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,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, Duke University, Durham, North Carolina, USA
1Correspondence: M.G.C.: Department of Cell Biology, Center for Models of Human Disease, IGSP, Box 3287, Duke University, Durham, NC, 27710, USA; E-mail: caron002{at}mc.duke.edu, or M.C.: Neuroscience Research Group, University of Quebec at Trois-Rivieres, C.P. 500, Trois-Rivieres, Quebec G9A 5H7, Canada. E-mail: cyrmi{at}uqtr.ca
SPECIFIC AIMS
An expansion in the CAG repeat of the IT15 (huntingtin) gene underlies the development of Huntington’s disease (HD), but the basis for the specific vulnerability of dopamine-receptive striatal neurons remains unclear. This study explored in vivo the role of dopamine transmission on the behavioral and cellular consequences of a mutant huntingtin protein by generation of a mouse model of HD exhibiting a persistent striatal hyperdopaminergic tone. This strain was generated by crossing the dopamine transporter knock-out mouse (DAT–/–), which exhibits a 5-fold elevation in extracellular dopamine levels in the striatum and locomotor hyperactivity, to a knock-in mouse model of HD containing 92 CAG repeats (HdhQ92/Q92).
PRINCIPAL FINDINGS
1. Motor deficits in hyperdopaminergic mice expressing mutant huntingtin
To investigate the behavioral consequences of pairing the dopaminergic hyperactivity phenotype (DAT–/–) with the mutant huntingtin protein genotype (HdhQ92/Q92), we followed locomotor activity in mice. No statistically significant signs of motor dysfunction were observed in DAT+/+, HdhQ92/Q92 in relation to aging. However, the enhanced dopamine transmission in the brain of HD mouse model (DAT–/–, HdhQ92/Q92) was associated with increased stereotypic activity at 6 months of age, followed by a progressive decline of their locomotor hyperactivity (Fig. 1
). Such robust biphasic motor alteration with aging has been reported in knock-in mouse models with higher numbers of CAG repeats (>94 CAG repeats) or in YAC and transgenic mouse models of HD known to exhibit more severe phenotypes. These observations indicate that enhanced dopamine transmission in the brain of HD mice with 92 CAG repeats does not influence the pattern of motor alterations, but rather influences the severity of its manifestation.
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2. Dopamine levels were unaltered in DAT–/–,HdhQ92/Q92 mice
To test whether the changes observed in locomotor activities in DAT–/–,HdhQ92/Q92 mice were related to alterations in dopamine transmission, we measured the total striatal levels of dopamine and its metabolites by HPLC; basal extracellular levels of dopamine were verified by an in vivo quantitative "low perfusion rate" microdialysis approach in the striatum of freely moving mice. No statistical difference was observed between mice. These findings suggest that the changes noted in the locomotor behavior of DAT–/–,HdhQ92/Q92 mice could not be explained by alterations in striatal dopamine levels beyond those of the DAT–/– mice.
