caspase-3,
caspase-11, and cytochrome c), the ETC group (ETC
complex I NADH–ubiquinone oxidoreductase 1 beta
subcomplex 10), and the anti-apoptotic gene group
(cyclin D1). Meth significantly induced the gene ex-
pression of apoptosis-related proteins caspase-3 (full-
length form, 35 kDa) and 48-kDa subunit caspase-11,
and showed a clear trend to increase cytochrome c.
Pretreatment with lithium or VPA attenuated Meth’s
increase in the expression of the 48-kDa subunit of
caspase-11 and caspase-3, and showed a trend to at-
tenuate Meth’s increase of cytochrome c expression.
Up-regulation of caspase-3, caspase-11, and cyto-
chrome c proteins was consistent with up-regulation
of the corresponding genes in the microarray finding.
In addition, Meth significantly reduced ETC complex
I NADH–ubiquinone oxidoreductase 1 beta sub-
complex 10, a protein crucial for mitochondrial res-
piration. Levels of b-actin showed no change. These
changes in protein levels were reflected in mRNA
levels in the microarray findings (Fig. 7).
Bcl-2, a common target of lithium and VPA, is
critical to the effects of lithium and VPA on
mitochondrial function
Among the mitochondrial proteins regulated by mood
stabilizers, Bcl-2 is known to be a key regulator of
mitochondrial function as well as a common target for
lithium and VPA in vivo (Chen et al. 1999). Here, we
further investigated whether Bcl-2 is the critical mol-
ecule in the up-regulation of mitochondrial function
by lithium and VPA. Western blot analysis showed
that long-term treatment with lithium or VPA at thera-
peutically relevant concentrations increased Bcl-2
levels in SH-SY5Y cell homogenates by 35% and 40%
respectively ; these levels reached their peak after 6 d
treatment (Fig. 8a). b-actin was used as a loading
control. In addition, in mitochondrial fractions of hu-
man neuroblastoma SH-SY5Y cells, Bcl-2 levels in-
creased to 339% of control after lithium treatment, and
to 426% of control after VPA treatment (Fig. 8a). Porin
was used as a loading control for mitochondrial frac-
tions.
Next, we investigated the role of Bcl-2 in enhancing
mitochondrial function after long-term treatment with
lithium or VPA. Bcl-2 siRNA in a vector, P-silencer,
was transfected into SH-SY5Y cells following treat-
ment with lithium (1.2 mM) or VPA (1.0 mM) for 3 d or
7 d. CFP was used as an indicator for the transfected
SH-SY5Y cells. After transfection of Bcl-2 siRNA or
scrambled siRNA (SCR) vectors, Bcl-2 siRNA signifi-
cantly reduced Bcl-2 protein levels (Fig. 8b), and also
attenuated MTR staining (Fig. 8c) ; these results sug-
gest reduced oxidization in mitochondria. MTR stain-
ing was also attenuated in the Bcl-2 siRNA-transfected
SH-SY5Y cells treated with lithium or VPA by about
30%, suggesting that Bcl-2 is one of the key players in
allowing lithium and VPA to increase mitochondrial
function (Fig. 8c).
150
125
100
75
50
25
0
Tyr
osi
ne
hyd
roxy
lase
(%
co
ntr
ol)
*
****
TH
Actin
Control Meth Li+Meth VPA+Meth
Fig. 6. Protective effect of lithium (Li) or valproate (VPA)
on tyrosine hydroxylase (TH), a functional enzyme in
dopaminergic neurons, after Meth injection in vivo. Chronic
treatment of rats with lithium or VPA for 4 wk was followed
by Meth injection for 1 d. Prefrontal cortical protein samples
underwent Western blot analysis with anti-TH antibody.
Actin was used as a loading control. Data are expressed as
mean¡S.E.M. (n=8 for each group, n=32 ; one-way ANOVA,
Tukey’s multiple comparison test : ** p<0.05 ; Student’s t test :
* p<0.05).
