The FASEB Journal • FJ Express Full-Length Article

Dopamine enhances motor and neuropathologicalconsequences of polyglutamine expanded huntingtin

Michel Cyr,*,1 Tatyana D. Sotnikova,† Raul R. Gainetdinov,† and Marc G. Caron†,1

*Neuroscience Research Group, University of Quebec at Trois-Rivieres, Trois-Rivieres, Quebec,Canada; and †Department of Cell Biology, Center for Models of Human Disease, IGSP, DukeUniversity, Durham, North Carolina, USA

ABSTRACT An expansion in the CAG repeat of theIT15 (huntingtin) gene underlies the development ofHuntington’s disease (HD), but the basis for the spe-cific vulnerability of dopamine-receptive striatal neu-rons remains unclear. To examine the potential role ofthe dopamine system in the emergence of pathologicalconditions in HD, we generated a double mutant mousestrain with both enhanced dopamine transmission andendogenous expression of a mutant huntingtin gene.This strain was generated by crossing the dopaminetransporter knock-out mouse, which exhibits a 5-foldelevation in extracellular dopamine levels in the stria-tum and locomotor hyperactivity, to a knock-in mousemodel of HD containing 92 CAG repeats. These doublemutant mice exhibited an increased stereotypic activityat 6 months of age, followed by a progressive decline oftheir locomotor hyperactivity. Expression of the mu-tated huntingtin did not alter dopamine or its metabo-lite levels in normal or dopamine transporter knock-outmice. However, the mutant huntingtin protein aggre-gated much earlier and to a greater extent in thestriatum and other dopaminergic brain regions in thehyperdopaminergic mouse model of HD. Furthermore,the formation of neuropil aggregates in the striatumand other regions of hyperdopaminergic HD mice wasobserved at 4 months of age, well before similar eventsoccurred in normal HD mice (12 months). Thesefindings indicate that dopamine contributes to thedeleterious effects of mutated huntingtin on striatalfunction, and this is accompanied by enhanced forma-tion of huntingtin aggregates.—Cyr, M., Sotnikova,T. D., Gainetdinov, R. R., Caron, M. G. Dopamineenhances motor and neuropathological consequencesof polyglutamine expanded huntingtin. FASEB J. 20,E1866–E1877 (2006)

Key Words: polyglutamine disease � basal ganglia � chorea� hyperkinetic disorders � Huntington’s disease � medium

spiny neurons

Huntington’s disease (hd) is a fatal neurodegenera-tive disorder usually manifested around midlife withchorea, dementia, and a variety of neuropsychiatricconditions such as agitation, mood disorders, irritabil-ity, and even psychosis (1–3). This hyperkinetic disor-der is directly related to the expansion of an unstable

CAG repeat in exon 1 of the IT15 gene (4). CAGrepeats beyond 36 predispose individuals to developHD with a complete penetrance. The expanded HDallele is autosomal dominant, and symptoms will inevi-tably appear in a gene carrier (5). However, phenotypicexpressions such as age of onset, disease duration, andthe emergence of physical and mental symptoms arehighly variable, even in individuals with the samelength of CAG repeats in their IT15 gene (5–8).

The protein product of the IT15 gene, huntingtin, isa protein normally found in the cytoplasm, where itassociates with vesicular structures and microtubules(9). The function of the huntingtin is not fully under-stood, and the critical processes determining deleteri-ous effects of its expanded form have yet to be defi-nitely identified. For example, despite ubiquitousexpression of huntingtin throughout the brain andother tissues, GABAergic neurons expressing dopa-mine (DA) receptors in the striatum predominantlydegenerate in HD patients, with layers of cerebralcortex being affected to a much lower extent andstriatal interneurons (mostly NAPDH- and ChAT-posi-tive) being spared (10–12). These observations aredifficult to reconcile unless there are factors in additionto the mutation in the IT15 gene that enhance thevulnerability of the striatal projection neurons.

DA input from the ventral midbrain neurons haslong been suspected to play a role in the pathophysiol-ogy of HD. Medium spiny GABA neurons in the stria-tum receive the densest dopaminergic innervations inthe brain, and DA is recognized for its potential toxicityto striatal neurons (13, 14). Clinical data support thiscontention, as DA-depleting agents reduce chorea inHD patients whereas administration of l-DOPA en-hances dyskinetic symptoms (15–21). Recently, in vitrostudies have documented that striatal primary culturecontaining a portion of the human huntingtin genewith expanded CAG repeats are more susceptible to

1 Correspondence: M.G.C.: Department of Cell Biology, Cen-ter for Models of Human Disease, IGSP, Box 3287, DukeUniversity, Durham, NC, 27710, USA; E-mail: caron002@mc.duke.edu or M.C.: Neuroscience Research Group, University ofQuebec at Trois-Rivieres, C.P. 500, Trois-Rivieres, Quebec G9A5H7, Canada. E-mail: cyrmi@uqtr.ca

doi: 10.1096/fj.06-6533fje

E1866 0892-6638/06/0020-1866 © FASEB

DA-related reactive oxygen species (ROS) and degen-eration induced by an excessive stimulation of DAreceptors (22, 23). However, whether changes in theefficacy of DA transmission can alter the consequencesof mutated huntingtin expression in a physiologicallyrelevant animal model of HD remain unclear.

To explore the role of DA in the behavioral andcellular consequences of an expanded CAG repeats inthe IT15 gene in vivo, we generated a mouse model ofHD with a persistently elevated striatal dopaminergictone. This strain was generated by mating the DAtransporter knock-out (DAT�/�) mice, which have5-fold elevation in striatal extracellular DA levels (24,25), to a knock-in mice with 92 CAG repeats (24, 26).Characterization of this unique hyperdopaminergicmouse model of HD directly demonstrated that persis-tently enhanced DA transmission exacerbates locomo-tor abnormalities in this mouse model of HD andaccelerates the formation of aggregates of mutanthuntingtin in the striatal projection neurons withoutprovoking neuronal death. This study implies that DAmay contribute to the striatal deterioration induced bymutant huntingtin and provides a theoretical basis forthe clinical utility of a strategy of suppressing DAneurotransmission in HD.

MATERIALS AND METHODS

Mice

The mouse strain with increased DA neurotransmission andendogenous expression of mutant IT15 gene (Hdh-Q92) hasbeen generated by mating mice with heterozygous deletion ofthe DA transporter (24) to mice with heterozygous targetedinsertion of a chimeric human-mouse exon 1 with 92 CAGrepeats (STOCK Hdhtm4Mem/J, The Jackson Laboratory, BarHarbor, ME, USA). These mice were intercrossed for sixgenerations. Mice were housed in an animal care facility at23°C on a 12 h light/12 h dark cycle with food and waterprovided ad libitum. Animal care was in accordance with theGuide for Care and Use of Laboratory Animals (NIH publication865–23, Bethesda, MD, USA) and approved by the Institu-tional Animal Care and Use Committee.

