1) Rats treated with 3-nitropropionic acid (3-NP), a model of Huntington's disease, exhibited weight loss, gait abnormalities, and striatal lesions. 2) The study found a dose-dependent reduction in complex-I activity in the cerebral cortex of 3-NP treated rats, as measured spectrophotometrically and by blue native-polyacrylamide gel electrophoresis. 3) Succinate driven State 3 respiration was significantly inhibited both in vivo and in isolated mitochondria from the cortex of 3-NP treated rats, suggesting complex-I dysfunction in addition to inhibition of complex-II and succinate dehydrogenase activity contributes to cortico-striatal lesions in this model

1. Mitochondrial NAD+
-linked State 3 respiration and complex-I
activity are compromised in the cerebral cortex of 3-nitropropionic
acid-induced rat model of Huntington’s disease
Mritunjay Pandey,*,
Merina Varghese,* Kizhakke M. Sindhu,* Sen Sreetama,*,
A. K. Navneet,* Kochupurackal P. Mohanakumar* and Rajamma Usha
*Laboratory of Clinical & Experimental Neuroscience, Division of Cell Biology & Physiology, Indian Institute of Chemical Biology,
Kolkata, India
Manovikas Biomedical Research and Diagnostic Centre, Kolkata, India
Huntington’s disease (HD) is an inherited progressive
neurodegenerative disorder, characterized by severe degen-
eration of the striatum and cerebral cortex, exhibiting motor
abnormalities, impaired cognitive functions and emotional
disturbances. Mitochondrial electron transport chain (ETC)
inhibition at complex-II (succinate dehydrogenase, EC
1.3.99.1; SDH) and complex-IV (cytochrome c oxidase,
EC 1.9.3.1) is reported in lymphoblasts (Sawa et al. 1999),
and in the brain samples of HD patients (Gu et al. 1996;
Browne et al. 1997). Increased level of lactic acid in cerebral
cortex of HD patients detected employing 1
H NMR or proton
magnetic resonance spectroscopy is yet another indication of
mitochondrial involvement in this disease (Jenkins et al.
1993; Harms et al. 1997). A suicidal inhibitor of SDH, 3-
nitropropionic acid (3-NP), when ingested from moldy
sugarcane, resulted in selective neuronal death and symptoms
similar to HD in humans (Ludolph et al. 1991). This led to
the widespread use of 3-NP as an animal model of HD,
which closely reproduced behavioral and neuropathological
features of the disease in rodents and primates (Beal et al.
1993). Although 3-NP-induced striatal and cortical cell death
Received April 11, 2007; revised manuscript received September 5,
2007; accepted September 8, 2007.
Address correspondence and reprint requests to Rajamma Usha,
Manovikas Biomedical Research and Diagnostic Centre, 482, Madudah,
Plot I-24, Sector-J, E. M. Bypass, Kolkata – 700 107, India.
E-mail: ushamvk@yahoo.co.in
Abbreviations used: 3-NP, 3-Nitropropionic acid; BN-PAGE, blue
native-polyacrylamide gel electrophoresis; BSA, bovine serum albumin;
DCIP, 2,6-dichlorophenol indophenol; DOPAC, 3,4-dihydroxyphenyl-
acetic acid; DTNB, 5,5¢-dithio-bis-nitrobenzoic acid; ETC, electron
transport chain; GFAP, glial fibrillary acidic protein; HD, Huntington’s
disease; HRP, horseradish peroxidase; HVA, homovanillic acid; MOPS,
3-(N-morpholino) propanesulphonic acid; NBT, nitroblue tetrazolium;
PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride;
RCR, respiratory control ratio; SDH, succinate dehydrogenase; TH,
tyrosine hydroxylase.
Abstract
Mitochondrial complex-I dysfunction has been observed in
patients of Huntington’s disease (HD). We assessed whether
such a defect is present in the 3-nitropropionic acid (3-NP)
model of HD. Rats treated with 3-NP (10–20 mg/kg i.p., for
4 days) exhibited weight loss, gait abnormalities, and striatal
lesions with increased glial fibrillary acidic protein immuno-
staining on fifth and ninth days, while increase in striatal
dopamine and loss of tyrosine hydroxylase immunoreactivity
were observed on fifth day following treatment. We report for
the first time a dose-dependent reduction in complex-I activity
in the cerebral cortex when analyzed spectrophotometrically
and by blue native-polyacrylamide gel electrophoresis follow-
ing 3-NP treatment. The citrate synthase normalized activities
of mitochondrial complex-I, -II, -(I + III) and -IV were de-
creased in the cortex of 3-NP treated rats. In addition, succi-
nate driven State 3 respiration was also significantly inhibited
in vivo and in the isolated mitochondria. These findings taken
together with the observation of a significant decrease in vivo
but not in vitro of State 3 respiration with NAD+
-linked sub-
strates, suggest complex-I dysfunction in addition to irre-
versible inhibition of complex-II and succinate dehydrogenase
activity as a contributing factor in 3-NP-induced cortico-striatal
lesion.
Keywords: BN-PAGE, cortico-striatal neurodegeneration,
electron transport chain, footprint analysis, GFAP, striatal
dopamine, succinate dehydrogenase.
J. Neurochem. (2008) 104, 420–434.
d JOURNAL OF NEUROCHEMISTRY | 2008 | 104 | 420–434 doi: 10.1111/j.1471-4159.2007.04996.x
420 Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors

2. has been investigated (La Fontaine et al. 2000; Galas et al.
2004), the exact pathways leading to selective neurodegen-
eration are elusive. It is known that chronic doses of 3-NP
cause oxidative stress (Schulz et al. 1996), excitotoxicity
(Kim et al. 2000), and apoptotic cell death in vitro (Pang and
Geddes 1997) and in vivo (Vis et al. 2001).
A deficit in the activity of mitochondrial NADH: ubiqui-
none oxidoreductase (EC 1.6.5.3; complex-I) has been
established in a number of neurodegenerative diseases, such
as idiopathic Parkinson’s disease (Mizuno et al. 1989;
Dawson and Dawson 2003), familial amyotrophic lateral
sclerosis (Jung et al. 2002; Rizzardini et al. 2006) and
Alzheimer’s dementia (Manczak et al. 2004). Complex-I
deficiency has also been reported in the animal (Mizuno
et al. 1988; Dabbeni-Sala et al. 2001) or in vitro models
(Casley et al. 2002) of some of these disorders. Unlike the
pattern in these neurodegenerative diseases, no change in
complex-I activity was found in the frontal or parietal
cortices, and in the cerebellum of HD brain samples, while a
significant reduction in the complex-II/III activity was
observed in the caudate and putamen (Browne et al. 1997).
However, two other clinical studies reported decreased
complex-I activity in platelets and muscle tissues of HD
patients (Parker et al. 1990; Arenas et al. 1998). In another
recent study, eleven subunits of mitochondrial complex-I
were found to have reduced expression in HD brain when
compared to age-matched controls (Weydt et al. 2006).
Clonal striatal cells with mutant huntingtin were found to
have lower State 3 respiration rates in the presence of NAD+
-
linked substrates when compared to wild-type (Milakovic
and Johnson 2005). Untreated HD patients have significantly
lower plasma coenzyme Q10 levels, which may be an
indication of complex-I defect in this disease (Andrich et al.
2004). Since comparatively low levels of complex-I inhibi-
tion (25% as against 70–80% of complex-III or -IV activity)
could bring about significant changes in oxidative phosphor-
ylation and reduced synthesis of ATP (Davey et al. 1998;
Brookes et al. 2002), we assessed the extent of the complex-I
dysfunction following severe SDH inhibition in a rat model
of HD. We also investigated mitochondrial respiration and
the enzyme activities of the mitochondrial ETC in the 3-NP
model of HD, and report here significant inhibition of the
complex-I activity and NAD+
-linked State 3 respiration.
Experimental procedures
Animals
Male Sprague–Dawley rats (20–24 weeks, with weights of 350–
400 g) used for the study were housed under standard conditions of
temperature (22 ± 1°C), humidity (60 ± 5%) and illumination (12 h
light/dark cycle). The experimental protocol met the National
CPCSEA Guidelines on the ‘Proper Care and Use of Animals in
Laboratory Research’ (Indian National Science Academy, New
Delhi, 2000) and was approved by the Animal Ethics Committee of
Indian Institute of Chemical Biology.
Materials
NADH, coenzyme Q0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone),
3-NP, rotenone, aminocaproic acid, catalase, dopamine (3,4-dihydr-
oxyphenylethylamine, DA) hydrochloride, 3,4-dihydroxyphenylace-
tic acid (DOPAC), homovanillic acid (HVA), leupeptin, 5,5¢-dithio-
bis-nitrobenzoic acid (DTNB), phenylmethylsulfonyl fluoride
(PMSF), 3-(N-morpholino) propanesulphonic acid (MOPS), sodium
deoxycholate, sodium orthovanadate, Nonidet P-40, Coomassie
brilliant blue R-250, tricine, 3,3¢-diaminobenzidine, bovine serum
albumin (BSA), EDTA, Triton X-100, pepstatin A, TRI reagent,
sephadex G-25, EGTA, heptane sulfonic acid and nitroblue
tetrazolium (NBT) were procured from Sigma (St Louis, MO,
USA). Chloral hydrate was obtained from Fluka, Germany. Acetyl
coenzyme A, cytochrome c, n-dodecyl-b-D-maltoside, mannitol and
dialyzed Ficoll were procured from MP Biomedicals (Aurora, OH,
USA). Avian myeloblastosis virus reverse transcriptase was pur-
chased from USB Corporation, Cleveland, OH, USA. Rabbit
tyrosine hydroxylase (TH) polyclonal antibody, biotinylated goat
anti-rabbit antibody and streptavidin–horseradish peroxidase (HRP)
conjugate were obtained from Chemicon (Temecula, CA, USA).
VDAC I, ND 4, COX-III, ND 5, glial fibrillary acidic protein
(GFAP) (all goat polyclonal antibodies), rabbit ND 6 polyclonal
antibody, and donkey anti-goat HRP antibody were purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Goat anti-
rabbit HRP antibody was purchased from Bangalore Genei,
Bangalore, India. Phenazonium methosulphate, 2,6-dichlorophenol
indophenol (DCIP), Coomassie brilliant blue G-250, tricine, bis-
Tris, oxaloacetate, ADP sodium salt, and 2,4-dinitrophenol were
supplied by Sisco Research Laboratories, Mumbai, India. Other
chemicals used were of analytical grade.
Drug treatment
Freshly prepared 3-NP in saline was adjusted to pH 7.4 using 5 mol/
L NaOH. Rats were treated in the mornings with 3-NP (10, 15,
20 mg/kg, i.p.) for 4 days and the control animals received saline
(pH 7.4) injections. Throughout the period of study, the animals
were monitored for changes in body weight and observed for any
alterations in general behavior. Footprint analyses were carried out
in animals on the fifth and ninth day of 3-NP treatment. For
biochemical experiments, the rats were killed at the end of fifth and
ninth days.
Footprint analyses
To quantify the gait abnormalities in 3-NP treated rats, we used the
method of Klapdor et al. (1997). Briefly, rats were made to walk on
an inclined gangway (100 cm · 12 cm · 10 cm with 30° inclina-
tion) leading to a darkened enclosure. The gangway was lined with
white paper and the fore- and hind-paws of the animals were dipped
in two different non-toxic watercolors to record the footprints. The
walking pattern was recorded twice for each animal, after which the
paints were washed off and the animals were toweled dry before
placing them back in the cage. The footprints were analyzed for
four parameters, viz., footprint length, stride length, stride width,
and toe spread (between the first and fifth or the second and fourth
digits).
Ó 2007 The Authors
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Complex-I inhibition in HD model | 421

