Uncoupling protein 2 protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity

Uncoupling protein 2 protects dopaminergic neurons from acute1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity

Bruno Conti,*,1 Shuei Sugama,�,1 Jacinta Lucero,* Raphaelle Winsky-Sommerer,�Sebastian A. Wirz,* Pamela Maher,§ Zane Andrews,¶ Alasdair M. Barr,* Maria C. Morale,*,**Covadonga Paneda,� Janell Pemberton,* Svetlana Gaidarova,* M. Margarita Behrens,*Flint Beal,�� Pietro Paolo Sanna,�� Tamas L. Horvath¶ and Tamas Bartfai*

* Harold L. Dorris Neurological Research Center, Scripps Research Institute, La Jolla, California, USA

�Department of Physiology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, Japan

�Department of Molecular Biology, Scripps Research Institute, La Jolla, California, USA

§Department of Cell Biology, Scripps Research Institute, La Jolla, California, USA

¶Department of Obstetrics and Gynecology, Yale University, New Haven, Connecticut, USA

**OASI1 , Institute for Research and Care on Mental Retardation and Brain Aging, Troina, Italy

��Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, USA

��Department of Neuropharmacology, Scripps Research Institute, La Jolla, California, USA

Abstract

Oxidative stress is implicated in the death of dopaminergic

neurons in sporadic forms of Parkinson’s disease. Because

oxidative stress can be modulated endogenously by

uncoupling proteins (UCPs), we hypothesized that specific

neuronal expression of UCP2, one member of the UCP family

that is rapidly induced in the CNS following insults, could

confer neuroprotection in a mouse model of Parkinson’s

disease. We generated transgenic mice overexpressing

UCP2 in catecholaminergic neurons under the control of the

tyrosine hydroxylase promoter (TH-UCP2). In these mice,

dopaminergic neurons of the substantia nigra showed a

twofold elevation in UCP2 expression, elevated uncoupling of

their mitochondria, and a marked reduction in indicators of

oxidative stress, an effect also observed in the striatum. Upon

acute exposure to 1,2,3,6-methyl-phenyl-tetrahydropyridine,

TH-UCP2 mice showed neuroprotection and retention of

locomotor functions. Our data suggest that UCP2 may rep-

resent a drug target for slowing the progression of Parkin-

son’s disease.

Keywords: MPTP, Parkinson, uncoupling protein 22 .

J. Neurochem. (2005) 93, 493–501.

Parkinson’s disease (PD) is a neurodegenerative diseasecharacterized by the loss of dopaminergic neurons of thesubstantia nigra pars compacta (SNpc). Neuronal degener-ation in PD can also be found in other brain areas includingthe locus coeruleus, hypothalamus and cerebral cortex. PD ischaracterized by resting tremors, rigidity, postural instabilityand bradykinesia with progression to dementia and auto-nomic dysfunctions. Although 5–10% of PD patients sufferfrom a genetic form of the disease (Polymeropoulos et al.1997; Kitada et al. 1998; Kruger et al. 1998) the majority ofPD cases, referred to as sporadic, are believed to be the resultof both genetic and environmental factors (for recent reviewsee Di Monte 2003). Studies on mechanisms underlying theloss of dopaminergic neurons in sporadic PD have shown acentral role for oxidative damage and disturbances in

mitochondrial function, particularly of complex I (reviewedin Greenamyre et al. 2001). Elevation in Fe2+ leading to the

Received November 9, 2004; revised manuscript received December 16,2004; accepted December 20, 2004.Address correspondence and reprint requests to Bruno Conti, Harold

L. Dorris Neurological Research Center, Department of Neuropharma-cology, The Scripps Research Institute, 10550 North Torrey Pines Road,SR307, La Jolla, CA 92037, USA. E-mail: [email protected] Bruno Conti and Shuei Sugama contributed equally to this work.Abbreviations used: BSA, bovine serum albumin; DNP, 2,4-dinitro-

phenylhydrazone; MPTP, 1,2,3,6-methyl-phenyl-tetrahydropyridine;PBS, phosphate-buffered saline; PD, Parkinson’s disease; ROS, reactiveoxygen species; SDS, sodium dodecyl sulfate; SN, substantia nigra;SNpc, substantia nigra pars compacta; TBARS, thiobarbituric acidreactive substances assay; TH, tyrosine hydroxylase; UCP, uncouplingprotein; VTA, ventral tegmental area.

Journal of Neurochemistry, 2005, 93, 493–501 doi:10.1111/j.1471-4159.2005.03052.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 493–501 493

generation of OH radicals and a decrease in glutathionelevels with consequent diminished H2O2 clearance is foundin post-mortem PD samples (for review see Olanow 1990,1993; Jenner and Olanow 1996). Therefore, reducingoxidative stress could represent a viable approach toprotecting dopaminergic neurons from degeneration in PD.