Behavioral assessments

Locomotor activity was evaluated using an automated Omni-tech Digiscan apparatus (AccuScan Instruments, Columbus,OH, USA) under illuminated conditions as described previ-ously (27). All behavioral experiments were performed be-tween 10:00 AM and 5:00 PM. Mice were observed individu-ally at 5 min intervals for 1 h. To ensure that the activityperiod was recorded rather than the reactivity period, weexcluded the first 15 min of each session from the assay.Locomotor activity was measured in terms of the total dis-tance covered, and stereotypy time refers to the total timeanimals exhibit stereotypic behavior (repetitive beam breaksof a given beam with an interval of less than 1 s).

Histological assessments

Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.)and perfused transcardially with ice-cold saline (0.9% NaCl),

followed by ice-cold 4% paraformaldehyde in 0.1 M boraxbuffer (PFA), pH 9.5. Brains were postfixed overnight in 4%PFA, immersed for a few hours in 10% (w/v) sucrose/4% PFAsolution, frozen in isopentane over dry ice, and kept at –80°C.Twenty micron tissue sections were prepared using a cryostatand kept in PBS solution at 4°C until used. Free-floatingsections were immunostained using the following primaryantisera directed against: polyglutamine (dilution 1:10000,1C2, MAB1574, Chemicon, El Segundo, CA, USA), GFAP(dilution 1:2000, MAB360, Chemicon), TH (dilution 1:1000,MAB5280, Chemicon), activated caspase-3 (dilution 1:20,AB3623, Chemicon), NeuN (dilution 1:200, MAB377, Chemi-con), and aggregated mutant huntingtin [dilution 1:2000,electron microscopy (EM)-48, gift from X. J. Li]. The sectionswere incubated with the appropriate labeled secondary anti-body (Ab) (Vector Laboratories, Burlingame, CA, USA). Notethat some of the brain sections were also incubated for 10 minat room temperature with a marker of cell nucleus, Hoechstdye 33342 (Molecular Probes, Eugene, OR, USA), at adilution of 1:10,000 in PBS. Sections were then slide-mountedusing vectashieldTM (Vector Laboratories) and cover slipped.A Zeiss confocal microscope (LSM-510) was used for sectionsviewing and images capturing. The EM48 immunostainingrevealed nuclear staining, nuclear aggregates, and neuropilaggregates. The categorizations were determined by countingthe number of nuclear staining or nuclear aggregates asrevealed by the EM-48 Ab for every 100 positive neuronslabeled by the NeuN Ab. For each structure, positive neuronssampled in a 0.31 �m3 (0.125 mm�0.125 mm�20 �m; 40 �objective) area in both hemispheres of three different sec-tions per animal were considered. Anatomical landmarkssuch as aspect, size, and situation of the anterior commis-sures, corpus callosum, and ventricles were used to ensurethat levels studied were similar within and between groups.The category definitions follow, with percents representingthe average number of nuclear staining or nuclear aggregatesper neurons: –, no cells (0%); �, few cells (1–25%); ��,scattered cells (25–50%); ���, numerous cells (50–75%);����, almost all cells (75–90%). Categorization of theneuropil aggregates was performed similarly to nuclear stain-ing or nuclear aggregates, except that the total numbers ofneuropil aggregates were counted in a 0.05 �m3 area (0.05mm�0.05 mm�20 �m; 100 � objective) in both hemispheresof three different sections per animal: –, no neuropil aggre-gates; �, few (1–10); ��, moderate (10–50); ���, dense(50–100); ���� very dense (100–150). All sections werecoded, and quantitative analyses of specimens were per-formed without the knowledge of genotypes.

For TUNEL assays (Roche Diagnostics, Mannheim, Ger-many), brain sections were mounted on microscope slides,desiccated under vacuum overnight, and fixed in 4% PFA.Slides were then treated with proteinase K (50 �g/ml in 0.1 MTris, pH 7.5, and 50 mM EDTA pH 8.0) and the protocolfrom the manufacturer (Roche Diagnostics) was followed.Note that a positive control was performed for every assay asrecommended by the manufacturer.

Biochemical and neurochemical analyses

For filter trapping assays, the mouse striatum was rapidlydissected out, frozen in liquid nitrogen, and kept at –80°Cprior to proteins extraction. Tissues were homogenized in aTris/SDS buffer (50 mM Tris, pH 7.5, 50 mg/ml SDS)supplemented with a cocktail of protease inhibitors (Sigma,St. Louis, MO, USA). Protein concentrations were measuredusing a DC-protein assay (Bio-Rad, Hercules, CA, USA). Equalamounts of proteins (200 �g) were transferred to nitrocellu-lose membranes (0.2 �, Schleicher and Schuell, Dassel,Germany) using a microfiltration apparatus (Bio-Dot, Bio-

E1867ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE

Rad). Proteins were detected using primary antibodies di-rected against polyglutamine (1C2) and mutant huntingtin(electron microscopy-48). Equal amounts of proteins (20 �g)were also separated on SDS/10% PAGE and transferred tonitrocellulose membranes in which the actin protein wasdetected as a loading control.

As described (28, 29), microdialysis probes of 2 mmmembrane length, 0.24 mm outer diameter (Cuprophane, 6kDa cutoff; model CMA-11; CMA Microdialysis, Solna, Swe-den) were implanted into the right striatum of anesthetizedmice (stereotactic coordinates: antero-posterior, 0.0 mm;dorso-ventral, –4.4 mm; lateral, 2.5 mm relative to bregma;ref. 30). Microdialysis experiments were performed at least24 h after implantation of the probe. A quantitative “lowperfusion rate” microdialysis approach (70 m/min) in freelymoving mice was used to directly compare basal extracellularlevels of DA (28). DA and its metabolite levels in dialysates orin striatal homogenates were measured by HPLC with elec-trochemical detection as described previously (28, 29).

Statistical analysis

Student’s two-tailed t test was used for statistical comparisonsbetween two groups and one-factor ANOVA followed by posthoc Fisher PLSD test, when appropriate, was applied for acomparison between more than two groups using GraphPadprism version 2.0 (Abacus concepts, Berkeley, CA, USA).

RESULTS

Behavioral manifestations in hyperdopaminergic miceexpressing mutant huntingtin

To investigate the behavioral consequences of pairingthe dopaminergic hyperactivity genotype (DAT�/�)with the mutant huntingtin protein genotype(HdhQ92/Q92), we followed the locomotor activity ofDAT�/�,HdhQ92/Q92 mice in comparison to litter-mate control mice that included DAT�/�, Hdh�/�,DAT�/�,HdhQ92/Q92, and DAT�/�, Hdh�/�mice. Two parameters of locomotor activity were ana-lyzed over a period of 1 year: horizontal activity definedas the total distance covered per hour in the open fieldand the stereotypy time, the total time that miceexhibited a stereotypic behavior (repetitive beambreaks of a given beam).

