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Mitochondrial NAD+
-linked State 3 respiration and complex-I
activity are compromised in the cerebral cortex of 3-nitropro...
has been investigated (La Fontaine et al. 2000; Galas et al.
2004), the exact pathways leading to selective neurodegen-
er...
Estimation of dopamine and its metabolites
Animals were killed on fifth or ninth day following 3-NP
administration (10, 15 ...
incubated in 50 mmol/L potassium phosphate buffer, pH 7.5 for
5 min at 37°C to activate the enzyme. The activity was monit...
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...
affected in the animals that received 20 mg/kg dose, but not
for those with the lower dose as compared to the controls. Th...
Effect of 3-NP on striatal dopamine and its metabolites
A significant dose-dependent increase in striatal DA levels
was obs...
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 Bra...
compared to the fifth day (Fig. 4a). Interestingly, in contrast
to mitochondria isolated from cortex, mitochondrial P2
frac...
compared to the controls, and the RCR was found to be
decreased by 44% (Table 2; representative tracings in Fig. 7
show th...
Discussion
In this study, we have investigated whether the ETC
dysfunction found in patients of HD is also present in the
...
suggesting an increase in reactive gliosis. Increased GFAP
expression has been consistently found in the 3-NP model of
HD ...
electrons in the ETC can affect the other. Bautista et al.
(2000) have shown a decline in activity of complex-I due to
its...
Galas M. C., Bizat N., Cuvelier L., Bantubungi K., Brouillet E.,
Schiffmann S. N. and Blum D. (2004) Death of cortical and...
activity, in striata of mice transgenic for the Huntington’s disease
mutation. Neurochem. Res. 29, 729–733.
Picklo M. J., ...
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  1. 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. 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 Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 420–434 Complex-I inhibition in HD model | 421
  3. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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