Dystonia is a disorder characterized by sustained muscle contractions, frequently causing twisting and repetitive movements, or abnormal postures (Jankovic and Fahn 1993). The most severe type of early onset generalized dystonia is an autosomal dominant disorder caused by a 3-bp deletion (ΔGAG) in the DYT1 gene that encodes torsinA (Ozelius et al. 1997). Symptoms of this disorder typically develop before the age of 21 years and include involuntary sustained muscle contractions that cause posturing of a foot, leg or arm, which frequently generalize to other body regions. The lifespan of affected individuals is not shortened, but the abnormal movements are debilitating and are difficult to treat.
The pathophysiologic mechanisms by which the ΔGAG mutation causes dystonia are unclear. There is no evidence for neuronal death in the limited number of postmortem examinations of DYT1 patients that have been conducted, although apparent enlargement of dopamine (DA) neurons (Rostasy et al. 2003) and the presence of ubiquitin inclusions (McNaught et al. 2004) have been reported. The affected protein, torsinA, shares homology with members of the AAA+ ATPase gene family (Lupas et al. 1997; Neuwald et al. 1999). Members of this family participate in numerous cellular functions, including protein folding and degradation, membrane trafficking and vesicle fusion (Ogura and Wilkinson 2001).
Genetics coupled with pharmacological studies have led to the hypothesis that at least some forms of dystonia may arise from disturbances in DA signaling. First, mutations within the TH gene (Knappskog et al. 1995), and the GTP cyclohydrolase gene (Ichinose et al. 1994), both of which limit DA synthesis result in a dystonic phenotype in humans. Second, a polymorphism within the DA D5 receptor gene has been associated with cervical dystonia (Placzek et al. 2001). Third, mechanical or ischemic lesions of the striatum and administration of pharmacological agents that block DA D2 receptors in vivo can result in a dystonic phenotype (Rupniak et al. 1986). Fourth, striatal D2 receptor binding in non-manifesting carriers of the DYT1 mutation is reduced (Asanuma et al. 2007).
In DYT1 dystonia, the available data on DA function are limited as there are very few DYT1 dystonia brains available for study, and until recently there has been a lack of relevant animal models for this disease. Previous work in a limited number of human DYT1 brains revealed an increase in the ratio of 3,4-dihydroxyphenylacetic acid (DOPAC), a DA metabolite, to DA (Augood et al. 2004). This increase suggests an abnormality in DA turnover, but the small number of cases available for study limited the strength of this conclusion.
Several published studies have focused on DA neurotransmission using different mouse models of DYT1 dystonia. The current animal models for DYT1 dystonia include various transgenic mice (Sharma et al. 2005; Shashidharan et al. 2005; Grundmann et al. 2007), in which expression of transgene is driven by assorted promoters resulting in expression of human mutant torsinA, and heterozygous ΔGAG knock-in mice and knock-out mice (Dang et al. 2005; Goodchild et al. 2005). Using transgenic DYT1 mouse, in which expression is driven by the cytomegalovirus promoter (Sharma et al. 2005), we have previously shown that the systemic administration of amphetamine-induced much less DA release in striatum of hMT1 mice as compared with hWT or control mice (Balcioglu et al. 2007). Reduced striatal tissue levels of DA metabolites–homovanillic acid and DOPAC and an increase in the ratio of DOPAC to DA in the same mouse model have also been reported (Zhao et al. 2008). Moreover, Pisani et al. (2006) showed that D2 receptor stimulation increased membrane depolarization in striatal cholinergic interneurons in the hMT1 mice compared with hWT mice. Dang et al. (2005) demonstrated reduced striatal tissue homovanillic acid levels in heterozygous Dyt1 ΔGAG knock-in mice, which express mouse mutant protein under the endogenous DYT1 promoter. They also showed reduced striatal tissue DOPAC levels in Dyt1 knock-down mice, which express lower level of mouse torsinA (Dang et al. 2005, 2006). Shashidharan et al. (2005) demonstrated reduced striatal DA levels in dystonic mice, which express mutant human torsinA under the neuronal enolase promoter. These findings all point to compromised DA neurotransmission in mouse models of DYT1 dystonia. Dopaminergic dysfunction in DYT1 dystonia and other forms of human dystonia has been reviewed in detail (Wichmann 2008). However, there is no information about the nature of the compromised DA system in dystonia. Pharmacological studies have implicated abnormalities in DA transporter, which is an important target for the action of amphetamine (Jones et al. 1998a; Sulzer et al. 2005). Thus, we studied the in vivo dynamics of DAT and the contribution of DAT to amphetamine-induced DA release along with behavioral consequences.