Effects of the HdhQ92/Q92 genotype were subtle onthe DAT�/� genotype (Fig. 1A, B). A between-geno-type analysis at each age revealed that the distancetraveled and stereotypy time were not statisticallydifferent between DAT�/�,HdhQ92/Q92 andDAT�/�, Hdh�/� mice (Fig. 1A, B). Nonetheless,the distance traveled was significantly lower in theDAT�/�,HdhQ92/Q92 mice at 12 months of agecompared with those at 2 months of age (within-genotype analysis, P�0.05, Fisher PLSD; Fig. 1A). Thedistance traveled and stereotypy time of DAT�/�,Hdh�/� mice or the stereotypy time of DAT�/�,HdhQ92/Q92 mice were unchanged with aging asrevealed by a within-genotype analysis (Fisher PLSD;Fig. 1A, B).

On the other hand, remarkable changes were ob-

served in the locomotor activity of DAT�/�,HdhQ92/Q92 mice. In these mice, the distance traveled wassignificantly lower at 6, 8, and 12 months of age thanthat of age-matched DAT�/�, Hdh�/� mice (Fig.1A). Moreover, a within-genotype analysis revealed asignificant decrease with aging in the distance traveledof DAT�/�,HdhQ92/Q92 mice at 6, 8, and 12 monthsof age compared with mice at 2 months of age (P�0.01,P�0.01, and P�0.005, respectively, Fisher PLSD; Fig.1A). No effect of aging was observed in DAT�/�,Hdh�/� mice (Fisher PLSD; Fig. 1A). Intriguingly,the stereotypy time did not follow this pattern ofchanges. In fact, it was significantly higher in DAT�/�,HdhQ92/Q92 mice at 6 and 8 months of age than inage-matched DAT�/�, Hdh�/� mice (Fig. 1B). Thewithin-genotype analysis revealed that only 6-month-oldDAT�/�,HdhQ92/Q92 mice showed higher stereo-typy than 2-month-old mice (P�0.01, Fisher PLSD; Fig.1B). Furthermore, stereotypy time at 12 months of agereturned to the values present at 2 months of age. Nostatistically significant effect of age was observed in

Figure 1. Locomotor alterations in a mouse model of HDwith enhanced dopamine transmission. A) Locomotor activityof DAT�/�,Hdh�/�, DAT�/�,HdhQ92/Q92, DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92 is shown as the hor-izontal distance traveled (meters/45 min) and (B) time spentin stereotypy (s/45 min). Activity was recorded every 5 minover a 45 min period in a novel environment in mice (n�9mice/group) 2, 4, 6, 8, and 12 months of age. Data representmean � sem for n � 9 mice/group. *P � 0.05, **P � 0.01 and***P � 0.005 vs. DAT�/�, Hdh�/�. Student’s t test.

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DAT�/�, Hdh�/� mice for either the distance trav-eled or stereotypy time (Fisher PLSD; Fig. 1A, B).

Dopamine levels were unaltered inDAT�/�,HdhQ92/Q92 mice

To test whether the changes seen in locomotor activi-ties in DAT�/�,HdhQ92/Q92 mice might be relatedto alterations in DA neurotransmission, the striatallevels of DA and its metabolites 3,4-dihydroxyphenyla-cetic acid (DOPAC) and homovanillic acid (HVA) weremeasured in mice of all genotypes at 8 months of age.No difference in DA, DOPAC, and HVA levels wasobserved between DAT�/�, Hdh�/� mice andDAT�/�,HdhQ92/Q92 mice or between DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92 mice (Fig 2A).However, there was a clear impact of DAT deletion onDA and HVA levels in both Hdh�/� and HdhQ92/Q92 mice. In addition, basal extracellular levels of DAwere verified by a quantitative in vivo microdialysis “lowperfusion rate” approach (28) in the striatum of freelymoving mice (Fig. 2B). In agreement with previousdata, DAT deletion resulted in a significant increase inextracellular DA in both Hdh�/� and HdhQ92/Q92mice (25). However, no statistical difference was ob-served between DAT�/�, Hdh�/� mice and DAT�/�,HdhQ92/Q92 mice or between DAT�/�,HdhQ92/Q92 and DAT�/�, Hdh�/� mice (Fig 2B). Thesedata suggest that the changes observed in the motorbehavior of DAT�/�,HdhQ92/Q92 mice cannot beexplained by alterations in striatal DA levels.

Mutant huntingtin aggregated prematurely in thestriatum of DAT�/�,HdhQ92/Q92 mice

The physical nature of the mutant huntingtin pro-teins was verified by immunofluorescence analysis inthe striatum of DAT�/�,HdhQ92/Q92 mice, usingthe anti-aggregated huntingtin EM-48 Ab (31). As acontrol, EM-48 labeling was verified in the striatumof DAT�/�, Hdh�/� mice and no positive signalwas detected (Fig. 3A). Similarly, no positive signalwas observed in the striatum of DAT�/�, Hdh�/�mice (Fig 3B). In striatal neurons of DAT�/�,HdhQ92/Q92 mice, a positive nuclear stainingknown to be associated with the emergence of micro-aggregates of mutant huntingtin (26, 32, 33) wasobserved at 8 months of age (Fig 3C, Table 1). Thisnuclear staining was observed more frequently at 12months of age (Fig 3C, Table 1). Remarkably, thenuclear staining of mutant huntingtin was detectedas early as 4 months of age (Fig. 3D, Table 1) in thestriatal neurons of DAT�/�,HdhQ92/Q92 mice. By8 months of age, in addition to nuclear microaggre-gates, nuclear aggregates of huntingtin were ob-served as well as neuropil aggregates (Fig. 3D, Table1, and Table 2). Nuclear aggregates were large,single, round, and similar to those first described forthe R6/2 mouse model and in the brain of HDpatients (34, 35). The neuropil aggregates were more

noticeable at 12 months of age, and nuclear staining aswell as the number of nuclear aggregates was increasedin the striatal neurons of DAT�/�, HdhQ92/Q92mice (Fig. 3D, Tables 1, 2).

In addition to the immunofluorescence study, weperformed a filter trapping assay on mouse striatalextracts followed by EM-48 immunoblotting. This assayallows the detection of nuclear aggregates of mutanthuntingtin (SDS-resistant) selectively. Furthermore, toverify the gene-dose effect of deleting the DA trans-porter on the formation of nuclear aggregates, weinvestigated the consequences of the HdhQ92/Q92

Figure 2. Levels of DA and its metabolites DOPAC and HVAare not altered in the striatum of DAT�/� mice with orwithout expression of the HdhQ92 gene. A) Striatal tissuelevels of DA, DOPAC, and HVA in DAT�/�,Hdh�/� (n�5mice), DAT�/�,HdhQ92/Q92 (n�5), DAT�/�,Hdh�/�(n�6), and DAT�/�,HdhQ92/Q92 (n�4). DA and HVAlevels in DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92were significantly altered vs. DAT�/�,Hdh�/� andDAT�/�,HdhQ92/Q92, respectively. B) Extracellular DAconcentrations measured by quantitative microdialysis “lowperfusion rate” approach in striatum of freely moving mice intheir home cages (n�5 mice/group). Extracellular DA levelsin DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92 weresignificantly altered vs. DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92, respectively. Mice were age-matched (8-months-old). Data represent mean � sem, ***P � 0.005 vs.DAT�/�,Hdh�/� and DAT�/�,HdhQ92/Q92. Student’st test.