We hypothesized that one way to achieve this goal was toelevate the expression of uncoupling proteins (UCPs) indopaminergic cells. UCPs are inner mitochondrial membraneproteins that allow leakage of protons from the intermem-brane space to the matrix, thereby reducing the production offree radicals at Complex I of the respiratory chain anddissipating the proton gradient in the form of heat (Lowelland Spiegelman 2000; Nishikawa et al. 2000). Five membersof the UCP family have been identified in mammalian cells(Bouillaud et al. 2001). UCP4 and 5 are found at bothconstitutive and inducible levels in several brain regionsincluding the substantia nigra (SN) where the expression ofUCP5 has been demonstrated (Sanchis et al. 1998; Maoet al. 19993 ; Kim-Han et al. 2001). In contrast, UCP2 isexpressed at a very low level in the dopaminergic neurons ofthe SN and throughout the CNS, but is readily induced in aregion-specific fashion following epileptic- or kainic acid-induced seizures, and entorhinal cortex lesions (Horvathet al. 2003; Bechmann et al. 2002; Clavel et al. 2003; Dianoet al. 2003; Sullivan et al. 2003). UCP2 expression in thehippocampus was found to be inversely proportional toactivation of caspase 3, suggesting that UCP2 may serve asan endogenous neuroprotective agent against apoptotic celldeath in neurons. Thus, increasing the expression of UCPsin specific regions of the CNS may play an important role inthe prevention of oxidative stress-mediated tissue damagesuch as that occurring in PD. To test this hypothesis, wegenerated transgenic mice specifically expressing murineUCP2 in catecholaminergic neurons by placing murineUCP2 cDNA under the control of the tyrosine hydroxylasepromoter (TH-UCP2). TH-UCP2 overexpressing mice wereexposed to acute treatment with 1,2,3,6-methyl-phenyl-tetrahydropyridine (MPTP), a by-product of a syntheticmeperidine derivative that causes a syndrome clinically andpathologically similar to that observed in PD (Tipton andSinger 1993; Przedborski et al. 2004). This study demon-strates that mice overexpressing TH-UCP2 show a reductionof oxidative stress markers in dopaminergic neurons andexhibit a significantly lower susceptibility to MPTP-inducedneuronal damage.

Materials and methods

Animals and treatments

All procedures were approved by the Scripps Research Institute

Animal Care and Use Committee. Transgenic mice expressing

murine UCP2 in catecholaminergic neurons were generated by

placing mouse UCP2 cDNA under the control of the 9 kb rat

tyrosine hydroxylase promoter (TH-UCP2) (a gift from Dr Tong Joh

and Dr Jin Son, Weill Medical College of Cornell University)

previously shown to confer specific expression in catecholaminergic

cells (Min et al. 1996). Mice were generated following standard

procedures at the TSRI transgenic core facility on a C57Bl/6

background and backcrossed for six generations on the same

background before establishing a colony. Food and water were

provided at libitum, light/dark cycle was 12 : 12 h with lights on at

06.00 and off at 18.00. Tissues were harvested between 09.00 and

11.00. Six-month-old male TH-UCP2 mice and their wild-type

littermates were used throughout the study.

For MPTP treatment, 6-month-old male TH-UCP2 (n ¼ 4) and

wild-type mice (n ¼ 4) received four intraperitoneal injections of

phosphate-buffered saline (PBS) or MPTP (10 mg/kg), with a 2 h

interval between injections and were killed for tissue analysis

90 min, 24 h or 7 days after injection (Kurkowska-Jastrzebska et al.1999).

Striatal MPP+ levels were determined by HPLC as previously

described (Matthews et al. 1999).

In situ hybridization and immunohistochemistry

Animals were deeply anesthetized with sodium pentobarbital

(120 mg/kg) and perfused transcardially with saline containing

0.5% sodium nitrite and 1000 U/100 mL heparin sulfate, followed

by cold 4% formaldehyde generated from paraformaldehyde in

0.1 M PBS (pH 7.2). The brains were post-fixed in the same solution

for 1 h and infiltrated with 30% sucrose overnight. Free-floating

sections (40 lm) were obtained on a freezing microtome. In situhybridization was performed as previously described (Sugama et al.2002) using 35S-labeled sense and antisense UCP2 riboprobes. For

immunohistochemistry, sections were washed for 30 min in 0.1 M

PBS and permeabilized with 0.2% Triton X-100 in 0.1 M PBS

containing 1% bovine serum albumin (BSA). Sections were washed

in PBS containing 0.5% BSA for 30 min and incubated overnight at

4�C with antibodies against TH (1 : 1000; Calbiochem, San Diego,

CA, USA). Sections were washed in PBS–BSA and incubated for

1 h with biotinylated goat anti-rabbit IgG (Vector Laboratories,

Burlingame, CA, USA) at 1 : 200.

