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Research Report

Effects of partial suppression of parkin on huntingtinmutant R6/1 mice

Isabel Rubioa,b, José Antonio Rodríguez-Navarroc, Cristina Tomás-Zapicob,d,Carolina Ruíza, María José Casarejosc, Juan Peruchob,c, Ana Gómezc, Izaskun Rodala,José J. Lucasb,d, María Angeles Menac, Justo García de Yébenesa,b,⁎aServicio de Neurología; Hospital Ramón y Cajal; Ctra Colmenar, Km 9.1, Madrid 28034, SpainbCIBERNED, Instituto de Salud Carlos III, Madrid, SpaincLaboratorio de Neurofarmacología, Hospital Ramón y Cajal, Madrid, SpaindCentro de Biología Molecular “Severo Ochoa”, CSIC/UAM y CIBERNED, Madrid 28049, Spain

A R T I C L E I N F O

⁎ Corresponding author. Servicio de NeurologíE-mail address: jgyebenes@yahoo.com (J.Abbreviations: TUNEL, TdT-mediated dU

glutathione; HSP-70, heat shock protein-70; CUPS, ubiquitin proteosomal system

0006-8993/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainres.2009.05.039

A B S T R A C T

Article history:Accepted 8 May 2009Available online 21 May 2009

Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion ofpolyglutamines which makes huntingtin more resistant to degradation. Parkin is anubiquitin ligase which promotes proteosomal degradation of abnormal proteins. Weinvestigated whether partial suppression of parkin increases HD phenotype. We studiedthe behavior and brain histology and biochemistry of the mice produced by interbreeding ofR6/1 (model of HD in mice) with Park-2−/− (parkin null mice): R6/1, WT (wild-type), PK+/−

(hemizygotic deletion of Park-2) and R6/1/PK+/−. R6/1 and R6/1/PK+/− mice had abnormalmotor and exploratory behavior. R6/1/PK+/− mice were more akinetic. These two groups ofmice had severe but similar loss of nigrostriatal dopamine neurons and monoamine levelsin striatum. R6/1/PK+/− mice had fewer huntingtin inclusions and a greater number ofTUNEL+ cells than R6/1 in striatum but there were no differences in the hippocampus.DARPP-32 protein was equally reduced in striatum of R6/1 and R6/1/PK+/− mice. Striatallevels of GSH were increased, of HSP-70 reduced and of CHIP unchanged in both R6/1 andR6/1/PK+/− mice. LC-3 II/I ratios were significantly increased in striatum of R6/1/PK+/− mice.Partial suppression of parkin slightly aggravates the phenotype in R6/1 mice, confirming apathogenic role of the UPS in the processing of mutant huntingtin. The absence of massiveadditional cellular lesions in R6/1/PK+/− mice suggests the existence of compensatorymechanisms, such as autophagy, for the processing of huntingtin.

© 2009 Elsevier B.V. All rights reserved.

Keywords:ApoptosisAutophagyDopamine neuronsHuntington's diseasePark-2 geneUbiquitin proteosomal system

a; Hospital Ramón y Cajal; Ctra Colmenar, Km 9.1, Madrid 28034, Spain. Fax: +34 91 3369016.G. de Yébenes).TP Nick-End Labeling; DARPP-32, dopamine and c-AMP-regulated phosphoprotein; GSH,HIP, c-terminal Hsc interacting protein; LC-3, microtubule-associated protein 1 light chain 3;

er B.V. All rights reserved.

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1. Introduction

Huntington's disease is a neurodegenerative disorder char-acterized by motor, cognitive and behavioral symptomsproduced by an abnormal expansion of CAG repeats in theN-terminal part of the gene of huntingtin (Gusella andMacdonald, 2006). The physiological role of huntingtin andthe pathogenic mechanisms that produce the disease areunknown. Mutant huntingtin, with an expanded polygluta-

Fig. 1 – Body weights and motor behavior of WT, R6/1, R6/1/PK+/−

Actitrack. (C) Hind limb stride of the footprint. (D) Motor coordinaexploratory activity expressed as the number of total entrances in(n=6 animals in each experimental group). Statistical analysis watest. *p<0.05, **p<0.01, ***p<0.001 vsWTmice; +p<0.05, ++p<0.01The two-way ANOVA shows interaction (***p<0.001) between thmutation of huntingtin is the main source of variation for the weR6/1 mice.

mine chain, produces fibrillary deposits, changes its normallocalization and produces cytoplasmic and intranuclearinclusions, changes gene transcription, alters synaptic trans-mission, impairs mitochondrial activity and activates cas-pases and other pro-apoptotic molecules (Khan et al., 2006;Solans et al., 2006; Zhai et al., 2005).