3. Estimation of dopamine and its metabolites
Animals were killed on fifth or ninth day following 3-NP
administration (10, 15 and 20 mg/kg, i.p), the striata were
micropunched (Palkovits and Brownstein 1983) and processed for
the analyses of DA, DOPAC, and HVA employing an HPLC-
electrochemical procedure (Muralikrishnan and Mohanakumar
1998). The tissue was sonicated in ice-cold 0.1 mol/L HClO4
containing 0.01% EDTA, centrifuged at 10 000 g for 5 min and the
supernatant (10 lL) was injected into an HPLC system (Merck
Hitachi, Germany) equipped with LaChrome L-3500A amperomet-
ric detector (Merck) and C18 ion pair analytical column
(4.6 mm · 250 mm; Ultrasphere IP; Beckman, Fullerton, CA,
USA), with a particle size of 5 lm and pore of 80 A˚ . The flow
rate was 0.7 mL/min and electrochemical detection was performed
at 0.74 V. The composition of the mobile phase was 8.65 mmol/L
heptane sulfonic acid, 0.27 mmol/L EDTA, 13% acetonitrile, 0.43%
triethylamine and 0.32% phosphoric acid.
Histochemical analyses in brain sections
TH and GFAP immunohistochemical reactions were carried out as
reported earlier (Saravanan et al. 2005). Following transcardial
perfusion with 50 mL of cold 100 mmol/L potassium phosphate
buffer, pH 7.4, and 50 mL of 4% (w/v) paraformaldehyde, brains were
fixed overnight and cryoprotected in 30% (w/v) sucrose. Forty micron
thick sections passing through the striatum were taken using a
cryotome (Thermo Shandon, Pittsburgh, PA, USA). The sections were
collected on poly-L-lysine-coated slides for cresyl violet staining. For
immunostaining, free-floating sections in phosphate buffered saline
(PBS) were incubated with 1% H2O2 for 10 min and blocked using
8% BSA and 0.02% Triton X-100 in PBS. The sections were
subsequently incubated with primary antibody for 16 h at 4°C (anti-
rabbit TH polyclonal 1 : 1000, anti-goat polyclonal GFAP 1 : 100).
For TH immunostaining, sections were incubated with biotinylated
secondary antibody (goat anti-rabbit IgG 1 : 500) for 2 h, washed and
incubated with streptavidin–HRP complex for 30 min. For GFAP
analysis, HRP-conjugated secondary antibody (donkey anti-goat IgG
1 : 300) was used. After mounting, the sections were viewed under a
stereomicroscope (Zeiss, Germany) and photographed.
Succinate dehydrogenase histoenzymological analysis
Control and 3-NP (20 mg/kg) treated rats were used for SDH
histochemical activity following the method of Brouillet et al. (1998).
Animals were anesthetized using chloral hydrate (400 mg/kg i.p.) and
transcardially perfused with 40 mL of cold 0.1 mol/L PBS, pH 7.4,
followed by 120 mL of cold 10% (v/v) glycerol in PBS. Twenty
micron thick frozen sections of the brain were dried at 25°C for
30 min, activated in PBS at 37°C for 10 min and then incubated with
100 lL of a reaction mixture containing 0.3 mol/L NBT, 0.05 mol/L
phosphate buffer, pH 7.4, and 0.05 mol/L sodium succinate at 37°C in
dark for 30 min. At the end of the reaction, sections were extensively
washed with the reaction buffer, mounted in glycerin jelly, examined
under the stereomicroscope and photographed.
Preparation of mitochondria
Preparation of mitochondria for enzyme assays
Cerebral cortex was processed for the preparation of pure mitochon-
drial fractions employing Ficoll density gradient as described by Lai
and Clark (1979) with slight modifications. All the procedures were
carried out at 4°C. In brief, tissues were homogenized in 12.5 volumes
of cold isolation buffer (20 mmol/L potassium phosphate, 0.15 mol/L
KCl, pH 7.6) and centrifuged at 1300 g for 3 min. The pellets were
resuspended in half of the original volume of isolation buffer,
centrifuged as above and the supernatants from the two centrifuga-
tions were pooled and laid over 10% (w/v) cold Ficoll solution. The
samples were centrifuged in a swing-out rotor at 66 000 g for 40 min.
The mitochondria-rich pellets were washed in isolation buffer at
9800 g for 10 min and reconstituted in the same buffer. The
mitochondria were used as such for the complex-IV assay. The
samples were freeze-thawed once and sonicated on ice at low energy
for 5 s to obtain the submitochondrial particles for other assays.
Preparation of mitochondrial P2 fraction
Mitochondrial P2 fractions were prepared from striata of control and
3-NP (20 mg/kg) treated rats as described earlier (Thomas and
Mohanakumar 2004). Animals were killed by decapitation and the
left and right striata of individual animals were homogenized
together in 10 volumes of ice-cold buffer containing 0.32 mol/L
sucrose, in 10 mmol/L potassium phosphate (pH 7.2). The homog-
enate was centrifuged at 1000 g for 10 min at 4°C. The pellet was
discarded and the supernatant was centrifuged at 10 000 g for
30 min at 4°C and the resulting pellet was washed in cold 50 mmol/
L Tris–HCl, pH 7.2 (centrifuged at 10 000 g for 30 min at 4°C). The
pellet was resuspended in cold 10 mmol/L potassium phosphate
buffer, pH 7.2 and used for enzyme assays on the same day.
Preparation of mitochondria for oxygen consumption studies
Mitochondria were isolated from cerebral cortices of control and 3-
NP (20 mg/kg) treated rats as per Clark and Nicklas (1970) for
mitochondrial respiration studies. Individual rat cerebral cortices
were dissected out on ice and homogenized in 20 volumes of ice-
cold mitochondrial isolation buffer containing 225 mmol/L manni-
tol, 75 mmol/L sucrose, 5 mmol/L MOPS, 1 mmol/L EGTA and
1 mg/mL BSA, pH 7.4 (adjusted with KOH). The homogenate was
centrifuged at 1800 g for 4 min at 4°C and the supernatant was
centrifuged at 12 200 g for 8 min at 4°C. The resulting pellet was
resuspended in 2 mL of 3% (w/v) Ficoll in isolation buffer, carefully
layered over 6% (w/v) Ficoll and centrifuged at 12 200 g for
15 min. The brown pellet containing mitochondria was then
resuspended in isolation buffer to give 15–20 mg protein/mL and
used for mitochondrial oxygen consumption studies.
The methodology used for the isolation of mitochondria for
western blots was the same as that used for the mitochondrial
respiration except that BSA was not used during the isolation. For
immunoblotting of complex-I subunits the pellet was resuspended in
radioimmunoprecipitation assay buffer containing 50 mmol/L Tris–
HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L PMSF, 1 lg/mL
leupeptin, 1 lg/mL pepstatin, 1 mmol/L sodium orthovanadate. The
pellet was kept on ice for 30 min with intermittent vortex at every
5 min interval and centrifuged at 15 000 g for 10 min at 4°C. The
supernatant was aliquoted and kept at )70°C until analysis.
Estimation of succinate dehydrogenase activity
Succinate dehydrogenase activity was assayed following the method
of Ackrell et al. (1978). The submitochondrial particles were pre-
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
422 | M. Pandey et al.