For double-labeling immunohistochemistry, sections were co-

incubated overnight in blocking solution containing commercially

available UCP2 (N-19, 1 : 500; Santa Cruz Biotechnologies, Santa

Cruz, CA, USA) and TH (1 : 500; Serotec, Kidlington, UK)

antibodies. Sections were then processed to develop UCP2

immunoreactivity by sequential incubations with a biotinylated

donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA,

USA) and avidin-biotinylated horseradish peroxidase complex

(ABC; Vector Laboratories). They were subsequently incubated in

1 : 100 fluorescein tyramide solution (TSA-direct) (NEN Life

Science Products, Perkin–Elmer Life Sciences, Boston, MA, USA).

For TH immunoreactivity, sections were further incubated for

45 min in Alexa Fluor 594-conjugated goat antirabbit IgG

(Molecular Probes, Inc., Eugene, OR). Sections were mounted

and analyzed by confocal microscopy. TH and UCP2 expression

levels were determined by recording the mean fluorescence per cell

using Adobe Photoshop’s histogram. The mean fluorescence in

green and red channels of 96 neurons from the substantia nigra was

analyzed through three sections per animal.

494 B. Conti et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 493–501

Real-time PCR

Quantitative PCR was performed on cDNA generated on RNA

extracted from substantia nigra/ventral tegmental area (SN/VTA)

dissected tissue using Roche LightCycler equipment and LightCy-

cler-Fast Start DNA Master SYBR Green I mix (Roche Molecular

Biochemicals, Indianapolis, IN, USA). Reactions were carried out

in a 10 lL volume using 0.5 lM primers and 2 mM MgCl2.The

sequences of the primers are as follows: for b-actin, 5¢-TACT-CCTGCTTGCTGATGC-3¢ and 5¢-CATGTACCCAGGCATTGCT-3¢; for UCP2, 5¢-GCTGGTGGTGGTCGGAGATA-3¢ and 5¢-ACA-GTTGACAATGGCATTACGG-3¢. PCR included an initial 10 min,

94�C step to activate Taq DNA polymerase, followed by 35–45

cycles of denaturation at 94�C, 10 s, annealing 58�C, 10 s, and

extension 72�C, 25 s. Standard curves were constructed using

purified and sequenced UCP2 and b-actin PCR fragments. Results

were analyzed by second derivative methods and were expressed in

arbitrary units normalized to the expression levels of the reference

gene, b-actin, quantified simultaneously with the target gene.

Mitochondrial uncoupling

Mitochondria were pooled from the SN/VTA (four animals per

group, total n ¼ 4 groups) and isolated by differential centrifuga-

tion. Briefly, the SN/VTAwas rapidly dissected and homogenized in

isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% fatty acid

free BSA, 20 mM Hepes, 1 mM EGTA, pH adjusted to 7.2 with

KOH). The homogenate was spun at 1300 g for 3 min, the

supernatant was removed and the pellet was resuspended in

isolation buffer and spun again at 1300 g for 3 min. The supernatant

from each sample was spun at 13 000 g for 10 min. After discarding

the supernatant, the pellet was washed with isolation buffer and spun

again at 13 000 g. Pellets were resuspended with isolation buffer

without EGTA and spun at 10 000 g for 10 min. The final

mitochondrial pellet was resuspended in 50 lL of isolation buffer

minus EGTA. Protein concentrations were determined with a BCA

protein assay kit (Pierce, Rockford, IL, USA).

Mitochondrial respiration was assessed using a Clark-type

oxygen electrode (Hansatech Instruments, King’s Lynn, UK) at

37�C with pyruvate and malate (5 and 2.5 mM) as oxidative

substrates in respiration buffer (215 mM mannitol, 75 mM sucrose,

0.1% fatty acid free BSA, 20 mM Hepes, 2 mM MgCl, 2.5 mM

KH2PO4, pH adjusted to 7.2 with KOH). Following the addition of

ADP and oligomycin, UCP-mediated proton conductance was

measured as increased fatty acid-induced respiration (Echtay et al.2002), which was then compared with state 4 respiration induced by

oligomycin, an inhibitor of H+ transporting ATP synthase. Typical

RCR values were � 4.