E1869ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE

mutation on DAT�/� background (DAT�/�,HdhQ92/Q92 mice), which normally have a 2-foldelevation in extracellular DA concentrations in thestriatum (25). Striatal aggregates of huntingtin weredetected in the DAT�/�,HdhQ92/Q92 at 12 monthsof age, in the DAT�/�,HdhQ92/Q92 at 8 and 12months of age, and in the DAT�/�,HdhQ92/Q92 at4, 8, and 12 months of age (Fig 4A). As a loadingcontrol, equal amounts of striatal extracts were sepa-rated by Western blot and immunoblotted for actinprotein (Fig 4B). A densitometric analysis of the EM-48immunoblot revealed a statistically significant effect ofage and genotype on the aggregation of mutant hun-tingtin (Fig 4C). Another Ab, the antipolyglutaminetract (1C2), which specifically recognizes large polyglu-tamine tracts (36), especially those that are expanded,confirmed that the aggregated form of the mutanthuntingtin was trapped in the membrane (Fig 4D).

Note that as negative controls, filter trapping assayswere also performed using the EM-48 and the 1C2antibodies in striatal extracts of DAT�/�, Hdh�/�and DAT�/�, and Hdh�/� mice; no signal wasdetected. The presence of huntingtin aggregates wasalso verified by this technique in the cerebral cortex(Fig 1E) and hippocampus (data not shown), and nosignal was noticed at any age or genotype.

Distribution of aggregates of mutant huntingtin in theDAT�/�,HdhQ92/Q92 mice

To explore the brain regional specificity of aggregateformation, an immunofluorescence analysis was per-formed in the whole brain of mice. As observed in thestriatum of the DAT�/�,HdhQ92/Q92 mice, nuclearstaining as well as nuclear aggregates appeared muchearlier in the olfactory tract, cerebral cortex, piriformcortex, striatum, hippocampus, dentate gyrus, and cer-ebellum in these mice compared with DAT�/�,HdhQ92/Q92 mice (Table 1, Fig. 5A–D). It is notewor-thy that all of these brain regions receive dense dopa-minergic input (37, 38). No positive signal was observedin the brain of DAT�/�, Hdh�/� and DAT�/�,Hdh�/� mice.

One fascinating result was the emergence of neuropilaggregates in the striatum, olfactory tract, externalsegment of globus pallidus and subtantia nigra parsreticulata of DAT�/�,HdhQ92/Q92 mice (Fig. 5E, F;Table 2). These aggregates were not observed in thebrain of DAT�/�,HdhQ92/Q92 (Fig. 5E, F; Table 2)or in DAT�/�,Hdh�/� and DAT�/�,Hdh�/�mice. In the striatum and olfactory tract, neuropilaggregates became visible together with the emergenceof nuclear staining and before the appearance ofnuclear aggregates (Tables 1 and 2). It is noteworthythat whereas few neuropil aggregates were seen in thesubtantia nigra at 4 months of age in the DAT�/�,HdhQ92/Q92 mice, they were already observed inmoderate number in the external segment of globuspallidus (Table 2).

To verify whether neuropil and nuclear aggregateswere located in DA neurons, we performed doubleimmunofluorescence in the brain of DAT�/�,HdhQ92/Q92 mice using EM-48 and a tyrosine hydrox-ylase Ab as a marker of dopaminergic neurons (Fig 5G).The neuropil aggregates observed in the subtantianigra and the globus pallidus were not found in thedopaminergic neurons (Fig. 5G). Moreover, no nuclearaggregates were detected in the DA neurons of thesubtantia nigra pars compacta (Fig 5G, left panel). Thepresence of neuronal death was investigated on brainsections of 12-month-old mice by immunofluorescenceusing the activated-caspase 3 Ab and the TUNEL assay.No positive signal was detected using both techniquesin all brain regions investigated (striatum, olfactorytract, cerebellum, hippocampus, and cerebral cortex)in the DAT�/�,Hdh�/�, DAT�/�,HdhQ92/Q92and DAT�/�,HdhQ92/Q92 mice (data not shown).

Figure 3. Early formation of huntingtin aggregates in thestriatum of DAT�/�,HdhQ92/Q92 mice. Examples of im-munofluorescence analysis using EM-48 Ab in the striatum ofmice A) DAT�/�,Hdh�/�; B) DAT�/�,Hdh�/�; C)DAT�/�,HdhQ92/Q92; and D) DAT�/�,HdhQ92/Q92 at4, 8, and 12 months of age. EM-48 Ab recognized selectivelythe aggregated form of huntingtin. Representative sectionsfor each age-matched group were selected from 6 coronalsections per mice (n�6 mice/group) within the interval �0.50 to � 0.10 from Bregma (30). Neuronal nuclei areimmunostained using NeuN antibodies (red) and aggregatesof the mutant huntingtin using EM-48 (green). The letter “i”is the symbol for nuclear staining, “ii” � nuclear inclusion,and “iii” � neuropil aggregate. Scale bar � 10 �m.

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DISCUSSION

Although the predisposition to develop HD is linkedto genetic factors, mechanisms underlying the emer-gence of specific neuropathological processes anddevelopment of symptoms in carriers of mutanthuntingtin are still unidentified. Here we show, usinga mouse model of HD with enhanced dopaminergictransmission, that the neuronal context plays a keyrole in influencing the effects of mutant huntingtin.We describe that the striatum’s dense dopaminergicinnervation may contribute significantly to the vul-nerability of this region in HD through its ability totrigger the formation of nuclear and neuropil aggre-gates of mutant huntingtin.