4. incubated in 50 mmol/L potassium phosphate buffer, pH 7.5 for
5 min at 37°C to activate the enzyme. The activity was monitored
spectrophotometrically at 600 nm for 1 min in a reaction mixture
containing 50 mmol/L potassium phosphate buffer, pH 7.5,
40 mmol/L sodium succinate, 750 lmol/L NaN3, 290 lmol/L
phenazonium methosulphate, and 50 lmol/L DCIP. The specific
activity of the enzyme was expressed as nmol DCIP reduced/min/
mg protein (e600 = 19.1/mmol/L/cm).
Determination of complex-I activity
The assay for complex-I activity was modified from the procedure
described by Shults et al. (1995). The reaction was carried out
spectrophotometrically at 340 nm for 3 min at 37°C in a solu-
tion containing 40–50 lg of the submitochondrial particles,
35 mmol/L potassium phosphate buffer, pH 7.4, 2.65 mmol/L
NaN3, 1 mmol/L EDTA, 5 mmol/L MgCl2, 200 lmol/L NADH,
and 100 lmol/L coenzyme Q0. The assay was carried out in the
presence and absence of 5 lmol/L rotenone in order to derive the
rotenone sensitive complex-I activity, which was expressed as nmol
NADH oxidized/min/mg protein (e340 = 6.23/mmol/L/cm).
In vitro effect of 3-NP was tested in sub-mitochondrial fraction
prepared from control cortex to determine whether the inhibition of
complex-I activity is a direct effect of the toxin or not. We pre-
incubated the mitochondrial preparation (40–50 lg protein) for
30 min at 37°C with 3-NP (1–1000 lmol/L in the final volume of the
assay mixture) and assayed for complex-I activity as described above.
NADH-cytochrome c oxidoreductase (complex-I plus -III) assay
Complex-I plus -III activity was assayed as described by Trounce
et al. (1996) with minor modifications. The reaction mixture
consisted of 50 mmol/L potassium phosphate buffer, pH 7.4,
containing 1 mmol/L EDTA potassium salt, 20 mmol/L NaN3,
50 lmol/L cytochrome c, 5–10 lg sub-mitochondrial protein,
which was incubated at 30°C for 1 min, before starting the reaction
by the addition of 100 lmol/L NADH. The reduction of cytochrome
c was monitored as increase in absorbance at 550 nm for 3 min in
the presence and absence of 5 lmol/L rotenone. The rotenone-
sensitive reduction of cytochrome c was expressed as nmol
cytochrome c reduced/min/mg protein (e550 of cytochrome
c = 19.0/mmol/L/cm).
Determination of complex-IV activity
The activity of complex-IV was measured as per Birch-Machin and
Turnbull (2001) with slight modifications. Briefly, each 1 mL
reaction mixture containing 20 mmol/L potassium phosphate buffer,
pH 7.0, 0.45 mmol/L n-dodecyl-b-D-maltoside and 1–5 lg mito-
chondrial protein was incubated for 2 min at 30°C and the reaction
was initiated by the addition of 25 lmol/L reduced cytochrome c.
The oxidation of cytochrome c was monitored as the decrease in
absorbance at 550 nm and the activity was expressed as nmoles of
cytochrome c oxidized/min/mg protein (e550 = 29.0/mmol/L/cm).
Cytochrome c was reduced using ascorbate, purified by Sephadex
G-25 chromatography and the concentration of reduced cytochrome
c was estimated spectrophotometrically before each assay.
Assay of citrate synthase (EC 4. 1. 3. 7) activity
The activity of the mitochondrial matrix enzyme citrate synthase
was assayed essentially according to the method of Trounce et al.
(1996). For each 1 mL reaction, submitochondrial protein (0.4–
1 mg) was incubated at 30°C with the assay mixture containing
0.1 mol/L Tris–HCl buffer, pH 8.0, 0.2 mmol/L acetyl coenzyme A
and 0.1 mmol/L DTNB. The reaction was started by the addition of
1 mmol/L oxaloacetate and 0.2% (v/v) Triton X-100 and the
reduction of DTNB was monitored at 412 nm for 1 min. Activity
was expressed as nmol DTNB reduced/min/mg protein (e412 = 13.6/
mmol/L/cm).
The mitochondrial complex-I, SDH, complex-I + -III and com-
plex-IV activities were normalized by dividing them with citrate
synthase activities.
BN-PAGE analysis
The cerebral cortices were pooled separately from control and 3-NP
(20 mg/kg) treated rats on the fifth day and samples for blue native-
polyacrylamide gel electrophoresis (BN-PAGE) analysis were
prepared as described by Schagger (1996). Tissues were homoge-
nized in 25 volumes of sample buffer-A containing 0.44 mol/L
sucrose, 1 mmol/L EDTA, 0.2 mmol/L PMSF and 20 mmol/L
MOPS, pH 7.2 at 4°C. The homogenates were centrifuged at
20 000 g for 20 min at 4°C. The pellets were resuspended (4 lL/mg
tissue) in sample buffer-B (1 mol/L amino caproic acid, 50 mmol/L
bis-Tris HCl, 1 lg/mL leupeptin, 1 lg/mL pepstatin, 5 mmol/L
PMSF, pH 7.0 at 4°C) containing freshly prepared 10% (w/v) n-
dodecyl-b-D-maltoside (2 lL/mg tissue). The samples were centri-
fuged at 100 000 g in a swing-out rotor for 15 min at 4°C and the
supernatant was used to perform BN-PAGE according to the method
of Schagger (1995). Sample was mixed in the ratio of 20 : 1 with
5% Coomassie brilliant blue G-250 in 1 mol/L aminocaproic acid
and loaded in a 5–11% polyacrylamide gradient gel of 1.5 mm
thickness. The electrophoresis was carried out in a Vertical Mini-Gel
apparatus (Bangalore Genei, India) at 100 V for 6–8 h at 4°C using
blue cathode buffer (50 mmol/L tricine and 15 mmol/L bis-Tris–
HCl, pH 7.4 with 0.002% Coomassie brilliant blue G-250) and
50 mmol/L bis-Tris–HCl, pH 7.0 as the anode buffer.
The in-gel activities were performed as per Jung et al. (2000).
For determining complex-I activity, the gel was incubated in
0.1 mol/L Tris–HCl, 0.14 mmol/L NADH and 1 mg/mL NBT, pH
7.4 for 6 h. The specificity of complex-I activity bands was
confirmed by pre-incubating a separate set with 20 lmol/L rotenone
for 30 min before the addition of 0.05 mmol/L NADH and 1 mg/
mL NBT. For complex-IV activity analysis, the gel was incubated
for 4 h in 0.05 mol/L potassium phosphate buffer pH 7.4 containing
0.5 mg/mL 3,3¢-diaminobenzidine, 2 lg/mL catalase, 1 mg/mL
cytochrome c and 75 mg/mL sucrose. A portion of the gel was
stained with Coomassie brilliant blue R-250 for 6–8 h. The wet gels
were scanned and the intensity of the protein bands corresponding to
the enzyme activities was measured from the scanned photographs
using ImageMaster Analysis 1D version 4 (Amersham Pharmacia
Biotech, Uppsala, Sweden). For densitometric analysis, the activity
bands of complex-I and complex-IV were divided by corresponding
protein bands in the Coomassie stained gel.
Mitochondrial respiration
Oxygen consumption was carried out in an oxygraph respirometer
(Hansatech, UK). Mitochondria were suspended at 0.6–0.8 mg/mL
in 0.5 mL of reaction buffer containing 95 mmol/L KCl, 75 mmol/L
mannitol, 25 mmol/L sucrose, 5 mmol/L KH2PO4, 20 mmol/L Tris–
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 423

5. HCl, 1 mmol/L EGTA and 1 mg/mL BSA, pH 7.4 (adjusted with
KOH) at 30°C. Mitochondrial respiration studies were carried out in
the presence of FAD+-linked substrate (5 mmol/L succinate) with
13 lmol/L rotenone or NAD+
-linked substrates (5 mmol/L each of
glutamate and malate). BSA was not used for the reaction with the
FAD+-linked substrate as rotenone is known to bind BSA
nonspecifically. State 3 (in the presence of 25 mmol/L ADP) and
State 4 (in the absence of ADP) respiration rates were calculated
from the slopes (monitored 7 min per trace), and expressed as ng
atom oxygen consumed/min/mg protein (Estabrook 1967). Respi-
ratory control ratios (RCRs) were derived as the ratio of the State 3
to subsequent State 4 respiration rates. For the in vitro assays
mitochondria were incubated with 3-NP (100–1000 lmol/L) in the
respiration buffer for 5 min prior to addition of substrates.
RT-PCR analysis for ND5 and ND6 subunits of complex-I
Total RNA was isolated from pooled cortex of control and treated
animals using TRI reagent following the manufacturer’s protocol.
The quality of the preparation was assessed by visualizing the
integrity of 28S and 18S rRNA bands on a denaturing gel and the
RNAwas quantitated by monitoring its absorbance at 260 nm. Equal
amounts (1 lg) of total RNA from each sample were reverse
transcribed using avian myeloblastosis virus reverse transcriptase
following the manufacturer’s recommendation. PCR amplification of
cDNA from the above reaction (1.5 lL of 10 times diluted cDNA)
using specific primers for mitochondrial genes ND5, ND6, and 12S
rRNAwas carried out in a MJ Research minicycler (MJ Research Inc.
Watertown, MA, USA). The Primer 3 program (Rozen and Skaletsky
2000) available online at http://frodo.wi.mit.edu was used for primer
design and the sequence of the primers are given in Table 1.
Optimization was performed for all the primer sets to determine
the cycle number within the logarithmic phase of amplification.
Multiplex PCR was carried out separately using the cDNA for ND5
or ND6 subunits with 12S rRNA in each reaction as an endogenous
control. PCR conditions included an initial denaturation at 95°C for
10 min, followed by 30 cycles of amplification reaction with
denaturation at 95°C, annealing at 62°C and extension at 72°C for
30, 30, and 40 s, respectively with a final extension for 5 min at
72°C. Fifteen lL of the PCR product was electrophoresed on an
ethidium bromide-containing 2% agarose gel. Bands were visual-
ized using ChemiDoc XRS gel documentation system (Bio-Rad,
Hercules, CA, USA) and semi quantitative analysis of RT-PCR
signals was carried out by densitometry using Quantity One
software (Bio-Rad Version 4.6.0). Values of targets were normalized
to those of 12S rRNA.
Immunoblot
Nearly 50 lg protein samples were mixed with Laemmli buffer
(Laemmli 1970) and heat denatured by boiling it for 5 min. Samples
were loaded on a 12.5% sodium dodecyl sulfate acrylamide gel,
electrophoresed, transferred to polyvinylidene difluoride membrane
and blocked with 10% (w/v) non-fat dry milk in Tris-buffered saline
containing 0.1% Tween 20. The membranes were probed separately
with anti-goat polyclonal antibodies of ND4 (1 : 500), ND5
(1 : 500), VDAC1 (1 : 500), COX-III (1 : 500) and rabbit anti-
ND6 polyclonal antibody (1 : 500). The blots were washed with
Tris-buffered saline containing 0.1% Tween 20. The blots were then
incubated with donkey anti-goat HRP antibody except for ND6
where the secondary antibody used was goat anti-rabbit HRP. The
blots were developed with 3,3¢-diaminobenzidine containing H2O2
and a densitometry has been performed employing ImageMaster 1D
Elite.
Estimation of proteins
Protein was estimated as described by Lowry et al. (1951), using
BSA as the standard.
Statistics
Student’s t-test was used to determine the significance and values of
p £ 0.05 were considered significant. For the footprint analysis, we
performed one-way ANOVA followed by Dunnett test to determine
the significance.
Results
All the animals that received the higher two doses of 3-NP
exhibited splayed paws and movement incoordination
(wobbling gait) from the fourth day. These abnormalities
were apparent from the third day onwards in the animals that
received the highest dose. However, the animals treated with
10 mg/kg exhibited no apparent behavioral abnormalities. A
significant loss of body weight was observed from the third
day of 3-NP administration in the animals that received
20 mg/kg dose of the neurotoxin (Fig. 1a).
Gait analysis
Animals treated with 3-NP (10 and 20 mg/kg) and controls
were tested for gait abnormalities daily from day 0 through 9
and representative data from 0, 5th, and 9th days are
provided. During the treatment period, the animals were
tested 30 min after the injections. We observed no changes in
the gait parameters between the controls (that received saline
injections) and the ‘0’ day animals (that received no
injections). Out of four parameters that were analyzed, the
footprint length and the stride length were significantly
Table 1 The sequences of the primers
used for the amplification of ND5, ND6 and
12S rRNA
Target cDNAs Primers Sequence
ND6 subunit Forward primer 5¢-ATCCGGAAACTTGAGGGTCT-3¢
Reverse primer 5¢-CCAGCCACCACTATCATTCA-3¢
ND5 subunit Forward primer 5¢-ATTGCAGCCACAGGAAAATC-3¢
Reverse primer 5¢-TGGTGATTGCACCAAGACAT-3¢
12S rRNA Forward primer 5¢-CACGGGACTCAGCAGTGATA-3¢
Reverse primer 5¢-TACCGCCAAGTCCTTTGAGT-3¢
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
424 | M. Pandey et al.