812 R. F. Bachmann et al.
Discussion
Mitochondrial dysfunction in the CNS appears to be a
pathogenic pathway for a variety of disorders as-
sociated with progressive atrophic/degenerative
changes, including Parkinson’s disease, Alzheimer’s
disease, Huntington’s disease, amyotrophic lateral
sclerosis (ALS), BPD, and schizophrenia (Browne &
Beal, 2004 ; Karry et al. 2004; Kato & Kato, 2000 ;
Mecocci et al. 1994 ; Orth & Schapira, 2001 ;
Stavrovskaya & Kristal, 2005). In the present study, we
investigated whether the mood stabilizers lithium and
VPA increase key mitochondrial functions and protect
against Meth-induced functional damage to mito-
chondria. We found that (i) long-term lithium or VPA
enhanced mitochondrial function as assessed by mi-
tochondrial membrane potential and mitochondrial
oxidation ; (ii) chronic lithium or VPA protected
against Meth-induced decreases in mitochondrial in-
dexes in the prefrontal cortex in vivo ; e.g. these agents
prevented Meth-induced changes in mitochondrial
Bcl-2/Bax ratio and COX activity ; (iii) a mitochondria-
relevant oligoarray showed that Meth up-regulated
the expression of 39 genes (>1.3-fold) and down-
regulated 20 genes. Lithium and VPA protected
against the alteration of some of these genes ; and (iv)
this regulation of mitochondrial functions by lithium
or VPA was partially mediated through Bcl-2 ; Bcl-2
knock-down attenuated the effects of lithium or VPA
on the regulation of mitochondrial functions.
Common effects of lithium and VPA on
mitochondrial function
Using multiple measures, we found a robust en-
hancement of mitochondrial function by long-term
treatment with lithium or VPA at therapeutically rel-
evant concentrations. Recent studies have shown that
lithium desensitizes brain mitochondria to calcium,
antagonizes permeability transition, and diminishes
cytochrome c release (Shalbuyeva et al. 2007) ; it also
inhibits mitochondria-generated reactive oxygen spe-
cies (ROS) and nitric oxide (NO) in an animal model of
ischaemia (Plotnikov et al. 2007). However, VPA has
been found to inhibit substrate-specific oxygen con-
sumption and mitochondrial ATP synthesis in rat he-
patocytes, and VPA metabolites have been found to
inhibit dihydrolipoyl dehydrogenase activity leading
to impaired oxidative phosphorylation (Luis et al.
2007). This is supported by evidence that VPA therapy
may cause inborn errors of metabolism (IEMs) and
acute liver toxicity, potentially because of its inter-
ference with mitochondrial b-oxidation (Silva et al.
2008).
Because mood stabilizers exhibit neuroprotective
properties against an array of insults in cells of neu-
ronal origin, this toxic effect seems cell-type specific.
Recent studies suggest that lithium and VPA may de-
crease the vulnerability of human neural, but not glial,
cells to cellular injury evoked by oxidative stress
possibly arising from putative mitochondrial dis-
turbances implicated in BPD (Lai et al. 2006). The stu-
dies found that pretreatment of SH-SY5Y cells for 7 d,
but not 1 d, with 1 mM lithium or 0.6 mM VPA sig-
nificantly reduced rotenone- and hydrogen per-
oxide-induced cytotoxicity, cytochrome c release, and
caspase-3 activation, and increased Bcl-2 levels ; these
results are in agreement with the findings of the pres-
ent study (Lai et al. 2006). Also key to this discussion is
VPA’s narrow therapeutic window. Although both
lithium and VPA have neuroprotective mechanisms
that are indirectly exerted through mitochondrial en-
hancement, clinical studies have shown that VPA in
the toxic range impairs mitochondrial b-oxidation in
patients (Eyer et al. 2005).
Lithium and VPA protect against Meth-induced
damage to mitochondrial function
To examine the utility of lithium- and VPA-induced
enhancement of mitochondrial function, we conduc-
ted Meth experiments in vivo, and found that both
lithium and VPA prevented Meth-induced reduction
of mitochondrial function. Meth is a neurotoxin
known to exert mitochondrially mediated toxicity, and
has been shown to directly inhibit COX, complex IV of
the ETC (Burrows et al. 2000), and succinate dehydro-
genase, complex II of the ETC (Brown et al. 2005). In
addition Meth induces apoptosis by activating the
mitochondrial cell death pathway (Cadet et al. 2005 ;
Deng et al. 2001, 2002) and has been shown to decrease
mitochondrial membrane potential (Riddle et al. 2006 ;
Wu et al. 2007) and increase ROS (Wu et al. 2007) fol-
lowed by apoptosis. In previous studies, Meth signifi-
cantly decreased the anti-apoptotic genes Bcl-2 and
Bcl-XL, which may contribute to its cytotoxic effect in
mouse neocortex (He et al. 2004 ; Jayanthi et al. 2001).