Thiobarbituric acid reactive substances assay

Tissue levels of lipid peroxidation were determined using the

thiobarbituric acid reactive substances (TBARS) assay (Keller

et al. 1998). Homogenates of dissected areas (1 : 10 w/v)

were prepared immediately before use in ice-cold aqueous

20 mM Tris–HCl buffer, pH 7.4 using a Teflon motor homogenizer.

Lipid peroxidation was stimulated in assays containing 100 lLhomogenate by addition of 10 lL of 100 lM Fe3+ (ammonium

ferric sulfate) solution, and the mixture was incubated for 30 min

at 37�C. The reaction was stopped by the addition of sodium

dodecyl sulfate (SDS; 100 lL of 8.1% w/v solution) and 750 lL

of 20% acetic acid (adjusted to pH 3.5 using NaOH). The

precipitated proteins were removed by centrifugation at 10 000 gfor 15 min. Aliquots (500 lL) of the clear supernatant were heated

in glass tubes capped with aluminum foil with an equal volume of

thiobarbituric acid solution (0.8% w/v, heated at 60�C to dissolve)

at 95�C for 30 min. Samples were cooled on ice and 100 lL of

each was pipetted into 96-well plates and the absorbance read at

532 nm using a Ceres UV 900C microplate reader. Blank values

representing samples containing 20 mM Tris–HCl were subtracted

from all readings. Each assay was performed in triplicate. Means

of triplicates from each assay were pooled to obtain final mean and

standard errors.

Protein carbonylation

Protein carbonyl levels in tissue samples were determined as

described by Sherer et al. (2003). Briefly, tissue samples were

homogenized in TPER Tissue Protein Extraction Reagent (Pierce)

supplemented with 1 mM dithiothreitol and protease inhibitors. The

homogenates were centrifuged for 10 min at 16 000 g4 at 4�C. Thesupernatant was collected and stored at )70�C. The protein content

of each sample was determined using the BCA Protein Assay kit

from Pierce with BSA as a standard.

Protein carbonyl levels were determined using dot blots and

the Oxyblot Protein Oxidation Detection Kit (Chemicon, Teme-

cula, CA, USA) according to the manufacturer’s protocol. In this

protocol, protein carbonyls are derivatized to 2,4-dinitrophenyl-

hydrazone (DNP) by reaction with 2,4-dinitrophenylhydrazine.

DNP-derivatized protein samples were analyzed using dot blots

and antibodies against DNP.

Determination of dopaminergic cell damage

Total numbers of TH-immunoreactive and Nissl-stained SNpc

neurons were counted in four mice per group by using stereology

as previously described (Coggeshall 1992; Gundersen 1992; Volpe

et al. 1998; Sugama et al. 2003). Non-neuronal cells were excludedby counting clearly defined nucleus, cytoplasm, and prominent

nucleolus by Nissl-staining. This procedure was carried out on three

to four sections at a periodicity of 160 lm in the SNpc. Average

neuron density was obtained by summing the number of neuron

profiles divided by the calculated volume. The total number of

neurons was calculated as the product of the neuron density and the

volume of SNpc as described previously (Coggeshall 1992;

Gundersen 1992; DeGiorgio et al. 1998; Volpe et al. 1998; Sugama

et al. 2003). With this procedure, the number of cells counted is not

affected by the volume of the SN or the size of the neurons. For the

quantification of TH, mean optical densities were obtained from

immunostained sections with an IBAS image analysis system as

previously described (Saji et al. 1996).

Testing gait abnormalities

Animals were tested for gait abnormalities, which are a frequently

reported effect of treatment with MPTP in mice (Sedelis et al.2001). Mice were trained to walk in a straight line on a plain white

strip of cardboard (9 · 35 cm). Following drug treatment, animals’

forepaws were dipped in green, non-toxic ink and their rear paws

were dipped in red, non-toxic ink. Mice were allowed to walk the

length of the strip, after which the distance between each stride was

measured (in cm), from heel to heel, and averaged for each animal.

Dopaminergic neurons and MPTP toxicity 495


Animals’ paws were gently cleaned following testing. Behavioral

analysis was conducted 7 days after the MPTP treatment.

Statistical analysis

Differences between groups for the various indices were compared

using standard statistical procedures (level of significance a¼ 0.05).

For direct comparisons between two groups, data were analyzed

using the Student’s t-test. For differences between three or more

groups, data were analyzed by ANOVA. When indicated by the ANOVA,

Fisher’s LSD post-hoc test was used to compare group differences.

Pearson’s correlational analysis was used to determine the associ-

ation between TH immunoreactivity and dopamine cell numbers.