6. affected in the animals that received 20 mg/kg dose, but not
for those with the lower dose as compared to the controls. The
stride length, the distance between two successive hind limb
prints (see Fig. 1c), was significantly decreased as compared
to the controls on the fifth and ninth days (Fig. 1d). The
footprint length, measured as the distance from the heel to the
tip of the third digit of the hind limb (see Fig. 1c), was
significantly increased in 3-NP-treated animals on both days
as compared to the control (Fig. 1e). However, the stride
width and toe spread remained unaffected for all the doses
that we studied. Interestingly, while footprints of the hind and
forelimbs were superimposed in the control animals (Fig. 1b)
and in the 10 mg/kg group, those of the animals in the 20 mg/
kg group were distinctly separated (Fig. 1c).
Ctrl
0
1.5
3
5
Days
Footprintlength(cm)
0
13
26
Stridelength(cm)
300
340
380
Weight(g)
9
*
*
10 mg/kg
0 1
Ctrl
10 mg/kg
20 mg/kg
2 3 4 5
Days
6 7 8
Footprint
length
Stride
length
9
*****
*
*
20 mg/kg
*
*
(a)
(d)
(e)
(b) (c)
Fig. 1 3-NP-induced changes in behavior were measured in rats
administered saline or 3-NP (10 or 20 mg/kg, i.p, once daily for
4 days). (a) Body weight (g) measured daily (0–9 days) is expressed
as mean ± SEM; closed triangle: control, open circle: 10 mg/kg 3-NP
and closed circle: 20 mg/kg 3-NP; *p £ 0.05 as compared to the
respective control (n = 9). (b–e) Foot print patterns were analyzed on
fifth and ninth days of treatment. A representative footprint pattern
obtained from (b) a control animal and (c) a 3-NP (20 mg/kg) treated
animal on day 5. The set of points, between which stride length and
footprint length are measured, have been marked out. (d) Stride length
and (e) footprint length measured in cm. Data represent mean ± SEM,
*p = 0.008 on fifth day and 0.007 on ninth day for the stride length and
p = 0.039 and 0.022 (Dunnett test) for the footprint length on the fifth
and ninth days respectively as compared to control (n = 9).
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 425

7. Effect of 3-NP on striatal dopamine and its metabolites
A significant dose-dependent increase in striatal DA levels
was observed on fifth day after 3-NP administration
(Fig. 2a). However, the striatal DA levels returned to control
levels on ninth day (Fig. 2a). We observed no change in the
level of striatal HVA (control value of 6.7 ± 0.50 pmol/mg
tissue) following 3-NP administration, whereas DOPAC level
(control value of 38.7 ± 1.14 pmol/mg) was significantly
decreased (20%, 13%, and 29% on the fifth day and 2, 21,
and 38% on the ninth day for 10, 15, and 20 mg/kg,
respectively). This is reflected in the DA turnover, which is
significantly decreased in animals treated with 3-NP as
compared to controls on fifth and ninth days (Fig. 2b).
Effect of 3-NP on histopathology
TH immunoreactivity was reduced on the fifth day in the
dorsolateral striatum of the animals treated with the 20 mg/
kg 3-NP dose and recovered by the ninth day (Fig. 3a–c).
However, we observed a comparatively higher staining for
TH in the rest of the striatum on the fifth day in the 3-NP
treated animals. The substantia nigra region showed no
change in the intensity of TH immunoreactivity in either
control or 3-NP treated rats (Data not shown).
Nissl staining also revealed severe lesions in the striatum
and the cortex of 20 mg/kg 3-NP-treated rats as compared to
controls on the fifth day (Fig. 3d and e), which were reduced
by the ninth day (Fig. 3f). A concomitant time-dependent
increase in the GFAP immunoreactivity was observed in the
lesioned area (Fig. 3g–i) after treatment, with more glial cell
activation on ninth day as compared to control animals.
Effect of 3-NP on SDH activity
Histoenzymological localization of SDH activity revealed a
significant decrease of the enzyme reaction in the striatum,
septal region, nucleus accumbens and the cortex in the 3-NP
(20 mg/kg)-treated animals on the fifth day (Fig. 3k) as
compared to the control (Fig. 3j). The activity recovered by
the ninth day (Fig. 3l). Biochemical assay for the SDH
activity, normalized to citrate synthase activity that remained
unaffected by the treatment, revealed a similar significant and
dose-dependent inhibition of the enzyme activity in the
purified mitochondrial fractions from the cortex of rats
treated with 3-NP. The inhibition was 33%, 63%, and 63%
on the fifth day and 39%, 44%, and 50% on the ninth day for
the 10, 15, and 20 mg/kg doses, respectively when compared
to control levels of 0.094 ± 0.004 and 0.111 ± 0.011 on fifth
and ninth days respectively (Fig. 4c). The inhibition was also
observed in mitochondrial P2 fractions prepared from the
striatum (data not shown).
Effects of 3-NP on the ETC enzyme activities
We observed no effect of 3-NP on complex-I activity in vitro
when the control mitochondrial preparation was pre-incu-
bated with the neurotoxin (Data not provided). However,
biochemical estimates and in-gel activity results showed a
significant inhibition of complex-I activity in the mitochon-
dria prepared from the cortex of animals treated with 3-NP. In
the biochemical assay, all the enzyme activities were
normalized to citrate synthase activity (control value:
555.15 ± 6.4 nmol DTNB reduced/min/mg protein), which
was unaffected by 3-NP treatment. Complex-I activity was
significantly decreased from the control value of
0.192 ± 0.007 by 27 and 61% on the fifth day for the 15
and 20 mg/kg 3-NP treated animals respectively. On the
ninth day, the enzyme activity showed a significant decrease
only for 20 mg/kg 3-NP (decreased by 35% from
0.19 ± 0.03 in controls; Fig. 4a). A significant recovery
was observed for the highest dose on the ninth day as
Ctrl
0
0.6
1.2
*
*
*
*
*
*
* *
10
3-NP (mg/kg)
DOPAC+HVA/DA
0
40
80(a)
(b)
DA(pmol/mg)
15 20
Ctrl 10
3-NP (mg/kg)
15 20
Day 5
Day 9
Fig. 2 Effect of 3-NP on striatal dopamine level and turnover was
assessed in control (ctrl) or 3-NP treated rats (10–20 mg/kg, i.p, once
daily for 4 days), killed on the fifth or ninth day of treatment. Striata
were micropunched and assayed for dopamine (DA), 3,4-dihydroxy-
phenyl acetic acid (DOPAC) and homovanillic acid (HVA) levels
employing HPLC-electrochemistry. DA turnover was calculated as the
ratio of (HVA + DOPAC):DA. (a) Striatal DA levels on fifth and ninth
day of treatment. (b) DA turnover in the striata of animals killed on fifth
and ninth day after 3-NP treatment. Control value of HVA and DOPAC
respectively were 6.7 ± 0.50 and 38.7 ± 1.14 pmol/mg tissue. Data
are mean ± SEM, *p £ 0.05 as compared to vehicle injected rats
(n = 6–7 in fifth day group; and n = 4 for the ninth day group).
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
426 | M. Pandey et al.

8. TH
Control
(a) (d) (g) (j)
(b) (e) (h) (k)
(c) (f) (i) (l)
3-NP (Day 5)
* *
3-NP (Day 9)
Cresyl violet GFAP SDH
Fig. 3 Brain sections from sham control (a,
d, g, j) or 3-NP (20 mg/kg) treated rats were
processed on the fifth day (b, e, h, k) or
ninth day (c, f, i, l) for (a, b, c) tyrosine
hydroxylase, (d, e, f) cresyl violet, (g, h, i)
glial fibrillary acidic protein and (j, k, l) suc-
cinate dehydrogenase activity. Represen-
tative sections from n = 4–5 animals are
shown at a magnification of 2.5 · . Stars
indicate the striatal lesions, the arrowheads
in e indicate the cortical lesion and those in
(f) and (i) point out the reactive gliosis in the
striatal lesion.
(a) (b)
(c) (d)
Fig. 4 Effect of 3-NP on mitochondrial
complex activities are measured in rats
administered saline (Ctrl) or 3-NP (10–
20 mg/kg, i.p, once daily for 4 days). Rats
were killed on the fifth or ninth day and pure
mitochondrial fractions prepared from the
cerebral cortex were used for the spectro-
photometric assay of the activity of (a)
rotenone sensitive complex-I, (b) rotenone-
sensitive complex-I + III, (c) succinate
dehydrogenase and (d) complex-IV. All the
activities are expressed as the ratio to the
citrate synthase (CS) activities. Data are
represented as mean ± SEM, *p £ 0.05 as
compared to control. #p £ 0.05 as com-
pared to the fifth day treated animal (n = 5–
6 in each group).
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 427