Notably, lithium and VPA exhibit neuroprotective
properties against an array of insults. Our in-vivo
studies showed that chronic treatment with lithium or
VPA attenuated the Meth-induced decrease of Bcl-2/
Bax ratio in the mitochondrial fraction of prefrontal
cortex.
Meth-induced toxicity probably occurs by increas-
ing oxidative stress and free radical production
through inhibition of COX. This inhibition eventually
leads to a decrease in mitochondrial respiration,
Lithium and valproate increase mitochondrial function 813
Table 1. Oligo-microarray analysis : Meth up-regulated genes
Acc. no. Title
Meth
vs. Con
Li+Meth
vs. Con
VPA+Meth
vs. Con
Li+Meth
vs. Meth
VPA+Meth
vs. Meth
Avg pa Avg pa Avg pa pb pb
Proapoptotic genes
NM_012922.1 Rattus norvegicus caspase-3 (Casp3) 1.5210 0.0025 0.9964 0.9453 0.9865 0.8515 0.0005 0.0004
M20622.1 Cytochrome c, somatic 1.4589 0.0089 1.0470 0.0905 1.0546 0.5345 0.0101 0.0115
NM_053736.1 Rattus norvegicus caspase-11 (Casp11) 1.4495 0.0001 0.6975 0.0001 0.8810 0.0265 0.0000 0.0000
Electron transfer chain
XM_343910.1 PREDICTED : Rattus norvegicus COX10 homolog, cytochrome
c oxidase assembly protein, heme A: farnesyltransferase
(yeast) (predicted)
2.0246 8.3002 1.3490 0.0086 1.0818 0.0034 0.0000 0.0000
XM_216378.2 Rattus norvegicus similar to NADH dehydrogenase 1.4437 0.0001 1.1437 0.1732 0.9241 0.2605 0.0242 0.0001
NM_012985.1 Rattus norvegicus NADH dehydrogenase (ubiquinone)
1 alpha subcomplex 5 (Ndufa5)
1.3732 0.0005 1.0380 0.5197 0.9871 0.6571 0.0003 0.0000
NM_030586.1 Rattus norvegicus cytochrome b5, outer mitochondrial
membrane isoform (omb5)
1.3627 0.0067 1.0179 0.8822 1.0065 0.9119 0.0406 0.0338
NM_017286.1 Rattus norvegicus cytochrome P450, subfamily 11A (Cyp11a) 1.3164 0.0001 1.0876 0.0918 1.0756 0.2917 0.0135 0.0093
XM_227084.2 Rattus norvegicus Nicotinamide nucleotide
transhydrogenase (NAD(P)+ transhydrogenase) (Nnt)
1.3039 0.0076 1.0891 0.4014 0.9819 0.7561 0.1726 0.0277
Chaperone interacting proteins
S78556.1 Grp75=75 kDa glucose regulated protein 1.4050 0.0031 1.3189 0.0027 1.5038 0.0038 0.8033 0.7501
XM_342763.1 FK506 binding protein 4, 59 kDa 1.3278 0.0037 0.9453 0.6196 0.9245 0.3784 0.0157 0.0108
NM_031122.1 Suppression of tumorigenicity 13 (colon carcinoma) (Hsp70
interacting protein)
1.3261 0.0001 0.9212 0.2403 0.9601 0.7394 0.0047 0.0104
NM_138911.2 Stress-induced-phosphoprotein 1 (Hsp70/ Hsp90-
organizing protein)
1.3227 0.0010 0.9106 0.1744 0.8961 0.1382 0.0002 0.0001
Enzymes for metabolism
NM_053607.1 Rattus norvegicus fatty acid coenzyme A ligase, long chain
5 (Facl5)
1.4552 0.0002 1.2884 0.0159 1.0781 0.5069 0.4196 0.0222
XM_217611.2 Rattus norvegicus similar to carbonic anhydrase 5b,
mitochondrial
1.3782 0.0079 1.1034 0.5435 1.0858 0.1091 0.2272 0.1898
XM_217181.2 Rattus norvegicus similar to serine beta lactamase-like
protein LACT-1
1.3739 0.0001 1.4016 0.0003 1.3328 0.0218 0.9675 0.9304
814R.F
.Bachm
annetal.