Results

Characterization of TH-UCP2 mice

Three TH-UCP2 transgenic mice founders were generated.Two of the lines could not be maintained as they failed toreproduce. Thus, all experiments were carried out on oneline. The possibility that clonal effects could contribute to thephenotype described is unlikely, yet cannot be excluded.

Expression of UCP2 mRNA in TH-UCP2 mice wasassessed by in situ hybridization and real-time PCR. In situhybridization performed using sense and antisense mouseUCP2 radiolabeled riboprobes demonstrated specific UCP2mRNA expression in the locus coeruleus and the SN/VTA(Fig. 1). No visible hybridization signal was detected inwild-type littermates. Measurement of the level of UCP2mRNA determined by real-time PCR demonstrated thatTH-UCP2 mice express nearly twice as much UCP2 mRNAas their wild-type littermates (UCP/actin mRNA arbitraryunits were 0.24 ± 0.03 in wild-type and 0.52 ± 0.09 in TH-UCP2; n ¼ 3, p < 0.05 by ANOVA).

To analyze the expression of UCP2 in the SN weperformed double immunohistochemistry with anti-UCP2and anti-TH sera. Fluorescence immunohistochemistry dem-onstrated the localization of UCP2 in TH neurons of theSNpc (Fig. 2a,b). Quantification of UCP2 immunofluores-cence in these neurons demonstrated that UCP2 levels arenearly doubled in TH-UCP2 mice compared with their wild-type littermates (arbitrary units: 26 ± 1 in wild-type and63 ± 1.1 in TH-UCP2; n ¼ 3 animals per condition with 96cells analyzed across three sections per animal, p < 0.001)(Fig. 2c).

To assess the biological significance of UCP2 overexpres-sion in these mice, the UCP-mediated proton conductance ofmitochondria from the SN/VTA and striatum of TH-UCP2mice and their wild-type littermates was compared. Nochanges in state 4 respiration were observed prior to additionof palmitate. The percentage increase in oxygen consumptionfollowing addition of palmitate with respect to oligomycin-induced state 4 respiration was elevated 200% in mitochon-dria from TH-UCP2 mice SN/VTA compared with 50% inwild-type mice (n ¼ 4, p < 0.001) (Fig. 3).

Effets of MPTP neurotoxicity in TH-UCP2 mice

To determine if UCP2 overexpression conferred neuropro-tection against acute dopaminergic-specific oxidative dam-age, TH-UCP2 and wild-type mice were challenged with thedopaminergic neurotoxin, MPTP. Following the final injec-tion, mice were killed after 90 min to determine MPP+levels, and after 7 days to assess neuronal damage/neuro-protection.

TH-UCP2 and wild-type mice generated similar levels ofMPP+ (wild-type: 4.9 ± 1.1 ng/mg of protein; TH-UCP2:5.2 ± 1.3 ng/mg of protein; n ¼ 6, n.s.) indicating thatneuronal UCP2 does not influence the formation of theneurotoxin.

Neuroprotection was determined by evaluating neuronalcell loss via determination of the number of TH-positivedopaminergic neurons compared with the total number ofNissl-stained neurons in the SNpc and by determination ofthe intensity of TH immunostaining in the striatum. Effectsof MPTP exposure on locomotor activity were also deter-mined.

Data from cell counts in the SN were analyzed by a two-factor ANOVA with genotype and drug treatment as mainfactors. The ANOVA indicated a highly significant main effectof drug treatment (F1,7 ¼ 77.87, p < 0.0001), indicating thepotent effects of MPTP on its ability to decrease the number

rTH promoter (~9,000 bp)

(a’) (b’)

(a) (b)

mUCP2

HindIII EcoRI SmaI EcoRI

ATG(417)

125 1578 Nidel

(1346)

Xhol

Nidel

BGHpoly(A)

TGA

Fig. 1 (Upper) Schematic representation of the construct used to

generate TH-UCP2 mice. The location of the restriction sites used for

cloning, as well as the exact position in bp of the start, stop codon, the

5¢- and 3¢-end of mouse UCP2 cDNA are reported. (Lower) Bright-field

photoemulsion autoradiogram following in situ hybridization with

mouse UCP2 antisense riboprobe showing that TH-UCP2 mice

express UCP2 in the locus coeruleus (a and a¢) and the substantia

nigra and ventral tegmental area (b and b¢). Bar ¼ 50 lm in (a) and (b).

Bar ¼ 100 lm in (a¢). Bar ¼ 200 lm in (b¢).