9. compared to the fifth day (Fig. 4a). Interestingly, in contrast
to mitochondria isolated from cortex, mitochondrial P2
fraction from the striatum of 3-NP (20 mg/kg) treated
animals showed no change in the complex-I activity on the
fifth or ninth day (data not shown). Complex-I + III activity
decreased by 46% and 48% for 15 mg/kg and 20 mg/kg
doses respectively from control value of 1 ± 0.07 on the fifth
day and by 62% for the highest dose from the control value
of 0.8625 ± 0.14 on the ninth day (Fig. 4b). Significant
decrease in the complex-IV activity was observed only for
the highest dose of 3-NP on fifth day (Fig. 4d; 40% from the
control value of 0.0753 ± 0.006).
The ratio of the pixel intensity of the complex-I activity
bands (Fig. 5a, lanes 3 and 4) when normalized to that of the
Coomassie-stained band representing complex-I (see Fig. 5a,
lanes 1 and 2) in the treated animals showed 29% reduction
as compared to that of the controls (Fig. 5b). Similarly, the
ratio of complex-IV activity (Fig. 5a, lanes 5 and 6) to the
protein band of complex-IV was reduced by 75% in the 3-NP
treated animals (Fig. 5b).
Effect of 3-NP on mRNA expression of complex-I subunits
RT-PCR analyses for the mitochondrial subunits ND5 and
ND6 from the cortex of control and 20 mg/kg 3-NP-treated
animals did not reveal any significant change in the
expression levels of the mRNA of these subunits for any
of the three doses (Fig. 6a and b).
Effect of 3-NP on mitochondrial respiration
State 3 respiration rate in presence of NAD+
-linked sub-
strates glutamate and malate on the fifth day was found to be
decreased by 31% in 3-NP-treated rats (20 mg/kg) as
Complex-I
Pixelintensity(AU)
3-NP
Ctrl50(b)
25
0
Complex-IV
*
*
(a)
Fig. 5 Saline (CON) and 3-NP treated (20 mg/kg) rats were killed and
the mitochondria from cerebral cortex were prepared for BN-PAGE.
(a) Representative BN-PAGE, showing protein staining (lanes 1 and 2),
activity staining for complex-I (lanes 3 and 4) and complex-IV (lanes 5
and 6). The positions based on activities of the various mitochondrial
complexes are indicated on the left. (b) Intensity of complex-I and -IV is
plotted as arbitrary units. The activity bands of complex-I (5a, lanes 3
and 4) were divided by the intensity of the protein bands of complex-I
(5a, lanes 1 and 2). Similarly, the activity bands of complex-IV (5a,
lanes 5 and 6) were divided by their protein bands (5a, lanes 1 and 2).
The experiment was done in triplicate using samples prepared from
three animals in each experiment. *p £ 0.05 as compared to control.
C
ontrol
10
m
g/kg
15
m
g/kg
20
m
g/kg
3-NP
12S rRNA
ND5
12S rRNA
ND5
ND6
Ctrl
0
0.8
1.6(b)
(a)
10 15
3-NP (mg/kg)
Pixelintensity(AU)
20
ND6
Fig. 6 Effect of 3-NP on expression of complex-I subunits was as-
sessed using total RNA prepared from the cerebral cortex of saline or
3-NP (10, 15 or 20 mg/kg, i.p. for 4 days) treated rats employing RT-
PCR for the assay of ND5 and ND6 subunits of complex-I and 12S
rRNA. (a) Representative gel showing the PCR products of cDNA
amplification for ND5 (upper panel), ND6 (lower panel) multiplexed
with 12S rRNA. (b) Levels of ND5 and ND6 normalized to 12S rRNA
represented as pixel intensities. The experiments were performed in
triplicate with 4 animals in each group.
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
428 | M. Pandey et al.

10. compared to the controls, and the RCR was found to be
decreased by 44% (Table 2; representative tracings in Fig. 7
show the respiration rate in presence of NAD+
-linked
substrates, glutamate and malate), while no significant
change was found in the State 4 respiration rates. There
was a significant decrease of 44% in the 2,4-dinitrophenol
uncoupled respiration in the 3-NP-treated rats when com-
pared to controls (Table 2). However, there was no effect on
the State 3 or 4 respiration with NAD+
-linked substrates by
3-NP in vitro.
State 3 respiration linked to FAD+
was significantly
inhibited in mitochondria isolated from 3-NP (20 mg/kg)-
treated rats (68%; Table 2). In contrast to the respiration with
NAD+
-linked substrates, the State 3 respiration rate measured
using FAD+
-linked substrate in mitochondria from normal
animals incubated with 3-NP in vitro showed a significant
decrease (State 3 respiration: 26% and 62%; RCR: 22% and
50% respectively for 100 lmol/L and 1 mmol/L of the toxin
as shown in Table 2).
Effect of 3-NP on complex-I and complex-IV subunits
Western blot analysis of complex-I subunits ND5, ND6, and
ND4 did not reveal any significant change in expression
because of 3-NP when compared to VDAC 1, which was
used as loading control (Fig. 8). However, complex-IV
subunit COX-III showed 31% decrease in its expression after
3-NP treatment (control 0.59 ± 0.05, treated 0.41 ± 0.05,
p £ 0.05, n = 12).
Table 2 Mitochondrial respiration in cortex with NAD+
-linked substrates glutamate and malate on fifth day after 3-NP (20 mg/kg) treatment
Parameter
In vivo In vitro
Control 3-NP Control 3-NP (100 lmol/L) 3-NP (1 mmol/L)
NAD+
-linked (glutamate/malate)
State 3 respiration 63.4 ± 4.4 44.0 ± 3.4* 53.0 ± 5.8 49.4 ± 1.7 54.7 ± 4.9
State 4 respiration 14.2 ± 1.3 17.5 ± 1.1 8.3 ± 1.2 10.3 ± 0.4 8.4 ± 1.6
RCR 4.5 ± 0.2 2.6 ± 0.3* 6.5 ± 0.4 4.8 ± 0.1 6.8 ± 0.9
DNP uncoupled respiration 93.6 ± 15.8 52.5 ± 4.3* 70.0 ± 13.3 63.2 ± 2.4 66.0 ± 1.1
FAD+
-linked (succinate)
State 3 respiration 63.8 ± 4.9 20.1 ± 2.1* 80.3 ± 5.8 58.7 ± 2* 30.4 ± 2*
State 4 respiration 17.0 ± 1.2 – 25.9 ± 2.7 24.2 ± 1.4 20.1 ± 2.4
RCR 3.4 ± 0.1 – 3.1 ± 0.1 2.4 ± 0.1 1.5 ± 0.1*
State 3 respiration is the ADP stimulated respiration, State 4 respiration is basal respiration and respiratory control ratio (RCR) is the ratio of State 3
respiration to State 4 respiration. State 3 and State 4 respiration rates are expressed as ng atom O/min/mg protein. Data are represented as
mean ± SEM, *p £ 0.05 as compared to control (n = 5).
Fig. 7 Effect of 3-NP on mitochondrial respiration. Representative
polarographic traces of oxygen consumption in mitochondria isolated
from rat brain cerebral cortex (final concentration of 0.6–0.8 mg pro-
tein/mL) of control animals (a) and from 3-NP (20 mg/kg) treated rats
on the fifth day (b) at 30°C in presence of NAD+
-linked substrates
glutamate and malate are provided. State 3 respiration was stimulated
by addition of 125 lmol/L ADP (final concentration) and was followed
by State 4 respiration when all the added ADP was phosphorylated. To
uncouple mitochondria, 2,4-dinitrophenol (DNP, 50lmol/L) was ad-
ded. The points of addition of mitochondria (M), ADP or DNP are
indicated by arrowheads.
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 429

11. Discussion
In this study, we have investigated whether the ETC
dysfunction found in patients of HD is also present in the
3-NP model of the disease in rats. A substantial decrease in
the activity of mitochondrial ETC at the level of complex-I in
the cerebral cortex of an animal model of HD has been
demonstrated for the first time in the present study. This is
also the first report on a significant decline in the cortical
mitochondrial respiration in the presence of NAD+
-linked
substrates in the 3-NP model. Taken together it may be
suggested that mitochondrial complex-I dysfunction possibly
contributes to the pathology of HD, which is known to
involve complex-II deficiency.
The consistent increase observed in footprint length and
significant decrease in stride length in 3-NP-treated animals
established gait abnormalities in this model. A visible loss of
stride length observed in the present study is in agreement
with an earlier report demonstrating similar gait abnormality
in the 3-NP model of HD in rat (Teunissen et al. 2001).
Striatal DA level may not be an accurate measure for an
animal model of HD, since reports on the striatal content of
this biogenic amine in HD or in animal models are
contradictory. In human post-mortem striatum, a loss (Kish
et al. 1987) or an increase (Spokes 1980) in DA content is
reported in HD. Beal et al. (1993) reported a significant
decrease following sub-acute intrastriatal infusion, but no
effect after chronic subcutaneous injections (5 days contin-
uous infusion at 20 mg/kg/day) of 3-NP on striatal DA level.
A significant, dose-dependent increase in the striatal DA
levels following chronic doses of 3-NP was observed in the
present study. Acute injection of 3-NP has been shown to
cause an increase in extraneuronal DA in the striatum (Fu
et al. 1995; Nishino et al. 1997). The increased DA levels
could be due to a decrease in its catabolism through the
mitochondrial enzyme monoamine oxidase-B since the
metabolite DOPAC was significantly decreased, which is
reflected in the decreased DA turnover. An increase in the
TH immunoreactivity observed in the areas of the striatum
other than the lesion site also supports the notion that DA
synthesis could be increased in this nucleus. DA has been
shown to modulate striatal cell death (Reynolds et al. 1998)
and the copious increases in the striatal DA level could be
one of the factors leading to striatal neurodegeneration in the
3-NP model of HD. While Nishino et al. (1997) reported no
damage in the neurons of the striatum and increased levels of
DA following multiple treatments with 3-NP, loss of
dopaminergic terminals, but not the perikarya, and decrease
in TH immunoreactivity in the striatum has been shown by
Blum et al. (2004). We found a loss of TH in the dorsolateral
striatum on the fifth day following 3-NP treatment, which
recovered by the ninth day in parallel with the striatal DA
levels. The dorso-lateral lesions in the striatum and adjoining
areas in the cortex, observed by Nissl staining following 3-
NP administration, indicate loss of cortical and striatal
neurons. Cortico-striatal neurons have been shown to play an
important role in 3-NP model of HD as decortication of rats
prevented the striatal cell death as a result of 3-NP (Beal
et al. 1993). The observed behavioral abnormalities, striatal
DA content and various histopathological parameters con-
form to the features reminiscent of HD, and validate the
model employed in the present study.
Succinate dehydrogenase histochemistry and spectropho-
tometric analyses after 3-NP treatment revealed a decrease of
the enzyme activity on fifth day with recovery by ninth day.
Brouillet et al. (1998) reported a recovery in the enzyme
activity (22–27%) at 48 h as compared to 6 h after a single
dose of 3-NP. Bizat et al. (2003) have shown that chronic
doses of 3-NP lead to 60–70% of SDH inhibition in cortex
within 3–5 days whereas acute doses lead to 40% inhibition
of SDH within 6 h, which decreased to 20% by 12 h after 3-
NP treatment. We found that SDH activity on fifth day was
decreased by 60% following 3-NP administration (Fig. 4c),
which is at par with an existing report (Brouillet et al. 1998)
and is reminiscent of that seen in HD (Gu et al. 1996). The
reversal of the activity decline may be due to clearance of 3-
NP through plasma and urine (Majak and McDiarmid 1990).
Moreover, 3-NP does not affect the SDH mRNA expression
(Page et al. 1998) suggesting that the steady rate of SDH
synthesis leads to a recovery of activity. We did not find any
preferential decrease of SDH activity in the lesion area in the
histochemical assay, which largely confirms earlier observa-
tions by Gould et al. (1985). GFAP immunoreactivity in the
lesion area increased progressively from fifth to ninth day
Fig. 8 Effect of 3-NP (20 mg/kg i.p., for 4 days and analyzed on fifth
day) on the expression of complex-I and complex-IV protein subunits
was assessed by western blotting. Fifty lg of protein was loaded in
each lane, electrophoresed and transferred to a polyvinylidene diflu-
oride membrane. Membranes were probed with antibodies for com-
plex-I subunits ND4, ND5, ND6 and complex-IV subunit COX-III. As a
loading control VDAC 1 was used. Mitochondria were prepared in two
batches from the cerebral cortex of 6 animals in each group and
representative blots from duplicate runs of each preparation are pre-
sented.
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
430 | M. Pandey et al.