XM_226211.2 Rattus norvegicus similar to thymidine kinase 2,
mitochondrial ; thymidine kinase 2
1.3371 0.0014 0.9054 0.3324 1.2136 0.1327 0.0133 0.6483
NM_130755.1 Rattus norvegicus citrate synthase (Cs) 1.3339 0.0016 1.0361 0.5139 1.0687 0.4587 0.0186 0.0377
XM_343764.1 Rattus norvegicus monoamine oxidase A (MAOA) 1.3314 0.0024 1.0768 0.1971 1.0671 0.5794 0.1072 0.0920
NM_053592.1 Rattus norvegicus Deoxyuridinetriphosphatase (dUTPase)
(Dut)
1.3307 0.0081 1.2282 0.1606 1.0645 0.3420 0.7732 0.1989
NM_019168.1 Rattus norvegicus arginase 2 (Arg2), 1.3269 0.0021 1.2234 0.0156 1.2514 0.0372 0.6399 0.7863
M_031720.2 Deiodinase, iodothyronine, type II 1.3203 0.0017 1.0729 0.1635 1.0878 0.4394 0.0838 0.1086
XM_223499.1 Rattus norvegicus similar to apurinic/apyrimidinic
endonuclease 2
1.3126 0.0002 1.0869 0.6406 1.0423 0.3923 0.3319 0.2132
XM_341628.1 Rattus norvegicus similar to malic enzyme 2,
NAD(+)-dependent, mitochondrial
1.3044 0.0047 0.9787 0.5792 1.0132 0.8618 0.0046 0.0110
Others
NM_017139.1 Proenkephalin 1.4176 0.0023 1.2298 0.0728 1.0861 0.5093 0.4531 0.1021
XM_214751.1 Rattus norvegicus similar to mitochondrial ribosomal protein
L18
1.6222 0.0016 1.2615 0.1107 1.0373 0.7301 0.1279 0.0090
XM_216010.2 Rattus norvegicus similar to mitochondrial ribosomal protein
L41
1.4442 0.0040 1.1631 0.3343 1.2224 0.0066 0.2158 0.3744
XM_344002.1 Rattus norvegicus similar to GA binding protein alpha chain
(GABP-alpha subunit) (transcription factor E4TF1-60)
(Nuclear respiratory factor-2 subunit alpha)
1.4272 0.0003 0.8527 0.0159 0.7524 0.0089 0.0000 3.6004
XM_343135.1 Unactive progesterone receptor, 23 kDa 1.3733 0.0039 1.1737 0.3904 1.0098 0.8864 0.5164 0.1301
XM_217179.2 Rattus norvegicus similar to ClpX protein 1.3669 0.0076 1.0847 0.3975 1.2241 0.1413 0.1949 0.6407
NM_053610.1 Rattus norvegicus peroxiredoxin 5 (Prdx5) 1.3587 0.0034 0.8464 0.1154 1.1156 0.3029 0.0019 0.1681
NM_053842.1 Mitogen-activated protein kinase 1 1.3463 0.0043 1.0694 0.3518 1.0466 0.5974 0.0561 0.0368
NM_053357.2 Catenin (cadherin-associated protein), beta 1, 88 kDa 1.3408 0.0063 1.0885 0.0469 1.1042 0.2799 0.0693 0.0923
XM_214491.2 Rattus norvegicus similar to mitochondrial ribosomal protein
L32
1.3403 0.0022 1.0475 0.4889 1.0457 0.5563 0.0210 0.0202
XM_236263.2 Rattus norvegicus similar to Ddx10 protein 1.3348 0.0032 1.1298 0.2182 1.1018 0.1154 0.1757 0.1116
NM_031818.1 Rattus norvegicus chloride intracellular channel 4 (Clic4) 1.3240 0.0056 1.0856 0.2837 0.9362 0.2581 0.0649 0.0023
XM_342712.1 Rattus norvegicus similar to mitochondrial ribosomal protein
L53
1.3203 0.0062 1.0861 0.0295 1.1565 0.1659 0.1077 0.3158
XM_221202.2 Rattus norvegicus similar to mitochondrial ribosomal protein
L12
1.3178 0.0040 1.0410 0.7843 1.1087 0.4890 0.2955 0.4894
M18331.1 Protein kinase C, epsilon 1.3142 0.0010 1.3292 0.0046 1.1009 0.3262 0.9902 0.1656
Meth, Methamphetamine treatment ; Li, lithium pretreatment ; VPA, valproate pretreatment ; Con, control.a p value of Student’s t test to compare each treatment and the corresponding control.b p value of one-way ANOVA Turkey post-hoc to compare lithium or VPA pretreatment vs. pure amphetamine treatment.