496 B. Conti et al.


of dopaminergic neurons. There was also a significantinteraction of genotype · drug treatment (F1,7 ¼ 6.66,p < 0.05), revealing that treatment with MPTP did not affectboth genotypes equally. Post-hoc analysis of the data showedthat following treatment with MPTP, the TH-UCP2 miceexhibited significantly less cell loss (7476 ± 468, or 27.9%cell loss for TH-positive neurons; 12537 ± 655 or 6.4% lossfor Nissl-stained neurons) than their wild-type littermates

(5766 ± 551, or 44.6% cell loss for TH-positive neurons;9697 ± 971, or 29% loss for Nissl-stained neurons)(p < 0.005) (Fig. 4). There was no significant differencebetween the number of cells in wild-type mice treated withvehicle (10368 ± 33) and TH-UCP2 mice treated withvehicle (10410 ± 399) (p ¼ 0.33), indicating that bothgenotypes have similar numbers of dopaminergic neurons.

ANOVA of striatal TH immunostaining also showed asignificant drug treatment · genotype interaction (F1,7 ¼9.95, p < 0.05)Post-hoc analysis confirmed that there was nodifference between wild-type and TH-UCP2 mice aftertreatment with vehicle, but wild-type mice displayed asignificantly greater loss of TH immunoreactivity in thedorsolateral striatum (wild-type: 45.65 ± 13.2; MPTP:21.55 ± 0.54) than the TH-UCP2 mice (wild-type:47.5 ± 9.2; MPTP: 36.49 ± 5.69) (p ¼ 0.06) after adminis-tration of MPTP. Pearson’s correlational analysis indicated astrong correlation (r ¼ 0.904) between dopamine cellnumbers in the midbrain and TH immunoreactivity in thedorsolateral striatum. The statistical significance of this linkwas confirmed using simple linear regression (F1,9 ¼ 40.36,p < 0.001).

To determine the functional significance of neuroprotec-tion against MPTP with respect to motor control, which is areflection of the integrity of the dopaminergic system,TH-UCP2 and wild-type mice were tested for gait abnor-malities. ANOVA indicated a marginally significant interactionof drug · genotype (F1,10 ¼ 3.96, p ¼ 0.08). The resultsindicate that stride length was reduced by MPTP treatment inwild-type mice (vehicle: 7.7 ± 0.7 cm; MPTP:7.02 ± 0.09 cm) but not in TH-UCP2 mice (vehicle:

0

100

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wt tg

*

wt tg

*

% s

tate

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espi

ratio

n

Fig. 3 Overexpression of UCP2 results in elevated uncoupling activ-

ity. The free fatty acid palmitate increased mitochondrial uncoupling

activity in the SN/VTA of TH-UCP2 mice. Data are expressed as

percent increase in oxygen consumption/oligomycin-induced state 4

respiration (n ¼ 4, *p < 0.001).

UCP2 TH

merge

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(a)

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merge

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Fig. 2 (Upper) Immunohistochemistry showing UCP2 (green), TH

(red) and colocalization (yellow) of UCP2 and TH immunoreactivity in

the SNpc of wild-type (a) and TH-UCP2 mice (b). (Lower) (c) Densi-

tometric analysis of fluorescence intensities for TH (red) and UCP2

(green) were performed in 96 SNpc cells per animal on three wild-type

and three TH-UCP2 (tg) animals (*p < 0.001, ANOVA followed by

Tukey’s test) with respect to UCP2 in wild-type animals. (d) Histogram

showing the relative level of UCP2 mRNA normalized for b-actin

mRNA in the SN of wild-type and TH-UCP2 (tg) mice determined by

real-time PCR (n ¼ 3, *p < 0.05).

0

2000

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wt TH-UCP2 wt TH-UCP2PBS PBS Acute MPTP Acute MPTP

Fig. 455 Dopaminergic cell loss following MPTP treatment is reduced in

TH-UCP2 mice. (Upper) Representative pictures of TH immunohisto-

chemistry in the SN of wild-type and TH-UCP2 transgenic mice 7 days

after acute MPTP treatment. Bar is 200 lm. (Lower) (Left) histogram

of TH-positive cell numbers versus neurotoxicity, (right) histogram of

Nissl-stained SNpc neurons demonstrating that mice overexpressing

UCP2 in catecholaminergic cells are partially protected from MPTP-

induced neuronal loss (*p < 0.05). CV, cresyl violet.



7.8 ± 0.22 cm; MPTP: 8.02 ± 0.37 cm). The post-hoc testindicated a significant difference between wild-type and TH-UCP2 mice (p < 0.05) following treatment with MPTP,suggesting a functionally significant neuroprotective effect intransgenic mice.