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We have found that DAT in hMT1 mice works less efficiently to remove extracellular DA in vivo in the striatum and that these mice travel less distance under basal conditions and following amphetamine administration.
We previously reported that expression of human mutant torsinA leads to an impairment of amphetamine-induced DA release. We were not able to attribute this effect to a reduction in the synthesis or storage of DA, because the striatal tissue levels of DA and its metabolites were not affected by expression of human mutant torsinA. Amphetamine is a complicated drug, which elicits its effect through variety of different targets, the most important being DAT (Sulzer et al. 2005; Williams and Galli 2006). Thus, we undertook no-net flux microdialysis (Jones et al. 1998b; Chefer et al. 2005) to study the efficiency of DAT in vivo in removing DA that was applied extracellularly in the striatum. The calculated lower extraction fraction indicated that DAT worked significantly less-efficiently to remove DA from the extracellular space in hMT1 mice, as compared with their non-transgenic littermates. As seen in the graph (Fig. 1), the slope of the curve was much lower for these mice suggesting that extracellular DA was not removed by DAT as efficiently compared with non-transgenic littermates. This finding could explain the impaired amphetamine-induced DA release in transgenic mice (Balcioglu et al. 2007). Less efficient DAT would take up much less amphetamine into cytosol/vesicles, and hence result in less release of DA into cytosol and the synapse. We previously studied binding of [3H]-mazindol (a ligand for DAT) in human mutant torsinA transgenic mice (Balcioglu et al. 2007) and did not report a significant difference in the density of total striatal DAT binding sites. It is important to note that the saturation binding experiments we performed addressed the total number of DAT protein and does not distinguish between functional surface and intracellular ligand binding sites.
Extracellular DA, on the other hand, is taken up by the functional membrane bound DAT. Thus no-net flux microdialysis experiments are more accurate in studying the functional status of DAT in vivo rather than comparing total number of DATs. It is plausible that either a lower number of functional membrane bound DAT or less-efficient membrane bound DAT might account for the difference in removing extracellular DA. Both possibilities are consistent with the predicted function of torsinA in which it is involved in the processing of proteins through endoplasmic reticulum (ER). Impaired processing of DAT through ER as a consequence of mutated torsinA might lead to either lower number or less efficient DAT on the membrane. In fact, torsinA is located primarily in the ER component of the secretory pathway (Hewett et al. 2000; Kustedjo et al. 2000; Liu et al. 2003) and is involved in processing proteins through that pathway (Torres et al. 2004; Hewett et al. 2007). The secretory pathway controls processing of a variety of proteins destined for cell membranes, organelles and the extracellular space, and is critical in vesicular release and reuptake of neurotransmitters (Ellgaard and Helenius 2003). Oligomerization of neurotransmitter transporters is a ‘ticket’ from the ER to the plasma membrane (Farhan et al. 2006). Consistent with a role in vesicular release of transmitters, torsinA has also been found in association with vesicles at synaptic terminals in human brain (Augood et al. 1999) and Drosophila neurons (Koh et al. 2004). In addition, other cellular data show that torsinA may modulate the function of vesicular monoamine transporter 2 and DAT (Torres et al. 2004; Misbahuddin et al. 2005) both of which are very important proteins in DA release and turnover. TorsinA is a member of the family of ATPases and may mediate ATP-dependent conformational remodeling reactions involving processing substrate proteins or protein complexes, such as DAT. Thus, it is possible that the altered amphetamine-induced DA release and DA reuptake arise from reduced surface expression and/or inefficient kinetics of DAT, which were not detected using ligand binding assay.
