Regular ArticleGeneration and characterization of Dyt1 ΔGAG knock-in mouse as a model for early-onset dystonia
Introduction
The mutant DYT1 gene has been associated with Oppenheim's dystonia, a neurological movement disorder characterized by uncontrollable and persistent muscle contractions of affected body parts with an onset time during childhood or adolescence (Fahn, 1988). DYT1 dystonia has an autosomal dominant inheritance, and the mutation has a penetrance of only 30–40% (Fahn, 1991). The mutation found in DYT1 of early-onset dystonic patients is a trinucleotide deletion (ΔGAG) that results in the removal of one member of a glutamic acid residue pair (ΔE302/303) in the carboxy region of torsinA protein (Ozelius et al., 1997). Unlike Parkinson's and Huntington's disease, dystonia is not considered a neurodegenerative disorder. Instead, a change in the neurochemistry of the brain has been implicated by studies that reported dopamine and dopamine metabolic level differences between dystonic patients and control subjects (Augood et al., 2002, Furukawa et al., 2000).
The exact function of torsinA is still unknown but is believed to have a chaperone function because the protein shares close sequence homology to members of the Clp protease/heat shock protein family, which is a member of the AAA+ ATPase superfamily. Members of this family perform a variety of chaperone activities for their interacting proteins (Ogura and Wilkinson, 2001, Ozelius et al., 1998). Chaperone activities of torsinA have been demonstrated to include neuroprotection against oxidative stress and the prevention of protein aggregate formation (Caldwell et al., 2003, Hewett et al., 2003, Kuner et al., 2003, McLean et al., 2002, Shashidharan et al., 2004). A recent cell culture study has also suggested that torsinA can modulate the activity of the dopamine transporter (Torres et al., 2004). A similar modulation of the dopamine transporter was also noted in a Caenorhabditis elegans model (Cao et al., 2005).
The functional change of torsinA caused by the mutation is still unknown. The mutation, however, has been seen to alter the cellular localization of the protein. Substantial evidence has identified the relocalization of the mutant torsinA to the nuclear envelope of cells (Bragg et al., 2004, Gonzalez-Alegre and Paulson, 2004, Goodchild and Dauer, 2004, Hewett et al., 2004, McNaught et al., 2004, Naismith et al., 2004, Shashidharan et al., 2005). In brain tissues of human patients who are carriers of the ΔGAG mutation, protein aggregates that are perinuclear have been identified (Goodchild and Dauer, 2004, McNaught et al., 2004). This nuclear localization has also been detected in brains of mice that overexpress the mutant torsinA (Shashidharan et al., 2005). A nuclear envelope protein, lamina-associated protein (LAP1), has been identified as a binding partner for torsinA that when mutated has an even greater interaction to it (Goodchild and Dauer, 2005).
To determine the involvement of torsinA in the development and neuropathology of dystonia, an animal model for DYT1 dystonia is imperative. Several have been produced and have provided valuable information about the disorder, but they all have limitations as a model for the disorder. C. elegans and Drosophila melanogaster overexpression models have shown that torsinA is involved in managing protein folding and the formation of the neuromuscular junction, respectively (Caldwell et al., 2003, Koh et al., 2004). Although the findings were insightful, undoubtedly a mammalian system is needed to further explore the mutated protein's role in context of a complex neurological system similar to that of human. Recently, two mammalian models for DYT1 dystonia were reported. Both were transgenic mice that overexpressed mutated human torsinA (Sharma et al., 2005, Shashidharan et al., 2005). While the motor behavioral and biochemical findings were compelling, the relevance of these and future findings from these transgenic mice to human patients is questionable for the following reasons. First, the expression of the mutated protein was driven either by a CMV or enolase promoter and not the native promoter of the DYT1 gene, which could lead to ectopic expression of the protein. Second, overexpressing the mutant protein to a non-physiological level can stress a system in unexpected ways. Together, the ectopic and overexpression of the human torsinA protein can readily lead to artifacts that may have conflated with the true phenotype.
We have generated a mouse model of Dyt1 ΔGAG dystonia that avoids all of these complications. Our knock-in mice have one normal Dyt1 allele and the other allele with ΔGAG. Here, we report that Dyt1 ΔGAG knock-in male mice display motor abnormalities and cellular characteristics similar to the dystonic phenotype that make this mouse line a useful animal model with which to study the pathophysiology of early-onset dystonia and test potential treatments.
Section snippets
Material and methods
All experimental procedures in this report were carried out in compliance with the USPHS Guide for Care and Use of Laboratory Animals and approved by the University of Illinois Institutional Animal Care and Use Committee.
Generation of Dyt1 ΔGAG mice
The targeting construct used to generate the line of mice had a Dyt1 gene that carried the ΔGAG mutation (Fig. 1A). Twenty-eight of 73 clones screened had homologously recombined the targeting construct, which was determined by Southern blot analysis on both sides (Fig. 1B). While all 28 contained the PGKneoSTOP sequence, only three clones contained ΔGAG. We predict that a highly efficient recombination site existed within intron 4 downstream of the PGK-neoSTOP cassette and before the ΔGAG site
Discussion
We have made a gene-targeted mouse model of Dyt1 ΔGAG to mimic the mutation found in DYT1 dystonic patients. The observed motor performance deficits and tissue aggregations indicate that the mouse model recapitulates some of the phenotypes seen in dystonic patients.
Our motor behavioral characterization of Dyt1 ΔGAG mice showed that when one copy of the Dyt1 gene is mutated, fine motor balance and coordination are impaired. The consistent muscle contractions of dystonic patients can prevent them
Acknowledgments
We express our thanks to Shinichi Mitsui, Morgan Pence, Krupa Patel, Jieun Jun, and Robert Yuen for their technical assistance and Yanyan Wang and Gary Iwamoto for helpful discussions. This work was supported by funds from the Dystonia Medical Research Foundation, Bachmann–Strauss Dystonia and Parkinson Foundation, Inc., the State of Illinois, and the Lucille P. Markey Charitable Trust. Behavioral equipment was funded by the Research Board of University of Illinois at Urbana-Champaign, the
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