We have observed that the locomotor activity of theDAT�/�,HdhQ92/Q92 mice is not significantly al-tered, although biphasic subtle motor alterations canbe discerned with an increase in locomotion andstereotypy at 6 months of age, followed by a decrease at12 months. However, in DAT�/�,HdhQ92/Q92 micethere is a progressive decrease in activity and a markedincrease in stereotypic behavior that becomes apparentat 6 months of age and returns to control values at 12months. This suggests that an enhanced DA transmis-sion in the brain of HD mouse model exacerbates theemergence of behavioral symptoms without affectingthe biphasic pattern of locomotor alterations withaging. An increase in repetitive movements at an earlyage followed by a decrease is documented in a knock-in

TABLE 1. Nuclear staining (NS) and nuclear aggregates (NA) in the brain of HdhQ92/Q92 mice with or without the DAT genotype

4-month-old 8-month-old 12-month-old

Brain region NS NS NA NS NADAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/�

Olfactory bulb - - - - - - - - - -Olfactory tract �/� �� � ��� - � �� ���� �/� ���Cerebral

cortexLayer II/III - - - �/� - - - �� - �/�Layer IV/V - - - - - - - - - -Piriform cortex - - - � - �/� �/� �� - �Striatum �/� �� � ��� - � �� ���� �/� ���Globus pallidusInternal

segment - - - - - - - - - -External

segment - - - - - - - - - -Subtantia nigra

parscompacta - - - - - - - - - -

HippocampusLayer CA1 - - - - - - - - - -Layer CA2/3 - - - - - - - � - -Dentate gyrus - - - - - - - � - -Cerebellum - - - � - - �/� �� - �/�

TABLE 2. Neuropil aggregates in the brain HdhQ92/Q92 mice with or without the DAT expression

4-month-old 8-month-old 12-month-old

Brain region DAT�/� DAT�/� DAT�/� DAT�/� DAT�/� DAT�/�Olfactory bulb - - - - - -Olfactory tract - �/� - � - ��Cerebral cortexLayer II/III - - - - - �/�Layer IV/V - - - - - -Piriform cortex - - - �/� - �Striatum - �/� - � - ��Globus pallidusInternal segment - - - - - -External segment - �� - ��� - ����Subtantia nigra - � - �� - ���HippocampusLayer CA1 - - - - - -Layer CA2/3 - - - - - -Dentate gyrus - - - - - -Cerebellum - - - - - -

E1871ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE

mouse model of HD with 94 CAG repeats, whenlocomotor activity is recorded during the dark phase ofthe diurnal cycle (33). In general, along with our data,no major signs of motor dysfunction have been re-ported in knock-in mouse models of HD with 92 CAGrepeats or with a similar number of CAG repeats inrelation to aging (26, 32, 39). On the other hand,robust biphasic motor alterations with aging are re-ported in knock-in mouse models with a high numberof CAG repeats (94 CAG repeats) in YAC and trans-genic mouse models as well as in a rat model of HD (34,40–44). It thus appears that the motor behavior of thedouble mutant mice is to some extent comparable withone of the other mouse models of HD with more severephenotypes. These observations indicate that enhancedDA transmission in the brain of HD mice does notinfluence the pattern of appearance of motor alter-ations, but rather the severity of its manifestation.

Formation of nuclear aggregates appears much ear-lier and to a greater extent in striatal neurons of HDmice with 5-fold enhanced DA levels in the DAT�/�background. Even a relatively moderate 2-fold elevationin extracellular DA in DAT�/� mice significantly

affects the formation of huntingtin aggregates. Nor-mally, nuclear aggregates can be observed at 15 to 18months of age in HdhQ92/Q92 mice alone or inHdhQ94/Q94 knock-in mice (26, 33). However, inclu-sion formation is believed to be the end stage of aprocess beginning with huntingtin translocation to thenucleus and continuing with the formation of microag-gregates (33, 34). The brain areas showing early nu-clear staining or microaggregates in the DAT�/�,HdhQ92/Q92 mice (i.e., the striatum and olfactorytract) all receive a dense dopaminergic innervation (37,38). As the mice aged, nuclear aggregates becamelarger and were seen in other areas of the brain such asthe piriform cortex, hippocampus, cerebellum, andsome layers of the cerebral cortex. These regions alsoreceive significant dopaminergic input (38). The obser-vation that brain regions with early formation of nu-clear aggregates of the mutant huntingtin proteins allreceive dense dopaminergic inputs has also been notedin knock-in mouse models of HD with 140 and 150 CAGrepeats (42, 45). Altogether, these observations demon-strate that in addition to the dose effect of extracellularDA levels on the formation of aggregates in the

Figure 4. Formation of huntingtin aggregates is influenced by the DAT genotype. A) Equal amounts of protein (200 �g)extracted from the striatum were transferred to nitrocellulose membranes by the filter trapping assay. Detergent-resistantproteins were detected using the primary antibodies EM-48 directed against mutant huntingtin. B) Equal amounts of protein (20�g) extracted from the striatum were separated on SDS/10% PAGE and immunoblotted for actin as a loading control. C)Densitometry analysis of the filter trapping assays using the EM-48 Ab. Data represent mean � sem, n � 4 mice/group; eachmeasure is from triplicate assay/mice. *P � 0.05, **P � 0.01, and ***P � 0.005 vs. DAT�/�,HdhQ92/Q92 with respective age;†P � 0.05 and ††P � 0.01 vs. respective 4-month-old; ‡P � 0.05, ‡‡P � 0.01, and ‡‡‡P � 0.005 vs. respective 8-month-old. ANOVAfollowed by Fisher PLSD test. D) The very same filter trapping membranes were probed with an antipolyglutamine Ab 1C2. E)The presence of detergent-resistant proteins from cortical extract was verified by a filter trapping assay using EM-48 Ab.

E1872 Vol. 20 December 2006 CYR ET AL.The FASEB Journal

HdhQ92/Q92 knock-in mice, the kinetics of aggrega-tion of mutant huntingtin is to some extent under theinfluence of dopaminergic transmission.

The nuclear aggregates of mutant huntingtin in theDAT�/�,HdhQ92/Q92 mice are observed without theemergence of signs of degeneration. It is well estab-lished that although there is formation of nuclearaggregates in several HD mouse models, none repro-duce the massive selective neuronal death that can beseen in the striatum of advanced HD patients (46). Thereasons for this are unknown, but may include therelatively short life span of the mouse. It is noteworthythat in these mouse models, nuclear aggregates areobserved at a time point when both behavioral andneuropathological changes, including neuronal loss,are present, suggesting that aggregates are not involvedin the initiation of neuronal loss (33, 43, 46, 47). Infact, although aggregates are the common hallmark ofnumerous polyglutamine disorders, studies in HD pa-tients showed little overlap between those cells exhibit-ing nuclear aggregates and cells that undergo neuro-degeneration, further supporting the lack ofcorrelation between aggregates and neuronal loss (31,48). Some studies even propose the idea that aggre-gates might be a protective mechanism, as inclusion

body formation seems to reduce the risk of neuronaldeath (47, 49). This is an interesting issue; we reportedearlier that a subpopulation of DAT�/� mice exhib-ited evidence of neuronal degeneration in the striatum(14). Whether aggregates of mutant huntingtin play arole in the absence of neuronal degeneration in theDAT�/�,HdhQ92/Q92 clearly requires further inves-tigation. On the other hand, ROS, induced by an excessof DA, is documented to make striatal neurons express-ing the mutant huntingtin gene in primary culturemore vulnerable to neuronal death (22, 23). However,despite the increased DA levels in DAT�/� mice,indirect observations suggest that an increase in ROS isunlikely in these mice, which might explain why neu-ronal death is not observed in DAT�/�,HdhQ92/Q92(14, 25, 50–52).