Extracellular DA could also be taken up by norepinephrine transporter. However, this seems to occur in areas where the distribution of DAT is sparse, such as the frontal cortex. Morón et al. (2002) clearly discussed that DA uptake in caudate nucleus of striatum primarily depended on the DAT unlike of frontal cortex. Because both non-specific and specific DAT inhibitors (cocaine and GBR 12 909, respectively) inhibited DA uptake into caudate synaptosomes from norepinephrine transporter knock-out mice, while they failed to do so in the frontal cortex synaptosomes from the same animals. Thus, the extinction coefficient of no-net flux microdialysis experiments with hMT1 mice represents activity of the DAT in the striatum to remove DA from extracellular environment.
Furthermore, we also explored the efficiency of NOM, a specific DAT inhibitor, to reduce amphetamine-induced DA release. We expected that if DAT works less than optimally in hMT1 mice, then NOM would have a different profile of inhibiting DAT. Twenty micromolar concentration appeared to block amphetamine’s effect by 84% in non-transgenic mice. The same concentration of NOM inhibited amphetamine-induced DA release by 48% in transgenic mice. The inhibition by NOM was statistically significant compared with amphetamine-treated non-transgenic mice whereas it was not in amphetamine-treated transgenic mice. This difference in NOM’s ability to inhibit amphetamine-induced DA release supports our contention that there is a difference in the activity of DAT in vivo in the presence of mutant torsinA. This suggests that DAT is involved in amphetamine-induced DA release in transgenic mice, and the less efficient inhibition by NOM in transgenic mice would be consistent with fewer and/or less efficient, possibly improperly processed, DAT at the dopaminergic terminal in hMT1 mice.
We also studied locomotion, very complicated behavior, in these mice. Even though there is no simple relationship between DA release and a specific behavioral profile, DA is crucial component in the motor activating effect of amphetamine. Amphetamine acutely increases extracellular DA in terminal DA fields by acting on DAT and this is thought to mediate the motor stimulation by amphetamine (Kuczenski and Segal 1992; Kuczenski et al. 1995). We observed that hMT1 mice travel significantly less following amphetamine administration, as well as under basal conditions, as compared with non-transgenic littermates. These results are consistent with altered dynamics of DAT in hMT1 transgenic mice with behavioral consequences. Reduced response to amphetamine-induced movement in transgenic mice would be in accord with impaired release of DA through DAT following its administration (Balcioglu et al. 2007). We also observed reduced locomotion under basal conditions. The fact that DAT functioned less efficiently in hMT1 mice is in accord with the notion that abnormalities in dopaminergic neurotransmission in mice have been associated with hypoactivity. In contrast, DAT knock-out mice show hyperlocomotion. The extent of the impaired DAT function may determine the behavioral outcome. While complete loss of DAT function leading to five times higher basal DA levels can cause hyperlocomotion (Giros et al. 1996), gradual loss of DAT function leading to no detectable change in basal DA levels may cause hypolocomotion. Moreover, these hMT1 transgenic mice have been evaluated for variety of behavioral phenotypes. They showed reduced motor learning performance in an accelerating rotarod paradigm (Sharma et al. 2005), prolonged traversal times on both square and round raised-beam tasks and more slips on the round raised-beam task and increased hind-base width (Zhao et al. 2008). Raised-beam task has been used to characterize motor dysfunction in murine models of ‘basal ganglia’ disorders such as Parkinson’s disease (Strome et al. 2006).
In conclusion, expression of human mutant torsinA expression appears to lead to compromise of DA neurotransmission, at least in part because of less efficient DAT. This would lead to impaired DA release under challenged conditions, such as amphetamine exposure, along with the impaired motor activation. Our working hypothesis is that mutant torsinA interferes with processing of proteins crucial to neurotransmission, such as DAT that is involved in DA release and re-uptake at the synapse. Changes (subtle and/or long lasting) in the function of such proteins may compromise the dynamics and regulation of DA release. Any triggering event (trauma, toxin, drug exposure, etc.) during the lifetime of DYT1 carriers may contribute to onset of dystonic symptoms because of the resulting compromise in regulation of DA release.