12. suggesting an increase in reactive gliosis. Increased GFAP
expression has been consistently found in the 3-NP model of
HD (Teunissen et al. 2001; Vis et al. 2001) as well as in
post-mortem HD brains (Selkoe et al. 1982). Increase in
reactive astrocytes at the site of injury is generally correlated
with neurodegeneration and reactive changes at the site of
injury.
The concept of mitochondrial involvement in HD has been
strengthened in recent years through accumulation of
unequivocal evidences from clinical findings and experi-
mental models of the disease. Existence of significant decline
in mitochondrial respiration and oxidative phosphorylation in
post-mortem HD brains and ATP depletion in muscle tissue
of living patients are reported (Koroshetz et al. 1997;
Schapira 1999; Tabrizi et al. 1999; Lodi et al. 2000).
Oxidative damage to mitochondrial DNA has also been
demonstrated in parietal cortex of HD brains (Polidori et al.
1999). Reductions in the activities of complex-II, -II/III, -IV
and aconitase (Shear et al. 1998; Tabrizi et al. 2000; Bizat
et al. 2003) leading to ATP depletion (Ludolph et al. 1991;
Alexi et al. 1998) have been reported in animal models of the
disease. Methylmalonate, a competitive inhibitor of SDH has
been shown to cause decrease in ETC complex activities
including complexes-I, -I + III, and -II + III ex vivo in
cortical homogenates and mitochondrial preparations from
rat brain (Brusque et al. 2002). Treatment with the SDH
inhibitor 3-NP has been shown to cause reactive oxygen
species generation (Schulz et al. 1996; Garcia et al. 2005),
mitochondrial membrane potential depolarization (Nasr et al.
2003; Garcia et al. 2005) and cytochrome c release from the
mitochondria (Antonawich et al. 2002). In agreement with
our findings (Fig. 4d), a decrease in complex-IV activity in
the rat brain was observed following intrastriatal injection
(Shear et al. 1998) or systemic administration of 3-NP (Page
et al. 1998). The latter study also demonstrated a decrease in
mRNA expression of COX-II and COX-IV subunits of the
complex-IV in cortex and striatum. We show here that
expression of COX-III subunit of the complex-IV is signif-
icantly decreased following 3-NP administration. Another
interesting outcome of our study was the decline in complex-
I + III activity, in addition to complex-I, which is of
importance as the electron transport to complex-III in rats
requires predominantly coenzyme Q9 which has been shown
to decline because of 3-NP (Matthews et al.1998).
However, involvement of mitochondrial complex-I of
ETC in 3-NP model of HD has not been reported till date.
Although lack of complex-I defect in the caudate and
putamen areas of HD post-mortem brain has been reported
(Browne et al. 1997), there are two reports that indicate
complex-I inhibition in platelets (Parker et al. 1990) and
muscle tissue (Arenas et al. 1998) of patients. Our study
clearly demonstrates significant dysfunction of the mito-
chondrial ETC at complex-I, -II, -I + III and -IV in the 3-NP
model of the disease. These findings indicate that in HD there
may be an involvement of complex-I defect in addition to
other mitochondrial ETC markers, especially complex-II. In
support of the significant decrease in complex-I activity
(Fig. 4a) following specific inhibition of complex-II by 3-NP,
we found a significant decrease in State 3 respiration (Fig. 7)
with NAD+
-linked substrates glutamate and malate. This
defect is also reflected in the 2,4-dinitrophenol uncoupled
respiration as shown in the present study. Kasparova et al.
(2006) have shown a decline in State 3 respiration in the
presence of glutamate in animals treated with 3-NP and
coenzyme Q10. Our results suggest that a decline in State 3
respiration with NAD+
-linked substrates and a significant
decline in RCR can lead to an energy deficit in mitochondria
which could be a possible cause of cell death in 3-NP model
of HD.
While Lee et al. (2005) have found significant decreases
in the mRNA levels of two of the mitochondrially encoded
complex-I subunits ND5 and ND6 in R6/2 transgenic mouse
model of HD, we could not find any significant changes in
the expression of these subunits after 3-NP either at mRNA
or protein level, which suggests that changes in other
subunits of complex-I or other mechanisms may lead to the
decrease in complex-I activity. Consistent with our finding of
decreased complex-I activity in the animal model, albeit by a
different mechanism, Weydt et al. (2006) recently reported
significant reductions in several nuclear DNA-encoded
complex-I subunits in HD brains compared to age-matched
controls when analyzed by microarray as well as real time
PCR assays. The contradiction of these findings with that of
Browne et al. (1997), where no significant functional
changes in complex-I were found in HD post-mortem brain
samples might arise due to at least two reasons. (i) The
reduced expression found by Weydt et al., reflected as a
complex-I functional deficit in the current study, may be a
feature of early stages of HD, which recovers with progress
of the disease and is undetectable at the later stages. It is
worth noting here that Browne et al. have used grade 3 to 4
HD brains for their study, while the group of Weydt has used
HD brains of 0 to 2 grade. (ii) Post-mortem changes or delay
in brain freezing might mask differences, if any, between
complex-I activities in control and HD brains without
affecting the mRNA levels.
Mitochondrial ETC inhibitors when administered chron-
ically for developing animal models of neurodegenerative
diseases exhibit secondary effects, which may have relevance
to neuronal cell death. A well known pesticide and an
inhibitor of mitochondrial complex-I, rotenone has been used
to create animal models of Parkinson’s disease (Betarbet
et al. 2000; Saravanan et al. 2005) as it reproduces patho-
logic features of the disease seen in humans. Rotenone, when
administered systemically has been found to inhibit mito-
chondrial complex-I as well as complex-II to the same extent
in rat brains (Panov et al. 2005). Our finding also indicates
that a functional decline at one of the entry points for
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 431