There is considerable evidence that aberrant DAmetabolism and transmission might underlie the clini-cal manifestations of HD. This idea was first proposedwhen asymptomatic relatives of individuals with HDdeveloped dyskinesias after being given l-DOPA (13,15, 16). Later it was reported that DA receptor antag-onists and release inhibitors were effective in treatingchorea, a major symptom in HD (17–21). Moreover,psychiatric manifestations of cortical and striatal dys-

Figure 5. The physical nature of mutant huntingtin is altered specifically in brain regions receiving dopaminergic input inDAT�/�,HdhQ92/Q92 mice. Examples of brain sections immunolabeled with EM-48 showing the aggregates of mutanthuntingtin in different regions of the brain (A–F) in 12-month-old DAT�/�,HdhQ92/Q92 and DAT�/�,HdhQ92/Q92 mice.Neuronal nuclei are immunostained using NeuN antibodies (red) and aggregates of mutant huntingtin using EM-48 (green).Scale bar � 100 �m. Representative sections were selected from 3 coronal sections per mice, 3 mice per genotype. G)Dopaminergic neurons in the substantia nigra and globus pallidus of DAT�/�,HdhQ92/Q92 were labeled using tyrosinehydroxylase (TH) Ab (red); aggregates of mutant huntingtin were labeled using the EM-48 Ab (green). Representative brainsections were selected from 4 coronal sections per mice (n�3). Scale bar � 10 �m.

E1873ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE

function can be observed in carriers of the HD muta-tion prior to the onset of overt clinical movementdisorder, and these manifestations can be reversed byDA receptor antagonists (53). These clinical observa-tions suggest that lowering the DA transmission couldbe beneficial in HD and indicate that “hyperactive” DAtransmission might contribute to development of thedisease. In contrast, some biochemical studies in HDbrain tissues and mouse models of HD suggest a“hypoactive” DA transmission. For instance, severalPET scanning studies show a reduction in levels ofstriatal D1 and D2 receptors in asymptomatic patientscarrying the mutation of the gene for huntingtin beforethe onset of overt clinical movement disorder (7,54–58). Whether these lower numbers of D1 and D2receptors are paralleled by an alteration in presynapticcomponents at an early stage or simply reflect degen-eration of striatal neuronal population is unclear.Nonetheless, despite the decrease in D1 and D2 recep-tors, D1 agonists elicited comparable induction of theimmediate early genes Zif268 and N10 and enhancedc-Fos and Jun B in a mouse model of HD, suggestingsupersensitivity of this receptor (59). There is also anincrease in the number of protein G and D5 DAreceptors, enhanced levels of Ca2�, and a neuronalhypersensitivity to application of NMDA in many pre-symptomatic HD mouse models (39, 60–63). Alto-gether, these data suggest that changes in DA transmis-sion emerge early during disease progression andmight include at least for a period of time an overacti-vation of the striatal neuronal elements.

In DAT�/� mice, the persistently enhanced DAtransmission in the striatum is associated with an earlydecrease in D1 and D2 receptors, followed by anincrease in the level of markers of DA receptors activa-tion (14, 24, 64). The sustained overactivation of DAreceptors in these mice has been documented to beassociated with subtle degeneration of striatal GABAer-gic neurons and motor alterations, in addition to anenhanced sensitivity to 3-nitropropionic acid, a mito-chondrial toxin known to induce striatal degeneration(14, 52, 65, 66). Whether the sustained DA receptoractivation in DAT�/� mice are responsible for theformation of aggregates of mutant huntingtin in thestriatal projection system of DAT�/�,Hdh Q92/Q92mice requires further investigation. However, in accordwith our contention, a recent study using striatal pri-mary cultures transiently transfected with CAG repeat-expanded fragments of huntingtin shows that DA-induced aggregates formation can be reversed by aselective D2 receptor antagonist and reproduced bydirect D2 DA receptor agonist (23). Other potentialmechanisms for the early aggregate phenotype in theDAT�/�,HdhQ92/Q92 mice may include alterationsin cdk5 and Akt activity, which have been described inan HD mouse model and in brains of patients. Cdk5and Akt activities are known to be altered in thestriatum of DAT�/� mice (14, 67), and a down-regulation in these proteins’ activity has been shown to

enhance the aggregation of mutant huntingtin (68–70).

In addition to nuclear aggregates of mutant hunting-tin, neuropil aggregates were observed in the striatum,the external segment of globus pallidus, and substantianigra pars reticulata of DAT�/�,HdhQ92/Q92 mice.That both neuropil and nuclear aggregates were ob-served simultaneously in the striatum of these mice isintriguing. Although further studies are needed todetermine whether striatal neuropil aggregates arefound in dendrites of striatal neurons or in the termi-nals of the corticostriatal axons, these neuropil aggre-gates may potentially affect the corticostriatal circuitry.For instance, a deleterious effect of these aggregates onsynaptic transmission and axonal transport has beendocumented in mouse models of HD (32, 60, 61, 71).On the other hand, the fact that the globus pallidusand substantia nigra pars reticulata contain neuropilbut not nuclear aggregates suggests that neuropil ag-gregates are found in the terminals of striato-pallidal(GPe) and -nigral axons. A higher density of neuropilaggregates are found in the external segment of globuspallidus (GPe), suggesting that neurons of the striato-pallidal pathway are affected before the striatonigral.Striatal deterioration is known to occur in the striatalprojection system that terminates in the external seg-ment of the globus pallidus (72), followed by losses inthe striatonigral pathway (73), whereas striatal projec-tions to the internal segment of the globus pallidus(GPi) remain relatively spared in HD (74, 75). In fact,neostriatal wiring models predict that the direct striato-nigral/-GPi pathway activation generates hyperkinesis(76, 77), a major behavioral symptom associated withHD. In DAT�/�,HdhQ92/Q92 mice, the formation ofrobust nuclear microaggregates and neuropil aggre-gates parallels the increase in stereotypy at 6 months ofage, whereas formation of nuclear aggregates is noticedwhen stereotypy and locomotion are significantly de-creased. Whether aggregates of huntingtin are directlyresponsible for the behavioral changes in DAT�/�,HdhQ92/Q92 mice requires further study. However,these data raise the interesting possibility that en-hanced DA transmission could accelerate the forma-tion of striatal neuropil aggregates, which in turn mayexert deleterious effects on synaptic transmission andexacerbate locomotor alterations in HD mouse models.