It has been proposed that mutant huntingtin impairs theproteasome system and alters the protein degradation. Mutanthuntingtin, therefore, accumulates in HD, as other proteins do

and PK+/− mice. (A) Body weight. (B) Motor activity in thetion expressed as the time spent in the Rota-Rod. (E) MotorYmaze at 6months. The values are expressed asmean±SEMs performed by one-way ANOVA followed by Newman–Keuls, +++p<0.001 vs PK+/−mice; ^p<0.05, ^^^p<0.001 vs R6/1mice.e genotype and ageing for weight evolution analysis. Theight reduction and the length of the hind limb stride in

Fig. 2 – The nigrostriatal dopamine pathway and monoamines and their metabolites in striatum of 7-month-old WT, R6/1,R6/1/PK+/− and PK+/− mice. (A–H) Representative microphotographs of TH+ immunostained cells in the substantia nigra. Lowpower, (2×, A–D, scale bar=100 μm) and high power (10×, E–H, scale bar=30 μm) view of the brain stem ofWT (A and E), R6/1 (Band F), R6/1/PK+/− (C and G) and PK+/− (D and H) mice immunostaining to TH antibody. (I) Average number of TH+ neurons arecounted per brain stem section. (J) Levels of TH protein/β-actin in striatum ofWT, R6/1, R6/1/PK+/− and PK+/−mice. (K) Dopamine,(L) norepinephrine, (M) serotonin, (N) 3,4-dihydroxy-phenyl-acetic acid, (O) homovanillic acid and (P) 5-hydroxy-indole-aceticacid. The results are expressed as themean±SEM (n=6 animals in each experimental group). Statistical analysiswas performedby one-way ANOVA followed by Newman–Keuls test. *p<0.05, ***p<0.001 vsWTmice; ++p<0.01, +++p<0.001 vs PK+/− mice. Themutation of huntingtin is themain factor for the differences in the number of TH+ positive cells and TH protein levels and for thelevels of monoamines and their metabolites in striatum.

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in Alzheimer's disease (AD) and Parkinson's disease (PD) (Benceet al., 2001; Bennett et al., 2007; Keller et al., 2000; Kitada et al.,1998; Ortega et al., 2007). Deposits of the different pathogenicproteins, enriched with ubiquitin and with components ofubiquitin proteosomal system (UPS), have been found in thesediseases (Lim et al., 2006; Ross and Pickart, 2004).

Parkin is an ubiquitin E3 ligase whose mutations producefamilial PD (Kitada et al., 1998; Lucking et al., 2000; Hoenicka etal., 2002). Parkin mutations and polymorphisms may increase

the risk of progressive supranuclear palsy (PSP), a diseasecharacterized by tau pathology (Morales et al., 2002; Ros et al.,2008). These findings suggest that abnormal parkin functionnot only produces parkinsonism but also may aggravate themolecular events that take place when abnormal proteinsaccumulate in neurons.

We hypothesized that if a compromised function of theUPSplays a role in thepathogenic effects ofmutanthuntingtin,any additional molecular impairment of this system would

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aggravate the phenotype and neuronal lesions which takeplace in HD.We have investigated the behavioral, biochemicaland cellular events which take place in R6/1 mice when, inaddition to enlarged CAG repeats, they have a partial deficiencyof the parkin function.

2. Results

2.1. Development and clinical phenotype

All animals developed normally and gained weight untilthreemonths of age. After that, and until sacrifice at 7months,weight gain continued in PK+/− and WT but decreased inR6/1/PK+/− and more severely in R6/1 (Fig. 1A) and the dif-ferences between these two groups were significant at 4 and5 months of age.