13. electrons in the ETC can affect the other. Bautista et al.
(2000) have shown a decline in activity of complex-I due to
its oxidation by hydroxyl radicals in vitro. Our finding of
complex-I inhibition may be an effect of hydroxyl radicals
generated due to 3-NP mediated SDH inhibition, since
complex-I (Saravanan et al. 2006, 2007) or complex-II
(Schulz et al. 1996; Wyttenbach et al. 2002; Perez-Severiano
et al. 2004; Garcia et al. 2005) inhibition has been shown to
generate reactive oxygen species. Complex-I activity has also
been shown to be decreased by S-nitrosation of some of its
subunits (Burwell et al. 2006), nitrotyrosine, peroxynitrite
(Riobo et al. 2001; Murray et al. 2003), and by a product of
lipid peroxidation, 4-hydroxy-2-nonenal (Picklo et al. 1999).
Another factor that passively supports our finding of
decreased activity of complex-I of the mitochondrial ETC
is the discovery that treatment of HD patients with coenzyme
Q10 decreased their cortical lactate concentration (Koroshetz
et al. 1997). Since decline of coenzyme Q10 in the plasma of
HD patients (Andrich et al. 2004), as well as loss of
coenzyme Q9 in 3-NP animal model of HD (Matthews et al.
1998) is known, it is reasonable to suggest that complex-I
could also be inhibited along with complex-II, where the
same cofactor is the electron acceptor. Further investigation
is underway to determine the factors responsible for com-
plex-I decline in 3-NP model of HD. Our observations
warrant further studies to determine the mechanism of
decrease of complex-I activity and its implications thereafter
in 3-NP model of HD.
Acknowledgments
The financial support from Department of Science and Technology,
Govt. of India vide Grant No. SR/FTP/LS-A-61/2001 (to UR) is
thankfully acknowledged. MP, MV and KMS are recipients of
Senior Research Fellowships from the Council of Scientific and
Industrial Research, Govt. of India and SS is in receipt of Senior
Research Fellowship from the University Grants Commission, India.
We are grateful to Mr. S. Guhathakurta for helping in PCR
experiment.
References
Ackrell B. A., Kearney E. B. and Singer T. P. (1978) Mammalian suc-
cinate dehydrogenase. Methods Enzymol. 53, 466–483.
Alexi T., Hughes P. E., Faull R. L. and Williams C. E. (1998) 3-Nitro-
propionic acid’s lethal triplet: cooperative pathways of neurode-
generation. Neuroreport 9, 57–64.
Andrich J., Saft C., Gerlach M., Schneider B., Arz A., Kuhn W. and
Muller T. (2004) Coenzyme Q10 serum levels in Huntington’s
disease. J. Neural Transm. Suppl. 68, 111–116.
Antonawich F. J., Fiore-Marasa S. M. and Parker C. P. (2002) Modu-
lation of apoptotic regulatory proteins and early activation of
cytochrome c following systemic 3-nitropropionic acid adminis-
tration. Brain Res. Bull. 57, 647–649.
Arenas J., Campos Y., Ribacoba R., Martin M. A., Rubio J. C., Ablanedo
P. and Cabello A. (1998) Complex-I defect in muscle from patients
with Huntington’s disease. Ann. Neurol. 43, 397–400.
Bautista J., Corpas R., Ramos R., Cremades O., Gutierrez J. F. and
Alegre S. (2000) Brain mitochondrial complex-I inactivation by
oxidative modification. Biochem. Biophys. Res. Commun. 27, 890–
894.
Beal M. F., Brouillet E., Jenkins B. G., Ferrante R. J., Kowall N. W.,
Miller J. M., Storey E., Srivastava R., Rosen B. R. and Hyman
B. T. (1993) Neurochemical and histologic characterization of
striatal excitotoxic lesions produced by the mitochondrial toxin
3-nitropropionic acid. J. Neurosci. 13, 4181–4192.
Betarbet R., Sherer T. B., MacKenzie G., Garcia-Osuna M., Panov A. V.
and Greenamyre J. T. (2000) Chronic systemic pesticide exposure
reproduces features of Parkinson’s disease. Nat. Neurosci. 3, 1301–
1306.
Birch-Machin M. A. and Turnbull D. M. (2001) Assaying mitochondrial
respiratory complex activity in mitochondria isolated from human
cells and tissues. Methods Cell Biol. 65, 97–117.
Bizat N., Hermel J. M., Boyer F., Jacquard C., Creminon C., Ouary S.,
Escartin C., Hantraye P., Kajewski S. and Brouillet E. (2003)
Calpain is a major cell death effector in selective striatal degen-
eration induced in vivo by 3-nitropropionate: implications for
Huntington’s disease. J. Neurosci. 23, 5020–5030.
Blum D., Galas M. C., Cuvelier L. and Schiffmann S. N. (2004) Chronic
intoxication with 3-nitropropionic acid in rats induces the loss of
striatal dopamine terminals without affecting nigral cell viability.
Neurosci. Lett. 354, 234–238.
Brookes P. S., Shiva S., Patel R. P. and Darley-Usmar V. M. (2002)
Measurement of mitochondrial respiratory thresholds and the con-
trol of respiration by nitric oxide. Methods Enzymol. 359, 305–319.
Brouillet E., Guyot M. C., Mittoux V., Altairac S., Conde F., Palfi S. and
Hantraye P. (1998) Partial inhibition of brain succinate dehydro-
genase by 3-nitropropionic acid is sufficient to initiate striatal
degeneration in rat. J. Neurochem. 70, 794–805.
Browne S. E., Bowling A. C., MacGarvey U., Baik M. J., Berger S. C.,
Muqit M. M., Bird E. D. and Beal M. F. (1997) Oxidative damage
and metabolic dysfunction in Huntington’s disease: selective vul-
nerability of the basal ganglia. Ann. Neurol. 41, 646–653.
Brusque A. M., Borba Rosa R., Schuck P. F. et al. (2002) Inhibition of
the mitochondrial respiratory chain complex activities in rat cere-
bral cortex by methylmalonic acid. Neurochem. Int. 40, 593–601.
Burwell L. S., Nadtochiy S. M., Tompkins A. J., Young S. and Brookes
P. S. (2006) Direct evidence for S-nitrosation of mitochondrial
complex-I. Biochem. J. 15, 627–634.
Casley C. S., Land J. M., Sharpe M. A., Clark J. B., Duchen M. R. and
Canevari L. (2002) Beta-amyloid fragment 25-35 causes mito-
chondrial dysfunction in primary cortical neurons. Neurobiol. Dis.
10, 258–267.
Clark J. B. and Nicklas W. J. (1970) Metabolism of rat brain mito-
chondria: preparation and characterization. J. Biol. Chem. 245,
4724–4731.
Dabbeni-Sala F., Di Santo S., Franceschini D., Skaper S. D. and Giusti P.
(2001) Melatonin protects against 6-OHDA-induced neurotoxicity
in rats: a role for mitochondrial complex I activity. FASEB J. 15,
164–170.
Davey G. P., Peuchen S. and Clark J. B. (1998) Energy thresholds in
brain mitochondria. Potential involvement in neurodegeneration.
J. Biol. Chem. 273, 12753–12757.
Dawson T. M. and Dawson V. L. (2003) Molecular pathways of neu-
rodegeneration in Parkinson’s disease. Science 302, 819–822.
Estabrook R. W. (1967) Mitochondrial respiratory control and polaro-
graphic measurement of ADP: O ratios. Methods Enzymol. 10,
41–47.
Fu Y., He F., Zhang S., Huang J., Zhang J. and Jiao X. (1995) 3-
Nitropropionic acid produces indirect excitotoxic damage to rat
striatum. Neurotoxicol. Teratol. 17, 333–339.
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
432 | M. Pandey et al.

14. Galas M. C., Bizat N., Cuvelier L., Bantubungi K., Brouillet E.,
Schiffmann S. N. and Blum D. (2004) Death of cortical and striatal
neurons induced by mitochondrial defect involves differential
molecular mechanisms. Neurobiol. Dis. 15, 152–159.
Garcia O., Almeida A., Massieu L. and Bolanos J. P. (2005) Increased
mitochondrial respiration maintains the mitochondrial membrane
potential and promotes survival of cerebellar neurons in an
endogenous model of glutamate receptor activation. J. Neurochem.
92, 183–190.
Gould D. H., Wilson M. P. and Hanar D. W. (1985) Brain enzyme and
clinical alterations induced in rats and mice by nitroaliphatic tox-
icants. Toxicol. Lett. 27, 83–89.
Gu M., Gash M. T., Mann V. M., Javoy-Agid F., Cooper J. M. and
Schapira A. H. (1996) Mitochondrial defect in Huntington’s dis-
ease caudate nucleus. Ann. Neurol. 39, 385–389.
Harms L., Meierkord H., Timm G., Pfeiffer L. and Ludolph A. C. (1997)
Decreased N-acetyl-aspartate/choline ratio and increased lactate in
the frontal lobe of patients with Huntington’s disease: a proton
magnetic resonance spectroscopy study. J. Neurol. Neurosurg.
Psychiatry 62, 27–30.
Jenkins B. G., Koroshetz W. J., Beal M. F. and Rosen B. R. (1993)
Evidence for impairment of energy metabolism in vivo in Hun-
tington’s disease using localized 1H NMR spectroscopy. Neurology
43, 2689–2695.
Jung C., Higgins C. M. and Xu Z. (2000) Measuring the quantity and
activity of mitochondrial electron transport chain complexes in
tissues of central nervous system using blue native polyacrylamide
gel electrophoresis. Anal. Biochem. 286, 214–223.
Jung C., Higgins C. M. and Xu Z. (2002) Mitochondrial electron
transport chain complex dysfunction in a transgenic mouse
model for amyotrophic lateral sclerosis. J. Neurochem. 83, 535–
545.
Kasparova S., Sumbalova Z., Bystricky P., Kucharska J., Liptaj T.,
Mlynarik V. and Gvozdjakova A. (2006) Effect of coenzyme Q10
and vitamin E on brain energy metabolism in the animal model of
Huntington’s disease. Neurochem. Int. 48, 93–99.
Kim G. W., Copin J. C., Kawase M., Chen S. F., Sato S., Gobbel G. T.
and Chan P. H. (2000) Excitotoxicity is required for induction of
oxidative stress and apoptosis in mouse striatum by the mito-
chondrial toxin, 3-nitropropionic acid. J. Cereb. Blood Flow Me-
tab. 20, 119–129.
Kish S. J., Shannak K. and Hornykiewicz O. (1987) Elevated serotonin
and reduced dopamine in subregionally divided Huntington’s dis-
ease striatum. Ann. Neurol. 22, 386–389.
Klapdor K., Dulfer B. G., Hammann A. and Van der Staay F. J. (1997) A
low-cost method to analyse footprint patterns. J. Neurosci. Meth-
ods 75, 49–54.
Koroshetz W. J., Jenkins B. G., Rosen B. R. and Beal M. F. (1997)
Energy metabolism defects in Huntington’s disease and effects of
coenzyme Q10. Ann. Neurol. 41, 160–165.
La Fontaine M. A., Geddes J. W., Banks A. and Butterfield D. A. (2000)
3-Nitropropionc acid induces in vivo protein oxidation in striatal
and cortical synaptosomes: insights into Huntington’s disease.
Brain Res. 858, 356–362.
Laemmli U. K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lai J. C. K. and Clark J. B. (1979) Preparation of synaptic and non-
synaptic mitochondria from mammalian brain. Methods Enzymol.
55, 51–60.
Lee J., Kim C. H., Simon D. K. et al. (2005) Mitochondrial creb
mediates mitochondrial gene expression and neuronal survival.
J. Biol. Chem. 280, 40398–40401.
Lodi R., Schapira A. H., Manners D., Styles P., Wood N. W., Taylor D. J.
and Warner T. T. (2000) Abnormal in vivo skeletal muscle energy
metabolism in Huntington’s disease and dentatorubropallidoluy-
sian atrophy. Ann. Neurol. 48, 72–76.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951)
Protein measurement with a Folin phenol reagent. J. Biol. Chem.,
193, 265–275.
Ludolph A. C., He F., Spencer P. S., Hammerstad J. and Sabri M. (1991)
3-Nitropropionic acid-exogenous animal neurotoxin and possible
human striatal toxin. Can. J. Neurol. Sci. 18, 492–498.
Majak W. and McDiarmid R. E. (1990) Detection and quantitative
determination of 3-nitropropionic acid in bovine urine. Toxicol.
Lett. 50, 213–220.
Manczak M., Park B. S., Jung Y. and Reddy P. H. (2004) Differential
expression of oxidative phosphorylation genes in patients with
Alzheimer’s disease: implications for early mitochondrial dys-
function and oxidative damage. Neuromolecular. Med. 5, 147–162.
Matthews R. T., Yang L., Browne S., Baik M. and Beal M. F. (1998)
Coenzyme Q10 administration increases brain mitochondrial con-
centrations and exerts neuroprotective effects. Proc. Natl Acad. Sci.
USA 95, 8892–8897.
Milakovic T. and Johnson G. V. (2005) Mitochondrial respiration and
ATP production are significantly impaired in striatal cells
expressing mutant huntingtin. J. Biol. Chem. 280, 30773–30782.
Mizuno Y., Suzuki K., Sone N. and Saitoh T. (1988) Inhibition of
mitochondrial respiration by 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine (MPTP) in mouse brain in vivo. Neurosci. Lett. 91,
349–353.
Mizuno Y., Ohta S., Tanaka M., Takamiya S., Suzuki K., Sato T., Oya
H., Ozawa T. and Kagawa Y. (1989) Deficiencies in complex-I
subunits of the respiratory chain in Parkinson’s disease. Biochem.
Biophys. Res. Commun. 163, 1450–1455.
Muralikrishnan D. and Mohanakumar K. P. (1998) Neuroprotection by
bromocriptine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine-induced neurotoxicity in mice. FASEB J. 12, 905–912.
Murray J., Taylor S. W., Zhang B., Ghosh S. S. and Capaldi R. A. (2003)
Oxidative damage to mitochondrial complex-I due to peroxynitrite:
identification of reactive tyrosines by mass spectrometry. J. Biol.
Chem. 278, 37223–37230.
Nasr P., Gursahani H. I., Pang Z., Bondada V., Lee J., Hadley R. W. and
Geddes J. W. (2003) Influence of cytosolic and mitochondrial Ca2+
,
ATP, mitochondrial membrane potential, and calpain activity on the
mechanism of neuron death induced by 3-nitropropionic acid.
Neurochem. Int. 43, 89–99.
Nishino H., Kumazaki M., Fukuda A. et al. (1997) Acute 3-nitroprop-
ionic acid intoxication induces striatal astrocytic cell death and
dysfunction of the blood-brain barrier: involvement of dopamine
toxicity. Neurosci. Res. 27, 343–355.
Page K. J., Dunnett S. B. and Everitt B. J. (1998) 3-Nitropropionic acid-
induced changes in the expression of metabolic and astrocyte
mRNAs. Neuroreport 9, 2881–2886.
Palkovits M. and Brownstein M. J. (1983) Microdissection of Brain
Areas by Punch Techniques. John Wiley & Sons, New York.
Pang Z. and Geddes J. W. (1997) Mechanisms of cell death induced by
the mitochondrial toxin 3-nitropropionic acid: acute excitotoxic
necrosis and delayed apoptosis. J. Neurosci. 17, 3064–3073.
Panov A., Dikalov S., Shalbuyeva N., Taylor G., Sherer T. and
Greenamyre J. T. (2005) Rotenone model of Parkinson disease:
multiple brain mitochondria dysfunctions after short term systemic
rotenone intoxication. J. Biol. Chem. 280, 42026–42035.
Parker Jr W. D., Boyson S. J., Luder A. S. and Parks J. K. (1990)
Evidence for a defect in NADH: ubiquinone oxidoreductase
(complex-I) in Huntington’s disease. Neurology 40, 1231–1234.
Perez-Severiano F., Santamaria A., Pedraza-Chaverri J., Medina-Campos
O. N., Rios C. and Segovia J. (2004) Increased formation of
reactive oxygen species, but no changes in glutathione peroxidase
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Complex-I inhibition in HD model | 433