Lithiu
mandvalproate
increase
mitochon
drialfunction
815
Table 2. Oligo-microarray analysis : Meth down-regulated genes
Acc. no. Title
Meth
vs. Con
Li+Meth
vs. Con
VPA+Meth
vs. Con
Li+Meth
vs. Meth
VPA+Meth
vs. Meth
Avg Pa Avg Pa Avg Pa pb pb
Antiapoptotic genes
NM_171992.2 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) 0.6284 0.0021 0.9187 0.3748 0.7825 0.0007 0.0221 0.2922
XM_215565.2 Cyclin A1 0.6883 0.0003 0.8556 0.0103 0.8301 0.0018 0.0252 0.0623
Enzymes for metabolism
AF111160.1 Glutathione S-transferase A3 0.5862 1.5701 0.8755 0.0712 0.7933 0.0018 0.0009 0.0152
NM_031039.1 Rattus norvegicus glutamic-pyruvate transaminase
(alanine aminotransferase) (Gpt)
0.5997 0.0010 0.9231 0.1189 0.9707 0.6027 0.0022 0.0005
NM_012569.1 Rattus norvegicus glutaminase (Gls) 0.6384 0.0003 0.8017 0.0382 0.9262 0.0484 0.1432 0.0060
NM_133598.1 Rattus norvegicus glycine cleavage system protein H
(aminomethyl carrier) (Gcsh)
0.6579 0.0071 0.8119 0.0412 0.8852 0.1318 0.3662 0.1262
NM_013177.1 Rattus norvegicus glutamate oxaloacetate transaminase
2 (Got2)
0.6714 0.0015 0.9570 0.3572 0.8828 0.0455 0.0029 0.0275
Y07534.1 Rattus norvegicus mRNA for mitochondrial vitamin
D(3) 25-hydroxylase
0.6728 0.0018 0.8476 0.0076 0.9235 0.0991 0.0614 0.0062
NM_012504.1 ATPase, Na+/K+ transporting, alpha 1 polypeptide 0.6762 0.0034 0.8418 1.9419 0.8228 0.0121 0.0968 0.1535
NM_017290.1 ATPase, Ca2+ transporting, cardiac muscle, slow
twitch 2
0.6783 0.0034 0.8843 0.0179 0.8708 0.0486 0.0478 0.0671
XM_228810.2 Rattus norvegicus similar to Nucleolar RNA helicase II
(Nucleolar RNA helicase Gu) (RH II/Gu)
(DEAD-box protein 21)
0.6793 0.0044 0.9502 0.3904 0.8003 0.0087 0.0172 0.3850
XM_214381.2 Rattus norvegicus similar to glutaminyl-tRNA
synthetase (glutamine-tRNA ligase) (GlnRS)
0.6811 0.0004 1.0574 0.3460 0.9798 0.8521 0.0049 0.0258
D90401.1 Rattus norvegicus mRNA for dihydrolipoamide
succinyltransferase
0.6912 0.0037 0.7964 1.7801 0.9125 0.1887 0.3894 0.0265
NM_012818.1 arylalkylamine N-acetyltransferase 0.6954 0.0001 0.8863 0.0111 0.9379 0.4414 0.0498 0.0113
816R.F
.Bachm
annetal.
proton leakage across the mitochondrial membrane,
and uncoupling. Uncoupling subsequently causes
energetic inefficiency and increases metabolic heat,
thereby increasing temperature. It has also been re-
ported that VPA (only at the high concentration of
10 mM) causes uncoupling of mitochondrial respir-
ation in liver cells (Jimenez-Rodriguezvila et al. 1985) ;
however, this may not apply to the low VPA concen-
tration (0.3–2.0 mM) used in the present study.
Meth was originally shown to inhibit COX activity
following administration in rats (Burrows et al. 2000),
suggesting that it has a direct inhibitory effect on
mitochondrial function exerted through incapacitation
of one of the enzymes responsible for oxidative phos-
phorylation and energy production. In the present
study, we found that Meth significantly decreased
COX activity in the homogenate of prefrontal cortex
and hippocampus. Chronic lithium preserved the ac-
tivity of mitochondrial COX (Fig. 5).
TH positive dopaminergic and norepinephrinergic
axons are distributed in prefrontal cortex (Divac et al.
1994 ; Lewis et al. 1998 ; Miner et al. 2003). Previous TH
immunostaining studies revealed that Meth is toxic to
dopaminergic neurons (Davidson et al. 2007 ; Deng
et al. 2007). Therefore, we also assessed levels of the
dopaminergic neuronal marker TH in prefrontal cor-
tex, because TH levels may reflect loss of dopaminer-
gic terminals. We found that TH levels in prefrontal
cortex were significantly reduced after Meth; notably,
chronic lithium or VPA pretreatment protected against
the Meth-induced TH reductions (Fig. 6).
Mitochondria-related groups of genes involved in the
protective effects of lithium and VPA against Meth
The mitochondria-related gene array studies provide
an overall picture of mitochondria-related gene chan-
ges involved in the protective effects of lithium and
VPA against Meth. Three major groups of mitochon-
drial genes – including apoptotic, ETC, and chaperone
interacting protein genes – were up-regulated byMeth
treatment ; Meth’s effect was significantly prevented
by lithium or VPA (Table 1). Mitochondrial functions,
such as oxidation or ATP production are performed by
groups of genes. As discussed above, Meth disturbed
energy production, therefore, it is not surprising that
five ETC genes were altered by Meth, including
NADH dehydrogenase, NADH dehydrogenase 1 al-
pha subcomplex 5, cytochrome b5, cytochrome P450,
and COX10 homolog cytochrome c. This Meth-
induced increase was attenuated by treatment with
lithium or VPA (Table 1). These effects on ETC genes
provide the molecular basis for the alteration inOthers
XM_219
938.2
Rattusnorvegicussimilar
tomitoch
ondrial
ribosomal
protein
L43
0.6579
0.0089
0.8894
0.24
890.9805
0.83
630.1973
0.0528
XM_213
242.1
Rattusnorvegicussimilar
toNADH
deh
ydrogen
ase
(ubiquinone)
1betasu
bcomplex,10,22
kDa;NADH
deh
ydrogen
ase(ubiquinone)
1betasu
bcomplex,
10
(22kDa,
PDSW)
0.6713
0.0004
0.9051
0.06
960.8381
0.00
380.0044
0.0442
XM_228
758.2
Rattusnorvegicussimilar
tomitoch
ondrial
inner
mem
branetran
slocase
componen
tTim
17b
0.6716
0.0013
0.7497
0.00
150.7381
5.65
580.5014
0.6031
NM_0
3105
1.1
Macrophag
emigrationinhibitory
factor
(glycosylation-inhibitingfactor)
0.6726
0.0041
0.8421
0.00
260.8571
0.04
150.1341
0.0962
NM_0
1259
0.1
Inhibin,alpha
0.6860
0.0006
0.8280
0.00
370.8782
0.00
210.0609
0.0095
NM_1
9875
0.1
Rattusnorvegicuscryptoch
rome1(photolyase-like)
(Cry1)
0.6965
0.0006
0.8114
0.00
490.8328
0.00
390.2088
0.1176
Meth,Metham
phetam
inetreatm
ent;Li,lithium
pretreatm
ent;VPA,valproatepretreatm
ent;Con,control.
apvalueofStuden
t’sttest
tocompareeach
treatm
entan
dthecorrespondingcontrol.
bpvalueofone-way
ANOVA
Turkey
post-hoc
tocomparelithium
orVPA
pretreatm
entvs.pure
amphetam
inetreatm
ent.