Markers of oxidative stress

Lipid peroxidation and protein carbonylation were measuredas indexes of the level of free radical production in untreatedanimals and in mice killed 24 h after the last MPTP injection.The results demonstrated that in untreated animals,TH-UCP2 overexpressors have a 49 and 28% reduction inlipid peroxidation in the SN/VTA and striatum, respectively,compared with wild-type controls (Fig. 5). Lipid peroxida-tion in SN/VTA and striatum was significantly and compar-ably increased by MPTP treatment in both TH-UCP2 andwild-type littermates animals. No statistically significantdifference was observed in the TBARS between the MPTP-treated wild-type and transgenic strains (Fig. 5).

Similar results were obtained for protein carbonylation.The level of protein carbonylation was reduced in untreatedTH-UCP2 mice compared with their wild-type littermates in

both SN/VTA and striatum (wild-type striatum: 12.3 ± 0.35,TH-UCP2 striatum: 6.6 ± 1.2, n ¼ 4, p < 0.01; wild-typeSN/VTA: 18.8 ± 0.13, TH-UCP2 SN/VTA: 16.2 ± 1.3, n ¼4, p < 0.05).

However, the level of SN/VTA and striatal proteincarbonylation was elevated similarly following MPTP treat-ment in both strains (wild-type striatum: 13.4 ± 2.4,TH-UCP2 striatum: 11.7 ± 0.8, n ¼ 4, n.s.; wild-type SN/VTA: 26.8 ± 0.8, TH-UCP2 SN/VTA: 33.1 ± 2.8, n ¼ 4,n.s.) (Fig. 5).

Discussion

UCP2 was identified as a homolog of the brown fatuncoupling protein UCP1 (Fleury et al. 1997). The tissuedistribution of UCP2 examined by in situ hybridization andimmunohistochemistry showed that it is mostly expressed inwhite adipose tissue, spleen, lung and stomach (Vidal-Puig2000; Vidal-Puig et al. 2000). In the CNS, it was found inthe brainstem, thalamus, and hypothalamus (Richard et al.1998; Horvath et al. 1999a, b). A neuroprotective action ofUCP2 was postulated when its level in the CNS was found tobe up-regulated following injury and to be inverselyassociated with caspase 3 activation (Bechmann et al.2002). Recent studies in vitro and on transgenic miceexpressing both human UCP2 and UCP3 under their ownpromoters demonstrated that UCP2 overexpression preven-ted neuronal death following seizure (Diano et al. 2003),oxygen glucose deprivation and ischemic stroke (Mattiassonet al. 2003). It was proposed that elevation of UCP2 levelswould enhance proton translocation thus leading to mitoch-ondrial depolarization with consequent reduction of Ca2+

influx into mitochondria and reduced ROS formation. Ourfindings demonstrate that overexpression of mitochondrialUCP2 in catecholaminergic neurons reduced markers ofoxidative stress and provided partial neuroprotection againstMPTP cytotoxicity.

After administration, MPTP is converted by astrocyticmitochondrial monoamine oxidase type B (MAO-B) into thepyridinium ion (MPP+), which is specifically taken up bydopaminergic neurons via the dopamine-reuptake system,causing a syndrome clinically and pathologically similar toPD (Tipton and Singer 1993). Because MPTP-inducedneurotoxicity is ultimately mediated by MPP+, any mech-anism that would alter MPTP metabolism reducing MPP+formation could confer protection against MPTP. However,striatal levels of MPP+ did not differ in TH-UCP2 and theirwild-type littermates indicating that UCP2 overexpressiondid not influence MPP+ formation. The observation thatMPTP treatment induced a similar elevation in markers ofoxidative stress in the dopamine transporter-containingregions of SN/VTA and striatum is also an indication thatneither MPTP metabolism nor MPP+ uptake are altered byUCP2 overexpression.

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Fig. 566 Markers of oxidative stress. (Upper) Lipid peroxidation meas-

ured by TBARS assay. (Lower) Protein carbonylation. Both markers

are significantly lower in the SN/VTA and the striatum of the TH-UCP2

overexpressors (tg) compared to their wild-type littermates (wt).

Twenty-four hours of MPTP treatment induced a significant increase

(*p < 0.01) (except for striatum of wild-type mice) in lipid peroxidation

and protein carbonylation in both brain regions of both lines. (n ¼ 4;

*p < 0.01; **p < 0.05).

498 B. Conti et al.


Nerve cell death in the MPTP model appears to proceed bya multifactorial process involving both ROS generated byvesicular dopamine displacement and mitochondrial dys-function at complex I (Halliwell and Gutteridge 1985;Olanow 1990, 1993; Lotharius and O’Malley 2000; Nakam-ura et al. 2000).