3. Mutant huntingtin aggregated prematurely in DAT–/–,HdhQ92/Q92 mice
The physical nature of the mutant huntingtin proteins was verified by immunofluorescence analysis in the striatum of DAT–/–,HdhQ92/Q92 mice, using the anti-aggregated huntingtin electron microscopy (EM)-48 antibody (Ab). In striatal neurons of DAT+/+,HdhQ92/Q92 mice, a positive nuclear staining known to be associated with the emergence of microaggregates of mutant huntingtin, was noticed at 8 months of age (few cells) and observed more frequently at 12 months of age (scattered cells). Remarkably, nuclear staining of mutant huntingtin was observed often at 4 months of age in striatal neurons of DAT–/–,HdhQ92/Q92 mice (scattered cells). By 8 months of age, in addition to nuclear microaggregate (numerous cells), nuclear aggregates of huntingtin were seen (few cells) and neuropil aggregates began to be noticeable (low number). The neuropil aggregates were more often observed at 12 months of age (moderate number) whereas robust nuclear staining as well as nuclear aggregates were observed in the striatal neurons of DAT–/–,HdhQ92/Q92 mice (numerous to almost all cells; Fig. 2
). In addition to the immunofluorescence study, we confirmed and extend these findings by a filter trapping assay on mouse striatal extracts, followed by immunoblotting with the EM-48 or the antipolyglutamine tract (1C2) that specifically recognizes large polyglutamine tracts. This assay allows the selective detection of nuclear aggregates of mutant huntingtin (SDS-resistant). To assess the gene-dose effect of deleting the dopamine transporter on the formation of nuclear aggregates, we also examined the DAT+/–, HdhQ92/Q92 group of mice that exhibit a 2-fold elevation in extracellular dopamine concentrations in the striatum. A densitometric analysis of the EM-48 and 1C2 immunoblot revealed a statistically significant effect of age and genotype on nuclear aggregation of mutant huntingtin. As documented earlier, the appearance of nuclear aggregates of mutant huntingtin in the striatum cannot explain the changes observed in the locomotor behavior of DAT–/–,HdhQ92/Q92 mice as they emerge subsequent to motor alterations. However, these findings demonstrate that enhanced dopaminergic transmission accelerates the formation of nuclear aggregates of mutant huntingtin.
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4. The regional distribution of neuropil aggregates of mutant huntingtin could underlie locomotor alterations in DAT–/–,HdhQ92/Q92 mice
To explore the brain regional specificity of aggregate formation and the presence of neuronal death, immunofluorescence analyses were performed in the brain of mice. Neuronal death was investigated in brain sections of 12-month-old mice by using an Ab raised against activated-caspase 3 in addition to TUNEL assays. No positive signal was detected using either technique in the brain of any group of mice. Besides confirming that mutant huntingtin protein aggregated much earlier and to a stronger extent in the striatum of DAT–/–,HdhQ92/Q92, we noted these aggregates were also located in other dopaminergic brain regions such as olfactory tract, piriform cortex, and nucleus accumbens. A fascinating result was the emergence of neuropil aggregates in the striatum, external segment of globus pallidus, and subtantia nigra pars reticulata of DAT–/–,HdhQ92/Q92 mice, but not in DAT+/+,HdhQ92/Q92 mice (Fig. 2)
. The number of neuropil aggregates was significantly elevated in these structures at 8 months, when locomotor behavior declined in DAT–/–,HdhQ92/Q92. To verify whether the nuclear or neuropil aggregates were located in nigrostriatal dopamine neurons, we performed a double immunofluorescence in the brain of DAT–/–,HdhQ92/Q92 mice using EM-48 and an Ab raised against tyrosine hydroxylase as a marker of dopaminergic neurons. Neuropil aggregates of the subtantia nigra and globus pallidus observed in DAT–/–,HdhQ92/Q92 mice were not localized in the dopaminergic terminals, and no nuclear aggregates were detected in the dopamine neurons of the subtantia nigra. These data suggest that the neuropil aggregates in the striatum, globus pallidus, and subtantia nigra of DAT–/–,HdhQ92/Q92 mice were likely contributing to the locomotor alterations seen in these mice. The fact that the brain regions with early formation of nuclear aggregates of mutant huntingtin all receive dense dopaminergic inputs is another indication that dopamine transmission did influence the kinetic of aggregation of this altered protein.
CONCLUSIONS AND SIGNIFICANCE
This study provides in vivo evidence that dopaminergic neurotransmission plays a role in the deleterious effects of mutated huntingtin proteins on striatal function and motor behavior by accelerating formation of mutant huntingtin aggregates (Fig. 3
). These findings may help us understand the role of the dopamine system in the pathophysiology of Huntington’s disease. Symptomatic Huntington’s disease patients can develop chorea, the major behavioral symptoms associated with Huntington’s disease, when brain levels of dopamine are increased after receiving L-dopa. In contrast, dopamine receptor antagonists and dopamine release inhibitors such as tetrabenazine are the most effective treatment to reverse chorea. Our findings corroborate these clinical observations and point to an unappreciated role of dopamine transmission in the striatal deterioration induced by mutant huntingtin.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6533fje
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