GABAergic/enkephalinergic striatal neurons thatproject to the external segment of the globus pallidusand the subtantia nigra are known to express highlevels of D2 receptors (76). Moreover, a higher densityof neuropil aggregates is found in the external segmentof globus pallidus (GPe) and the substantia nigra, twobrain regions where D2 receptors are concentrated.Since neuropil aggregates are found in the externalsegment of globus pallidus (GPe) and in the subtantianigra of DAT�/�,HdhQ92/Q92 mice at an early age,one could speculate that the D2 receptor might play arole in the early formation of aggregates. In accord withthis, recent in vitro findings demonstrate that a D2receptor agonist increases aggregate formation of mu-

E1874 Vol. 20 December 2006 CYR ET AL.The FASEB Journal

tated huntingtin in all cellular compartments of striatalprimary cultures, including neurites, soma, and nuclei(23). Altogether, these findings argue for a role of D2receptor stimulation in aggregate formation.

DA modulation of striatal activity is a primary physi-ological event in basal ganglia nuclei. Therefore,changes in postsynaptic phenotype expression, cytoar-chitectural properties of dendrites and their spines,and losses in the DA receptors associated with theseneuronal elements will have serious consequences forsynaptic communication in the striatum and for theoutput of the basal ganglia as a whole (32, 60, 61, 71,76, 77). Here we directly demonstrate that enhancedDA transmission potentiates locomotor alterations andincreases the occurrence of nuclear and neuropil ag-gregates in the striatum and other brain regions receiv-ing the dopaminergic input in a mouse model of HD.Until now there was no clear indication that decreasingefficacy of DA transmission (by using DA antagonists ordepleters) could affect the rate of illness progression inHD (78–80). Our study unravels a role for DA in thestriatal deterioration induced by mutant huntingtin invivo and suggests a molecular basis for clinical strategyof reducing activity of the DA system in HD.

This work was supported in part by National Institutes ofHealth grant NS19576 (to M.G.C.) and a Hereditary DiseaseFoundation research grant (to M.C.). During part of thiswork, M.C. was the holder of a Huntington’s Disease Societyof America fellowship. We thank S. Suter for excellenttechnical assistance. EM-48 Ab was a gift from Dr. X. J. Li(School of Medicine, Emory University, Atlanta, GA, USA).

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Received for publication May 22, 2006.Accepted for publication July 17, 2006.

E1877ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE

The FASEB Journal • FJ Express Summary

Dopamine enhances motor and neuropathologicalconsequences of polyglutamine expanded huntingtin

Michel Cyr,*,1 Tatyana D. Sotnikova,† Raul R. Gainetdinov,† and Marc G. Caron†,1

*Neuroscience Research Group, University of Quebec at Trois-Rivieres, Trois-Rivieres, Quebec,Canada; and †Department of Cell Biology, Center for Models of Human Disease, IGSP, DukeUniversity, Durham, North Carolina, USA

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6533fje

SPECIFIC AIMS

An expansion in the CAG repeat of the IT15 (hunting-tin) gene underlies the development of Huntington’sdisease (HD), but the basis for the specific vulnerabilityof dopamine-receptive striatal neurons remains un-clear. This study explored in vivo the role of dopaminetransmission on the behavioral and cellular conse-quences of a mutant huntingtin protein by generationof a mouse model of HD exhibiting a persistent striatalhyperdopaminergic tone. This strain was generated bycrossing the dopamine transporter knock-out mouse(DAT�/�), which exhibits a 5-fold elevation in extra-cellular dopamine levels in the striatum and locomotorhyperactivity, to a knock-in mouse model of HD con-taining 92 CAG repeats (HdhQ92/Q92).

PRINCIPAL FINDINGS

1. Motor deficits in hyperdopaminergic miceexpressing mutant huntingtin

To investigate the behavioral consequences of pairingthe dopaminergic hyperactivity phenotype (DAT�/�)with the mutant huntingtin protein genotype(HdhQ92/Q92), we followed locomotor activity inmice. No statistically significant signs of motor dysfunc-tion were observed in DAT�/�, HdhQ92/Q92 inrelation to aging. However, the enhanced dopaminetransmission in the brain of HD mouse model(DAT�/�, HdhQ92/Q92) was associated with in-creased stereotypic activity at 6 months of age, followedby a progressive decline of their locomotor hyperactiv-ity (Fig. 1). Such robust biphasic motor alteration withaging has been reported in knock-in mouse modelswith higher numbers of CAG repeats (�94 CAG re-peats) or in YAC and transgenic mouse models of HDknown to exhibit more severe phenotypes. These ob-servations indicate that enhanced dopamine transmis-sion in the brain of HD mice with 92 CAG repeats doesnot influence the pattern of motor alterations, butrather influences the severity of its manifestation.

2. Dopamine levels were unaltered inDAT�/�,HdhQ92/Q92 mice

To test whether the changes observed in locomotoractivities in DAT�/�,HdhQ92/Q92 mice were relatedto alterations in dopamine transmission, we measuredthe total striatal levels of dopamine and its metabolitesby HPLC; basal extracellular levels of dopamine wereverified by an in vivo quantitative “low perfusion rate”microdialysis approach in the striatum of freely movingmice. No statistical difference was observed betweenmice. These findings suggest that the changes noted inthe locomotor behavior of DAT�/�,HdhQ92/Q92mice could not be explained by alterations in striataldopamine levels beyond those of the DAT�/� mice.

3. Mutant huntingtin aggregated prematurely inDAT�/�,HdhQ92/Q92 mice

The physical nature of the mutant huntingtin proteinswas verified by immunofluorescence analysis in thestriatum of DAT�/�,HdhQ92/Q92 mice, using theanti-aggregated huntingtin electron microscopy(EM)-48 antibody (Ab). In striatal neurons of DAT�/�,HdhQ92/Q92 mice, a positive nuclear staining knownto be associated with the emergence of microaggregatesof mutant huntingtin, was noticed at 8 months of age(few cells) and observed more frequently at 12 monthsof age (scattered cells). Remarkably, nuclear staining ofmutant huntingtin was observed often at 4 months ofage in striatal neurons of DAT�/�,HdhQ92/Q92mice (scattered cells). By 8 months of age, in additionto nuclear microaggregate (numerous cells), nuclearaggregates of huntingtin were seen (few cells) andneuropil aggregates began to be noticeable (low num-