Progressive motor and behavioral abnormalities weredetected in R6/1 and R6/1/PK+/− after three months of age(Fig. 1B–E). Global motor activity, as measured as the total dis-tance run in the Actitrack, was reduced in R6/1/PK+/− (Fig. 1B).The length of the hind limb stride, was reduced in PK+/− and,more severely in R6/1 and R6/1/PK+/− (Fig. 1C). We have reportedthathind limbstridewas reduced inoldPK−/−mice but therewasno information of this parkinsonian sign in PK+/− heterozygote,suggesting that this test is very sensitive for hypoactivity(Rodriguez-Navarro et al., 2007). Motor coordination, analyzedas time in the Rota-Rod, was altered in both R6/1 and R6/1/PK+/−

without differences between these two groups (Fig. 1D). Motorexploratory activity, as measured as the number of entrancesin the Y maze was significantly reduced in the double mutants(Fig. 1E). In summary, R6/1/PK+/− have more severe behavioralabnormalities in most but not all tests than R6/1 mice.

2.2. The nigrostriatal dopamine pathway and the striatallevels of monoamine metabolites

There was a severe loss of nigrostriatal dopamine neurons inmidbrains of R6/1 and R6/1/PK+/− (Fig. 2 A–H) to roughly 50% ofthe numbers observed in WT and PK+/− mice (Fig. 2I). Tyrosinehydroxylase (TH) protein levels were greatly decreased instriatum of R6/1 and R6/1/PK+/− with respect to WT and PK+/−

mice (Fig. 2J). The levels of dopamine (DA), norepinephrine,serotonin (5-HT) and their corresponding metabolites wereseverely reduced in the striatum of both R6/1 and R6/1/PK+/−

mice in comparison with the levels of both WT and PK+/− mice(Fig. 2 K–P). No difference was observed in these parametersbetween R6/1 and R6/1/PK+/− mice.

Fig. 3 – Representative microphotographs of huntingtin inclusioimmunostaining and DARPP-32 Western blot in striatum of 7-huntingtin inclusions in striatum. Low power, (2×, A–D, scaleview of the brain stem of WT (A and E), R6/1 (B and F), R6/1/Phuntingtin antibody. (I) Average number of huntingtin positivelow power (10×, J–M, scale bar=30 μm) and high power (20×, NDARPP-32 antibodies. WT (J and N), R6/1 (K and O), R6/1/PK+/− (Lprotein, as measured by Western blot in striatum. Values are eperformed by one-way ANOVA followed by Newman–Keuls tesvs R6/1 mice.

2.3. Huntingtin inclusions and cell death in brain areas

Huntingtin inclusions were detected only in striatum of R6/1and R6/1/PK+/− mice (Fig. 3 A–H). Surprisingly, the number ofthese inclusions was smaller in R6/1/PK+/− than in R6/1 mice(Fig. 3 F, G and I). The total number of neurons was slightly butnot significantly decreased in striatum of R6/1 and R6/1/PK+/−

mice (data not shown). Similarly, the levels of Neu-N (a nonselective protein marker of neurons) were slightly but notsignificantly reduced in striatum of R6/1 and R6/1/PK+/− mice(data not shown). Thenumber of neurons in hippocampuswassimilar in all experimental groups of animals. The number ofhuntingtin inclusions was similar in R6/1 and R6/1/PK+/−

hippocampi (data not shown).The number of dopamine and c-AMP-regulated phospho-

protein (DARPP-32)+ cells, the medium size and small efferentspiny striatal neurons which are the main pathological targetin HD, was greatly reduced in both R6/1 and R6/1/PK+/− mice(Fig. 3J-Q) and a severe loss of DARPP-32 protein was alsoobserved in striatum of these two huntingtin mutant mice(Fig. 3R), but there were no differences between both groups.PK+/−mice have slightly but not significantly increased DARPP-32 levels.

Representative photomicrographs of TUNEL (TdT-mediateddUTP Nick-End Labeling) (Fig. 4A) and bisbenzimide (Fig. 4B)stainedandcolocalization (Fig. 4C) instriatumofR6/1/PK+/−miceare shown. The number of TUNEL+ cells was significantlyincreased in R6/1/PK+/−, suggesting that the apoptotic celldeath was greatly increased in striatal cells of the R6/1/PK+/−

(Fig. 4D).The number of TUNEL+ cells was also increased in the

hippocampus of both R6/1 and R6/1/PK+/− mice but, withoutdifferences between these two groups (Fig. 4E). The striatalratio of pro/anti-apoptotic proteins, Bax/Bcl2, was signifi-cantly increased in R6/1 and furthermore in R6/1/PK+/− mice(Fig. 4F).

2.4. Molecular mechanisms of cellular protection anddeath in R6/1 and R6/1/PK+/− mice

The striatal levels of glutathione (GSH) were increased in R6/1and R6/1/PK+/− mice (Fig. 5A), suggesting an increasedoxidative stress in these animals and compensatory effectelevation of GSH. There were no differences in the levels ofthe mitochondrial cytochrome C protein (data not shown).The heat shock protein-70 (HSP-70) was slightly increased inPK+/− and reduced in R6/1 and R6/1/PK+/− mice (Fig. 5B),suggesting a lack of chaperone protection in these animals.

ns, counts of inclusion bearing neurons, DARPP-32month-old mice. (A–H) Representative microphotographs ofbar=100 μm) and high power (10×, E–H, scale bar=30 μm)K+/− (C and G) and PK+/− (D and H) mice immunostaining tocells in striatum. (J–Q) Representative microphotographs at–Q, scale bar=20 μm) striatum of these mice stained withand P) and PK+/− (M and Q). (R) Levels of DARPP-32/β-actinxpressed as mean±SEM (n=6). The statistical analysis wast. ***p<0.001 vs WT mice;+++p<0.001 vs PK+/− mice; ^p<0.05

Fig. 4 – Apoptotic cells and pro- and anti-apoptotic proteins in striatum of WT, R6/1, R6/1/PK+/− and PK+/− of 7-month-old mice.Microphotographs of striatum showing TUNEL+ cells (A) in striatum of R6/1mouse, their corresponding cells nuclei stainingwith bisbenzimide (B) and co-marked (C). Bar=20μm,magnification 20×. Average number of TUNEL+ cells are counted per brainsection in striatum (D) and (E) in hippocampus of the four experimental groups. (F) Ratio of pro-apoptotic/anti-apoptotic(Bax/Bcl2) proteins in striatum of the four experimental groups. The values are expressed as mean±SEM (n=6). The statisticalanalysis was performed by one-way ANOVA followed by Newman–Keuls test. *p<0.05, ***p<0.001 vs WT mice; +++p<0.001 vsPK+/− mice.

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The levels of c-terminal Hsc interacting protein (CHIP) didnot change in any of the experimental groups (Fig. 5C). Thelevels of microtubule-associated protein 1 light chain 3(LC-3 I) did not change in any of the experimental groups.However, LC-3 II, a protein considered a marker of autop-hagy, and the LC-3 II/LC-3 I ratios were increased in R6/1/PK+/− mice (Fig. 5D).

3. Discussion

Our study reveals that partial suppression of parkin in R6/1mice results in aggravation of motor and behavioral deficitsand in an increased number of apoptotic cells in striatum. R6/1and R6/1/PK+/− mice have similar levels of damage of the

Fig. 5 – GSH, HSP-70, CHIP and LC-3 in striatum of WT, R6/1, R6/1/PK+/− and PK+/− of 7-month-old mice. (A) GSH levels.Representative Western blot and mean values of HSP-70/β-actin (B), CHIP/β-actin (C) and LC-3 II/LC-3 I (D) in striatum of eachexperimental group. The values are expressed as mean±SEM (n=6). Statistical analysis was performed by one-way ANOVAfollowed by Newman–Keuls Test. *p<0.05, **p<0.01, ***p<0.001 vs WT mice; +++p<0.001 vs PK+/− mice.

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nigrostriatal dopamine neurons, similar levels of striatalmonoamine depletion, and levels of chaperones. R6/1/PK+/−

mice had fewer huntingtin inclusions and slightly higherlevels of LC-3 II proteins than R6/1 but there was no differencebetween the two groups in the number of DARPP-32 positivecells. No differences were observed in any of the parametersinvestigated between R6/1 and R6/1/PK+/− mice in hippocam-pus. With the limitations derived from the mixing of animalswith different genetic backgrounds, our data suggest, insummary, that partial impairment of the ubiquitin–proteoso-mal system, by reduction of the levels of parkin partiallyaggravates the deficits observed in R6/1 mice and, therefore,that impairment of the UPS may be important in thepathogenesis of HD. We have not observed, however, that

the partial suppression of parkin in R6/1 mice producesmassive additional striatal neuronal loss or any additionaldamage in other brain regions. It is possible that the stress ofparkin deficiency is partially compensated by the activation ofother compensatory mechanisms. Mutant huntingtin, withlong polyglutamine chains, forms β-structures and aggregatesas amyloid (Marchut and Hall, 2007). Cellular mechanisms ofprevention of protein aggregation include enhanced produc-tion of chaperones and autophagy (Paul, 2007). Chaperoneswere not different in R6/1 and R6/1/PK+/− mice, but LC-3 IIprotein levels were increased in R6/1/PK+/− mice.

Alteration of theUPShas been described inpatientswithHD,and in cellularmodels and inmice after viral transfer ofmutanthuntingtin (Bennett et al., 2007; Hunter et al., 2007; Wang et al.,

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2008). There is, however, a controversy about the role of the UPSin HD, which is due to the differences between the differentmodels of thedisease and thedifferentmethodsof evaluationofthe UPS function (Ortega et al., 2007; Valera et al., 2005).

The most compelling evidence in support of a pathogenicrole forparkinsuppression inhuntingtinmutantmice is the factthat these animals dohavehigher levels of apoptosis,measuredasnumberof TUNEL+ cells and theBax/Bcl2 ratio. Thedifferenceis statistically significantwith respect to the other experimentalgroups in striatum and with respect to WT and PK+/− mice inhippocampus. The level of apoptosis doesnot correlatewith thenumber of huntingtin+ inclusions, since R6/1/PK+/− do havefewer inclusions instriatumand the same inhippocampus thanR6/1mice. Both groups R6/1 and R6/1/PK+/− mice had a severedrop-out DARPP-32 expression in striatum in the absence ofmassiveneuronal loss, as it has been reported inmicemodels ofHD. The increased level of apoptosis in striatum of R6/1/PK+/−

mice does not correlate with a more severe drop-out of striatalneurons, of non selective neuronal protein markers, such asNeu-N, or of the most selective marker of the target efferentstriatal neurons DARPP-32. In the first two cases the lack ofcorrelation could be explained by shrinkage of the striatum inR6/1 and R6/1/PK+/− mice. With respect to DARPP-32 we haveobservedup-regulation thisprotein inPK−/− (Rubio, unpublishedobservation). In this study we observed a modest but nonsignificant elevationofDARPP-32 levels in striatumofPK+/−withrespect to WT and we could not exclude that there was up-regulation mediated compensation of DARPP-32 in striatum ofR6/1/PK+/− with respect to R6/1 mice.

In summary, partial suppression of parkin aggravates theclinical phenotype and the effects of mutant huntingtin instriatal cells. The effect of parkin reduction in other brainregions was less severe or absent. These findings suggest thatin R6/1 mice partial impairment of the UPS further aggravatesthe effects of mutant huntingtin on target cells but the impactof partial parkin suppression is restricted to certain neuronsand limited, perhaps by the development of compensatorymechanisms, mainly the elevation of LC-3 II/LC-3 I ratio, andindex of increased autophagy. If so, the pharmacologicalstimulation of autophagy may be a potential therapeuticapproach in HD.

4. Experimental procedures

4.1. Breeding, development and behavior of mutant mice

Breeding of the animals was performed according to thefollowing schema. The numbers in brackets represent theexpected/found frequencies of genotypes expressed in % ofthe offspring.

Huntingtinmice ðR6=1Þ + wild−type ðWTÞR6=1 � WT

AF1 : R6=1 50=43ð Þ; WT 50=57ð Þ

Huntingtinmice R6=1ð Þ + parkinnull PK−=−� �

R6=1 � PK−=−

AF1 : R6=1=PKþ=− 50=58:1ð Þ; PKþ=− 50=41:9ð Þ

R6/1 male founders, expressing exon 1 of the humanhuntingtin gene with 115 CAG repeats were donated by Prof.Lucas (Mangiarini et al., 1996). PK−/− founders were donated byAventis Pharma (Vitry-sur-Seine, France) (Itier et al., 2003).The different experimental groups were obtained from R6/1male (CBA/C57BL6 background), wild-type (C57BL6 back-ground) female and PK−/− (C57BL6/129SV background) femalemice intercross. The different genotypes were defined asbelow. Procedures using laboratory animals were in accor-dance with the European Union Directives. All efforts weremade to minimize the number of animals used and theirsuffering.

Twenty-four male mice, 6 mice for each group, were usedfor the biochemical and for behavioral studies and eight more,2 per group, for histology. The animals were sacrificed at7 months of age by cervical dislocation.

4.2. Determination of the genotype

Genomic DNA was extracted from mouse tail after proteinaseK digestion (16 h at 55 °C) in lysis buffer according to themanufacturer's instructions (High Pure PCR template prepara-tion kit, Roche, Barcelona, Spain). For genotyping WT and PK−/

− mice 150 ng of genomic DNA were denatured for 3 min at94 °C and subjected to 35 cycles of 1min at 94 °C, 1min at 53 °C,and 1 min at 72 °C, followed by 5 min of a final extension at72 °C. PCRwas performed in a final volume of 40ml containing1 U of Taq DNA polymerase (Promega, 5 U/ml), 1 mM dNTP(4×0.25mM), 2.5mMMgCl2, 5mMTris–HCl (pH 8.0) and 1ml ofspecific sense and antisense primers at 50 ng/μl. For genotyp-ing WT and PK−/− mice we use the following primers: pk1F: 5′-T G C T C TGGGGT T CG T C - 3 ′ a n d p k R : 5 ′ - T C CA -CTGGCAGAGTAAATGT-3′ for WT and pk2F: 5′-TTGTTTTG-CCAAGTTCTAAT-3′ and pkR for PK−/− mice.

For genotyping R6/1 mice 150 ng of genomic DNA weredenatured for 3min at 94 °C and subjected to 35 cycles of 1minat 94 °C, 1 min at 55 °C, and 1min at 72 °C, followed by 5min ofa final extension at 72 °C. PCRwas performed in a final volumeof 25 μL containing 0.75 U of Taq DNA polymerase (Promega,Madrid, Spain; 5 U/μL), 0.2mMdNTP, 1.5mMMgCl2, 5mMTris–HCl (pH 8.0), 0.5 μL of DMSO and 1.5 μL of specific sense andantisense primers at 10 μM.

The specific primers were HD-1D: 5′-CCGCTCAGGTTCTG-CTGCTTTTA-3′ and HD-1R: 5′TGGAAGGACTTGAGGGACTC-3′.

Twenty microlitres of the PCR reaction products wereanalyzed by electrophoresis in a 1.8% agarose gel that wassubsequently stained with ethidium bromide for visualizationof DNA bands. DNA molecular weight markers (Roche, Spain)were used to provide a size reference for the test reactions.The sizes of PCR products which allow for the identification ofthe different genotypes are the following: 170 bp for mutanthuntingtin, 297 bp forWTparkin and 285 bp for deleted parkin,respectively.

4.3. Behavioral studies

Exploratory behavior and motor activity were measured in 4-,5- and 6-month-old mice in open field and activity cages asdescribed (Gonzalez et al., 2005; Itier et al., 2003; Serrano et al.,2005). Motor coordination was evaluated in the Rota-Rod; the

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cylinder rotates at an initial speed of 4 rpm and acceleratesgradually on 30 s to a maximum of 40 rpm. The Y maze wasmade of PVC, each arm was 21 cm long, 40 cm high and 4 cmwide. The arms converged to a triangular central part. Eachmouse was placed at the triangular part and allowed to movefreely through the maze for 5 min. The number of entries intothe different arms was recorded visually. Alternation wasdefined as successive entry into the three arms. Alternationbehavior (%) was calculated as the ratio of actual to possiblealternations (defined as the number of arm entriesminus two)multiplied by 100.