15. activity, in striata of mice transgenic for the Huntington’s disease
mutation. Neurochem. Res. 29, 729–733.
Picklo M. J., Amarnath V., McIntyre J. O., Graham D. G. and Montine
T. J. (1999) 4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial
respiration at multiple sites. J. Neurochem. 72, 1617–1624.
Polidori M. C., Mecocci P., Browne S. E., Senin U. and Beal M. F.
(1999) Oxidative damage to mitochondrial DNA in Huntington’s
disease parietal cortex. Neurosci. Lett. 272, 53–56.
Reynolds D. S., Carter R. J. and Morton A. J. (1998) Dopamine modu-
lates the susceptibility of striatal neurons to 3-nitropropionic acid in
rat model of Huntington’s disease. J. Neurosci. 18, 10116–10127.
Riobo N. A., Clementi E., Melani M., Boveris A., Cadenas E., Moncada
S. and Poderoso J. J. (2001) Nitric oxide inhibits mitochondrial
NADH: ubiquinone reductase activity through peroxynitrite for-
mation. Biochem. J. 359, 139–145.
Rizzardini M., Lupi M., Mangolini A., Babetto E., Ubezio P. and Can-
toni L. (2006) Neurodegeneration induced by complex I inhibition
in a cellular model of familial amyotrophic lateral sclerosis. Brain
Res. Bull. 69, 465–474.
Rozen S. and Skaletsky H. J. (2000) Primer3 on the WWW for general
users and for biologist programmers, in Bioinformatics Methods
and Protocols: Methods in Molecular Biology, (Krawetz S. and
Misener S., eds), pp. 365–386. Humana Press, Totowa, NJ, USA.
Saravanan K. S., Sindhu K. M. and Mohanakumar K. P. (2005) Acute
intranigral infusion of rotenone in rats causes progressive bio-
chemical lesions in the striatum similar to Parkinson’s disease.
Brain Res. 1049, 147–155.
Saravanan K. S., Sindhu K. M., Senthil Kumar K. S. and Mohanakumar
K. P. (2006) L-Deprenyl protects against rotenone-induced, oxi-
dative stress-mediated dopaminergic neurodegeneration in rats.
Neurochem. Int. 49, 28–40.
Saravanan K. S., Sindhu K. M. and Mohanakumar K. P. (2007) Mela-
tonin protects against rotenone-induced oxidative stress in a
hemiparkinsonian rat model. J. Pineal Res. 42, 247–253.
Sawa A., Wiegand G. W., Cooper J., Margolis R. L., Sharp A. H.,
Lawler Jr J. F., Greenamyre J. T., Snyder S. H. and Ross C. A.
(1999) Increased apoptosis of Huntington disease lymphoblasts
associated with repeat length-dependent mitochondrial depolar-
ization. Nat. Med. 5, 1194–1198.
Schagger H. (1995) Quantification of oxidative phosphorylation enzymes
after blue native electrophoresis and two-dimensional resolution:
normal complex-I protein amounts in Parkinson’s disease conflict
with reduced catalytic activities. Electrophoresis 16, 763–770.
Schagger H. (1996) Electrophoretic techniques for isolation and quan-
tification of oxidative phosphorylation complexes from human
tissues. Methods Enzymol. 264, 555–566.
Schapira A. H. (1999) Mitochondrial involvement in Parkinson’s dis-
ease, Huntington’s disease, hereditary spastic paraplegia and
Friedreich’s ataxia. Biochim. Biophys. Acta 1410, 99–102.
Schulz J. B., Henshaw D. R., MacGarvey U. and Beal M. F. (1996)
Involvement of oxidative stress in 3-nitropropionic acid neuro-
toxicity. Neurochem. Int. 2, 167–171.
Selkoe D. J., Salazar F. J., Abraham C. and Kosik K. S. (1982) Hun-
tington’s disease: changes in striatal proteins reflect astrocytic
gliosis. Brain Res. 245, 117–125.
Shear D. A., Dong J., Gundy C. D., Haik-Creuger L. K. and Dunbar
G. L. (1998) Comparison of intrastriatal injections of quinolinic
acid and 3-nitropropionic acid for use in animal models of Hunt-
ingtons disease. Prog. Neuro-Psychopharmacol. Biol. Psychiatry
22, 1217–1240.
Shults C. W., Nasirian F., Ward D. M., Nakano K., Pay M., Hill L. R.
and Haas R. H. (1995) Carbidopa/levodopa and selegiline do not
affect platelet mitochondrial function in early parkinsonism. Neu-
rology 45, 344–348.
Spokes E. G. (1980) Neurochemical alterations in Huntington’s chorea: a
study of post-mortem brain tissue. Brain 103, 179–210.
Tabrizi S. J., Cleeter M. W., Xuereb J., Taanman J. W., Cooper J. M. and
Schapira A. H. (1999) Biochemical abnormalities and excitotox-
icity in Huntington’s disease brain. Ann. Neurol. 45, 25–32.
Tabrizi S. J., Workman J., Hart P. E., Mangiarini L., Mahal A., Bates G.,
Cooper J. M. and Schapira A. H. (2000) Mitochondrial dysfunction
and free radical damage in the Huntington R6/2 transgenic mouse.
Ann. Neurol. 47, 80–86.
Teunissen C. E., Steinbusch H. W., Angevaren M., Appels M., de Bruijn
C., Prickaerts J. and de Vente J. (2001) Behavioural correlates of
striatal glial fibrillary acidic protein in the 3-nitropropionic acid rat
model: disturbed walking pattern and spatial orientation. Neuro-
science 105, 153–167.
Thomas B. and Mohanakumar K. P. (2004) Melatonin protects against
oxidative stress caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine in the mouse nigrostriatum. J. Pineal Res. 36, 25–32.
Trounce I. A., Kim Y. L., Jun A. S. and Wallace D. C. (1996) Assess-
ment of mitochondrial oxidative phosphorylation in patient muscle
biopsies, lymphoblasts, and transmitochondrial cell lines. Methods
Enzymol. 264, 484–509.
Vis J. C., Verbeek M. M., de Waal R. M., ten Donkelaar H. J. and
Kremer B. (2001) The mitochondrial toxin 3-nitropropionic acid
induces differential expression patterns of apoptosis-related mark-
ers in rat striatum. Neuropathol. Appl. Neurobiol. 27, 68–76.
Weydt P., Pineda V. V., Torrence A. E. et al. (2006) Thermoregulatory
and metabolic defects in Huntington’s disease transgenic mice
implicate PGC-1alpha in Huntington’s disease neurodegeneration.
Cell Metab. 4, 349–362.
Wyttenbach A., Sauvageot O., Carmichael J., Diaz-Latoud C., Arrigo
A. P. and Rubinsztein D. C. (2002) Heat shock protein 27 prevents
cellular polyglutamine toxicity and suppresses the increase of
reactive oxygen species caused by huntingtin. Hum. Mol. Genet.
11, 1137–1151.
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434
Ó 2007 The Authors
434 | M. Pandey et al.