The levels of lipid peroxidation and protein carbonylation,the two markers of reactive oxygen species (ROS) formationused in this study, were reduced in the SN and the striatum ofuntreated TH-UCP2 mice compared with wild-type litter-mates. These data are in agreement with the postulated roleof UCP2 in reducing mitochondrial production of ROS(Mattiasson et al. 2003). Consistent with what has beenreported by other groups (Rojas and Rios 1993; Zou et al.2000; Kaur et al. 2003) lipid peroxidation and proteincarbonylation were increased in the SN and striatum afterMPTP treatment. However, despite the neuroprotectiveaction of UCP2 overexpression in MPTP toxicity, we sawno changes in short-term indicators of oxidative stressfollowing MPTP treatment. Lipid peroxidation and proteincarbonylation are indicators of mitochondrial as well ascytoplasmic oxidative stress. It is thus possible that the ROSformation observed at 24 h is mainly due to the ROSgenerated by vesicular dopamine displacement (Lothariusand O’Malley 2000). Although it is also possible that UCP2could act at minimizing ROS by-products at different time,our data demonstrate that treatment that fails to prevent theearly accumulation of ROS, but rather specifically targets themitochondria, is neuroprotective.

Other studies with UCP2 (Mattiasson et al. 2003) haveshown that under conditions of stress, UCP2 overexpressioncan lead to limited mitochondrial membrane depolarizationthereby reducing the rate of both mitochondrial calciumuptake and ROS generation. Increases in mitochondrialcalcium uptake and ROS generation are tightly coupled andare associated with nerve cell death (Tan et al. 1998).Following MPTP treatment, neuronal death occurs slowlyover a period of 7 days. This suggests that mitochondrialROS generation and calcium uptake could also occur slowlybut eventually accumulate to a level sufficient to fatallydamage the cell. Thus, it is possible that by reducing the rateof mitochondrial calcium uptake and ROS generation, UCP2overexpression keeps the levels of mitochondrial ROS andcalcium within tolerable limits. Further study will test thesehypotheses.

Transgenic mice were generated using the 9 kb rat THpromoter previously shown to confer tissue specific basal andinducible expression in mice (Min et al. 1996). UCP2expression in the SN of TH-UCP2 mice was shown tocolocalize with TH immunoreactivity demonstrating the cell-specificity of the expression. Nevertheless, colocalizationwas not absolute, as some TH-positive neurons in the SN didnot show UCP2 expression. This suggests that although therat TH-promoter confers specific expression in mouse

catecholaminergic neurons, it may not drive expression inall cells of the SN. Alternatively, although the TH-UCP2construct lacked the ORF1 postulated to inhibit UCP2mRNA translation, the existence of post-transcriptionalcontrol of UCP2 in those cells cannot be excluded. NoTH-negative/UCP2-positive neurons were observed, indica-ting that the promoter utilized lacked ectopic expression. TH-UCP2 mice showed a twofold increase in the levels of UCP2mRNA and protein, which resulted in effective uncoupling,reduction in markers of oxidative stress and partial neuro-protection to acute MPTP treatment. Whether it is possiblefor these cells to sustain a higher degree of UCP2 expression,uncoupling and neuroprotection in vivo remains to bedetermined. Our data show that a twofold increase ofUCP2 expression did not compromise the energetic require-ment for catecholaminergic cell development and survival,yet proved sufficient to confer partial neuroprotection.

UCP2 has, to date, been investigated mainly in non-neuronal cell types (i.e. adipocytes, pancreatic b cells) whereits expression was found to be regulated at thetranscriptional, post-transcriptional and post-translationallevels (Bouillaud et al. 2001).

Our data indicate that the mechanisms regulating UCP2expression and activity are present in dopaminergic neuronswhere UCP2 transcription was followed by protein synthesisand activity. Thus, drugs stimulating UCP2 transcription,translation or activity could have therapeutic relevance inPD. Such an approach seems particularly attractive as ourstudy showed that only a twofold increase in UCP2 mRNAlevels was sufficient to confer neuroprotection. In addition,some of the pharmacological agents may already be avail-able. For instance, pioglitazone, a peroxisome proliferators-activated receptor c agonist shown to confer neuroprotectionagainst MPTP toxicity (Breidert et al. 2002) was shown toelevate UCP2 transcription in skeletal muscle (Shimokawaet al. 1998). Furthermore, coenzyme Q10, also shown toprotect against MPTP treatment in mice (Beal et al. 1998)and primates (Horvath et al. 2003), is an activator of UCP2(Echtay et al. 2000). Thus, UCP2 may represent a novel drugtarget for slowing the progression of PD.

Acknowledgment

This study was supported by Ellison Foundation.

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Uncoupling protein 2 protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity