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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).


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 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.


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







**

#

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.


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


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).


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,


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


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


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


L53

1.3203 0.0062 1.0861 0.0295 1.1565 0.1659 0.1077 0.3158


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


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


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.


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).


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.


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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