4.4. Histological studies

The animals used for these studies were anaesthetizedintraperitoneally with ketamine, 50 mg/ml, diazepam, 1 mg/ml, and atropine, 1 mg/ml, (5:4:1) and perfused with 4%paraformaldehyde. The brain was immersed in paraformal-dehyde for 24 h included in paraffin, sectioned at a thicknessof 4 μm, and stained for haematoxylin/eosine (H&E) andNissl's stain. Tyrosine hydroxylase, huntingtin and DARPP-32antibodies from Chemicon (Temecula, CA, USA) were used forimmunohistochemistry. Mouse polyclonal anti-TH antibodywas diluted 1:2000, mouse monoclonal anti-huntingtin anti-body was diluted 1:200 and rabbit polyclonal anti-DARPP-32was diluted 1:1000. Anti-rabbit and anti-mouse secondaryantibodies were from Dako (Glostrup, Denmark). The numberof TH immunoreactive neurons in the substantia nigra and thehuntingtin immunoreactive neurons in the striatum andhippocampus were counted in an Olympus Bx51 stereologicalmicroscope (Olympus, Ballerup, Denmark) using a Cast Gridsoftware. TH cells and huntingtin aggregates were quantifiedin 8–9 regularly spaced sections in eachmouse brain, coveringthe entire surface of the antero-posterior extent of thesubstantia nigra or the hippocampus respectively. Countingof cells was performed independently by two blind observersbut no stereological methods were used.

4.5. TUNEL assay

TUNEL (TdT-mediated dUTP Nick-End Labeling) detectionsystem for apoptosis measures the fragmented DNA of cellsby incorporating fluorescein-12-dUTP⁎ at the 3′–OH ends of theDNA by using the enzyme Terminal deoxynucleotidyl Trans-ferase (TdT). For this assay, cerebral sections containing thestriatum and the hippocampus were fixed in 4% paraformal-dehyde and permeabilized with 0.2% Triton X-100. Thefluorescein-12-dUTP-labeled DNAof apoptotic cells was visua-lised by fluorescence microscopy (positive cells with greenfluorescence). The number of TUNEL+ cells was counted in thestriatum and hippocampus. Cells were counted in pre-definedparallel strips by means of a counting reticule in the ocularpiece of the microscope. Brain sections incubated with bufferin the absence of TdT enzyme were used as negative control.

4.6. Brain dissection, tissue preparation and monoaminedetermination

After decapitation, the brains were dissected as described(Carlsson and Lindqvist, 1973). The brain parts were processed

and analyzed as described (Garcia Ruiz et al., 1995). Thestriatum was used for the measurement of dopamine and itsmetabolites, 3-methoxy-tyramine (3-MT), 3,4-dihydroxypheny-lacetic acid (DOPAC) and homovanillic acid (HVA), noradrena-line (NA), serotonin and its metabolite, 5-hydroxy-indole-aceticacid (5-HIAA).

4.7. Measurement of glutathione and protein analysis

Total glutathione (GSx) levels were measured according toTietze (Tietze, 1969). Protein levels weremeasured byWesternblot as described (Rodriguez-Navarro et al., 2007). β-actin wasused as a control of charge. Rabbit polyclonal anti-DARPP-32antibody diluted 1:1000, mouse monoclonal anti-TH antibodydiluted 1:5000, mouse monoclonal anti-Neuronal nuclei (Neu-N) antibody diluted 1:1500 were from Chemicon; mousemonoclonal anti-Bcl2 (1:500), rabbit polyclonal anti-Bax(1:500) and mouse monoclonal anti-HSP-70 (1:1000) werefrom Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbitpolyclonal anti-CHIP (1:1000) was from Abcam (Cambridge,UK); rabbit polyclonal anti-LC-3 (1:1000) was from MBL(Nagoya, Japan), mouse monoclonal anti-cytochrome C(1:800) was from BD biosciences. Mouse monoclonal anti-β-actin antibody diluted 1:5000 was from Sigma.

Goat anti-mouse-horseradish peroxidase and anti-rabbit-horseradish peroxidase secondary antibodies diluted 1:1500were from Amersham. β-actin secondary antibody was ananti-mouse phosphatase alkaline conjugate diluted 1:3000from Sigma.

4.8. Statistical analysis

Analysis of the data was performed by one- and two-wayanalysis of variance (ANOVA) followed by the Newman–Keulsmultiple comparison test or Bonferroni test. Differences wereconsidered statistically significant when p<0.05.

Acknowledgments

This study has been supported in part by grants from theSpanish Minister of Health, Instituto de Salud Carlos III, FIS2007/725 and CIBER 2006/05/0059. The authors thank MatildeSerrano for excellent technical assistance and Mrs. ClaireMarsden for editorial help.

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