1 Correspondence: M.G.C.: Department of Cell Biology, Cen-ter for Models of Human Disease, IGSP, Box 3287, DukeUniversity, Durham, NC, 27710, USA; E-mail: caron002@mc.duke.edu, or M.C.: Neuroscience Research Group, Univer-sity of Quebec at Trois-Rivieres, C.P. 500, Trois-Rivieres, QuebecG9A 5H7, Canada. E-mail: cyrmi@uqtr.ca

doi: 10.1096/fj.06-6533fje

25410892-6638/06/0020-2541 © FASEB

ber). The neuropil aggregates were more often ob-served at 12 months of age (moderate number)whereas robust nuclear staining as well as nuclearaggregates were observed in the striatal neurons ofDAT�/�,HdhQ92/Q92 mice (numerous to almost all

cells; Fig. 2). In addition to the immunofluorescencestudy, we confirmed and extend these findings by afilter trapping assay on mouse striatal extracts, followedby immunoblotting with the EM-48 or the antipolyglu-tamine tract (1C2) that specifically recognizes largepolyglutamine tracts. This assay allows the selectivedetection of nuclear aggregates of mutant huntingtin(SDS-resistant). To assess the gene-dose effect of delet-ing the dopamine transporter on the formation ofnuclear aggregates, we also examined the DAT�/�,HdhQ92/Q92 group of mice that exhibit a 2-foldelevation in extracellular dopamine concentrations inthe striatum. A densitometric analysis of the EM-48 and1C2 immunoblot revealed a statistically significant ef-fect of age and genotype on nuclear aggregation ofmutant huntingtin. As documented earlier, the appear-ance of nuclear aggregates of mutant huntingtin in thestriatum cannot explain the changes observed in thelocomotor behavior of DAT�/�,HdhQ92/Q92 miceas they emerge subsequent to motor alterations. How-ever, these findings demonstrate that enhanced dopa-minergic transmission accelerates the formation ofnuclear aggregates of mutant huntingtin.

Figure 1. Locomotor activity of DAT�/�,Hdh�/�, DAT�/�,HdhQ92/Q92, DAT�/�,Hdh�/�, and DAT�/�,HdhQ92/Q92 mice is shown as the horizontal distance traveled(meters/45 min). Activity was recorded every 5 min over 45 minin a novel environment (n�9 mice/group) at 2, 4, 6, 8, and 12months of age. Data represent mean � sem for n � 9 mice/group. **P � 0.01 and ***P � 0.005 vs. DAT�/�, Hdh�/�.Student’s t test.

Figure 2. The physical nature of mutant huntingtin is alteredspecifically in brain regions receiving dopaminergic input inDAT�/�,HdhQ92/Q92 mice. Examples of brain sectionsimmunolabeled with EM-48 showing the aggregates of mu-tant huntingtin in the striatum (A), substantia nigra (B), andglobus pallidus (C) of DAT�/�,HdhQ92/Q92 and DAT�/�,HdhQ92/Q92 mice. Neuronal nuclei are immunostained usingNeuN antibodies (red) and the aggregates of mutant huntingtinusing EM-48 (green). Representative sections were selectedfrom 3 coronal sections per mice, 3 mice per genotype. Scalebar � 100 �m.

Figure 3. Enhanced dopamine (DA) transmission in aknock-in mouse model of HD containing 92 CAG repeats(DAT�/�,HdhQ92/Q92) exacerbates the deleterious ef-fects of mutated huntingtin protein on striatal function andmotor behaviors by accelerating the formation of mutanthuntingtin aggregates. Schematic diagrams represent thestriatum, globus pallidus, and subtantia nigra of 8-month-oldDAT�/�,HdhQ92/Q92 and DAT�/�,HdhQ92/Q92 miceimmunostained with EM48 Ab. Nuclear staining (large circu-lar dots) and neuropil aggregates (small oval dots) were notfound in the subtantia nigra pars compacta (SNc), whichprojects to the striatum (STR). In DAT�/�, HdhQ92/Q92,nuclear staining was noticed in only a few cells of the striatum.On the other hand, nuclear staining and neuropil aggregateswere both observed in abundance in the striatum of DAT�/�,HdhQ92/Q92 mice. A few nuclear aggregates (asterisks)were also observed in this structure. Neuropil aggregates, butnot nuclear aggregates, were found in the globus pallidus(GP) and the subtantia nigra pars reticulata (SNr). Thesechanges in the physical nature of mutant huntingtin wereassociated with a decline in locomotor behaviors in DAT�/�,HdhQ92/Q92 mice.

2542 Vol. 20 December 2006 CYR ET AL.The FASEB Journal

4. The regional distribution of neuropil aggregates ofmutant huntingtin could underlie locomotoralterations in DAT�/�,HdhQ92/Q92 mice

To explore the brain regional specificity of aggregateformation and the presence of neuronal death, immu-nofluorescence analyses were performed in the brain ofmice. Neuronal death was investigated in brain sectionsof 12-month-old mice by using an Ab raised againstactivated-caspase 3 in addition to TUNEL assays. Nopositive signal was detected using either technique inthe brain of any group of mice. Besides confirming thatmutant huntingtin protein aggregated much earlierand to a stronger extent in the striatum of DAT�/�,HdhQ92/Q92, we noted these aggregates were alsolocated in other dopaminergic brain regions such asolfactory tract, piriform cortex, and nucleus accum-bens. A fascinating result was the emergence of neuro-pil aggregates in the striatum, external segment ofglobus pallidus, and subtantia nigra pars reticulata ofDAT�/�,HdhQ92/Q92 mice, but not in DAT�/�,HdhQ92/Q92 mice (Fig. 2). The number of neuropilaggregates was significantly elevated in these structuresat 8 months, when locomotor behavior declined inDAT�/�,HdhQ92/Q92. To verify whether the nu-clear or neuropil aggregates were located in nigrostri-atal dopamine neurons, we performed a double immu-nofluorescence in the brain of DAT�/�,HdhQ92/Q92mice using EM-48 and an Ab raised against tyrosinehydroxylase as a marker of dopaminergic neurons.Neuropil aggregates of the subtantia nigra and globuspallidus observed in DAT�/�,HdhQ92/Q92 mice

were not localized in the dopaminergic terminals, andno nuclear aggregates were detected in the dopamineneurons of the subtantia nigra. These data suggest thatthe neuropil aggregates in the striatum, globus palli-dus, and subtantia nigra of DAT�/�,HdhQ92/Q92mice were likely contributing to the locomotor alter-ations seen in these mice. The fact that the brainregions with early formation of nuclear aggregates ofmutant huntingtin all receive dense dopaminergic in-puts is another indication that dopamine transmissiondid influence the kinetic of aggregation of this alteredprotein.

CONCLUSIONS AND SIGNIFICANCE

This study provides in vivo evidence that dopaminergicneurotransmission plays a role in the deleterious effectsof mutated huntingtin proteins on striatal function andmotor behavior by accelerating formation of mutanthuntingtin aggregates (Fig. 3). These findings may helpus understand the role of the dopamine system in thepathophysiology of Huntington’s disease. SymptomaticHuntington’s disease patients can develop chorea, themajor behavioral symptoms associated with Hunting-ton’s disease, when brain levels of dopamine are in-creased after receiving l-dopa. In contrast, dopaminereceptor antagonists and dopamine release inhibitorssuch as tetrabenazine are the most effective treatmentto reverse chorea. Our findings corroborate these clin-ical observations and point to an unappreciated role ofdopamine transmission in the striatal deteriorationinduced by mutant huntingtin.

2543ROLE OF DOPAMINE IN HUNTINGTON�S DISEASE