Lithium and valproate increase mitochondrial function 817
mitochondrial functions, and support the mitochon-
drial oxidation and membrane potential alterations we
observed in vitro. In the apoptotic group, it is notable
that mRNA and protein levels of caspase-3, a pro-
apoptotic marker used to detect early apoptosis (Belloc
et al. 2000), were significantly enhanced by Meth;
lithium or VPA prevented these increases, suggesting
that they offer protection against mitochondrially
mediated cell apoptosis.
Several mitochondria-related genes were down-
regulated by Meth, including anti-apoptotic genes and
enzymes involved in metabolism; Meth’s effects were
prevented by lithium and VPA. Both lithium and VPA
pretreatment attenuated the Meth-induced down-
regulation of the anti-apoptotic genes cyclin D1 and
cyclin A1 (Table 2), suggesting a mechanism of neu-
roprotection. Overall, these microarray results help to
explain the mechanism whereby lithium and VPA are
involved in the protection of mitochondrial functions.
Bcl-2 is involved in the regulation of mitochondrial
function induced by lithium and VPA
Finally, to elucidate the mechanisms underlying the
enhancement of mitochondrial functions, we ex-
amined the role of Bcl-2. It is now clear that Bcl-2 is a
protein that robustly enhances mitochondrial func-
tion, thereby inhibiting both apoptotic and necrotic
cell death induced by diverse stimuli (Adams & Cory,
1998 ; Bruckheimer et al. 1998 ; Merry & Korsmeyer,
1997). Recent studies suggest that lithium’s effects on
Bcl-2 may be mediated through the expression of p53
(Chen & Chuang, 1999). Previous studies from our
laboratory and others have shown that lithium and
VPA increase cellular levels of Bcl-2 (Bush & Hyson,
2006 ; Chen et al. 1999 ; Ghribi et al. 2002 ; Zhang et al.
2003). However, mitochondrial Bcl-2 and its impact on
mitochondrial function remain unclear. In the present
study, we found that treatment with lithium or VPA
produced a marked (>300%) increase in mitochon-
drial Bcl-2 levels.
We therefore used a variety of independent
measures to determine whether the lithium- or VPA-
induced increases in Bcl-2 levels did, in fact, translate
into enhanced mitochondrial function. Both lithium
and VPA treatment increased cellular respiratory rate,
enhanced mitochondrial membrane potential, and in-
creased mitochondrial oxidation. In addition, Bcl-2
siRNA significantly knocked down Bcl-2 gene ex-
pression and clearly reduced mitochondrial oxidation
parameters, suggesting that Bcl-2 is a key modulator of
mitochondrial function regulation by lithium and
VPA. It is likely that several cellular mechanisms are
involved in mediating Bcl-2’s protective effects, in-
cluding sequestering the proforms of caspases, in-
hibiting the effects of caspase activation, producing
antioxidant effects, enhancing mitochondrial calcium
uptake, and attenuating the release of calcium and
300
250
200
150
100
50
0
**
*****
*
*
Control
MethMeth+LiMeth+VPA
Caspas
e-3
Caspas
e-11
(48 k
Da)
Caspas
e-11
(20 k
Da)
Cytoch
rom
e c
NDUFB10
Cyclin
D1
ACTB
Fig. 7. Protein expression levels (Western blot analysis) of five target genes selected from oligoarray analysis. Both lithium (Li)
and valproate (VPA) pretreatment partially prevented Meth’s effects on the expression of five genes chosen for mitochondrial
microarray profiling analysis. b-actin (ACTB) was used as a loading control. Mean¡S.E.M. are shown for relative protein levels
based on densitometry analysis (n=8 animals for each group; one-way ANOVA and Tukey HSD post-hoc : * p<0.05, ** p<0.01.
NDUFB10:NADH–ubiquinone oxidoreductase 1 beta subcomplex, 10).
818 R. F. Bachmann et al.
cytochrome c from mitochondria (reviewed in Adams
& Cory, 1998 ; Bruckheimer et al. 1998 ; Li et al. 1999 ;
Sadoul, 1998).
Taken together, the evidence suggests that the
mood stabilizers lithium and VPA have a common
effect on the regulation of mitochondrial functions
CFP
MTG
SCR Bcl-2 SiRNA VPA+SCR VPA+Bcl-2 SiRNA Li+SCR Li+Bcl-2 SiRNA
MTR
Overlay
(c)
0
50
100
150
200
250
Con SCR Bcl-2SiRNA VPA/SCR VPA/Bcl-2 Li/SCR Li/Bcl-2
* *
#
MT
R (
% o
f co
ntr
ol)
(d)
Con Bcl-2siRNASCR
Con Bcl-2siRNASCR
Bcl
-2 (
% o
f co
ntr
ol) *
*
500
400
300
200
100
0
Bcl
-2 (
% o
f co
ntr
ol)
Con Li VPA Con Li VPA
Homogenates Mitochondria
Bcl-2Porin
Bcl-2Actin
*
*
**
ConLiVPA
120
100
80
60
40
20
0
(a) (b)
Fig. 8. Bcl-2 plays an important role in the effect of lithium (Li) and valproate (VPA) on mitochondrial function. (a) Lithium and
VPA up-regulated Bcl-2 protein in SH-SY5Y cells in both total protein homogenates and mitochondrial fractions. SH-SY5Y cells
of 80% confluency were treated with lithium (1.0 mM) or VPA (1.0 mM) for 6 d. Western blot analysis with anti-Bcl-2 antibody
was performed to determine the protein level in cell homogenates and mitochondrial fraction. Data are expressed as
mean¡S.E.M. (N=3, n=5 for each group; one-way ANOVA, Tukey’s multiple comparison test : * p<0.05). (b) Effects of Bcl-2
siRNA transfection on Bcl-2 protein levels. Bcl-2 siRNA significantly attenuated Bcl-2 expression in SH-SY5Y cells after 2 d of
transfection. Data are expressed as mean¡S.E.M. ; Student’s t test : * p<0.05 (n=4). (c) Effects of Bcl-2 knock-down with Bcl-2
siRNA on lithium- or VPA-evoked mitochondrial oxidation revealed by MitoTracker Red (MTR) staining. SH-SY5Y cells were
exposed with or without VPA or lithium for 6–7 d after transfection with P-silencer containing siRNA sequence against Bcl-2 or
scrambled sequence. MTR and MitoTracker Green (MTG) staining showed that Bcl-2 siRNA significantly attenuated
mitochondrial oxidation in cultured SH-SY5Y cells ; only the transfected cells were quantified as indicated by cyan fluorescent
protein (CFP, blue). (d ) Bcl-2 siRNA, but not the scrambled siRNA (SCR), significantly attenuated MTR staining after treatment
with VPA (1.0 mM) for 6 d or with lithium (1.2 mM) for 6 d (N=2–4, n=20–29 ; one-way ANOVA, Tukey’s multiple comparison
test : # p<0.05 ; Student’s t test : * p<0.05). Data are expressed as mean¡S.E.M.
Lithium and valproate increase mitochondrial function 819
in vitro and in the CNS. This modulation of mito-
chondrial function may ultimately be useful in pro-
tecting mitochondria from pathological mitochondrial
dysfunctions in the brain. The anti-apoptotic protein
Bcl-2 appears to be one of the key players in this
regulation of mitochondrial function. Indeed, this
protective effect is quite profound, as highlighted by
the fact that in this study, multiple functional groups
of mitochondria-related genes were protected by lith-
ium and VPA in the CNS in vivo.
Note
Supplementary material accompanies this paper on
the Journal’s website (http://journals.cambridge.org).
Acknowledgements
We acknowledge the support of the Intramural
Research Program of the National Institute of Mental
Health. We thank Ioline Henter and Holly Giesen
for their invaluable editorial assistance. This work
was undertaken under the auspices of the NIMH
Intramural Program; Dr Manji is now at Johnson and
Johnson Pharmaceutical Research and Development.
Statement of Interest
None.
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