Overexpression of human wildtype torsinA and human ΔGAG torsinA in a transgenic mouse model causes phenotypic abnormalities

Abstract

Primary torsion dystonia is an autosomal-dominant inherited movement disorder. Most cases are caused by an in-frame deletion (GAG) of the DYT1 gene encoding torsinA. Reduced penetrance and phenotypic variability suggest that alteration of torsinA amino acid sequence is necessary but not sufficient for development of clinical symptoms and that additional factors must contribute to the factual manifestation of the disease. We generated 4 independent transgenic mouse lines, two overexpressing human mutant torsinA and two overexpressing human wildtype torsinA using a strong murine prion protein promoter. Our data provide for the first time in vivo evidence that not only mutant torsinA is detrimental to neuronal cells but that also wildtype torsinA can lead to neuronal dysfunction when overexpressed at high levels. This hypothesis is supported by (i) neuropathological findings, (ii) neurochemistry, (iii) behavioral abnormalities and (iv) DTI-MRI analysis.

Introduction

Primary torsion dystonia (DYT1 dystonia) is an autosomal-dominant inherited neurodevelopmental disorder. Most cases are caused by a 3 base pair (GAG) deletion in the coding region of the DYT1 gene (TOR1A), which is up to now the only known mutation responsible for the development of the disease (Ozelius et al., 1997). DYT1 dystonia manifests as abnormal, involuntary twisting movements most likely due to dysfunction of selective CNS motor circuits. Onset of symptoms is usually restricted to a period between 1 and 28 years of age and penetrance is reduced to 30% (Fahn, 1988, Ozelius et al., 1999), thus it is supposed that other genetic or environmental factors beyond the GAG deletion itself must have an impact on the factual manifestation of the disease. The underlying reason for the more common sporadic forms of dytonia is still unknown however there is increasing evidence, especially from recent association studies investigating polymorphisms in the torsinA gene and in regulatory regions (Clarimon et al., 2005, Kamm et al., 2006, Kock et al., 2006), that genetic variability in the expression of wildtype gene might be a risk factor for the sporadic disease (Clarimon et al., 2005).

The specific physiological role of torsinA still remains uncertain. It belongs to the AAA+ (ATPase associated with different cellular activities) protein family, and has been shown to be involved in cellular response to stress (Hewett et al., 2003, Kuner et al., 2003), in neurite outgrowth (Ferrari-Toninelli et al., 2004, Hewett et al., 2006), and dopaminergic transmission (Augood et al., 2002, Augood et al., 2003, Torres et al., 2004). Furthermore, it is still unknown how mutant torsinA disrupts to cellular function of neurons and leads to the development of an exclusively neurological disease. High expression levels in dopaminergic neurons of the basal ganglia (Augood et al., 2003, Konakova et al., 2001) together with findings from functional imaging studies (Eidelberg et al., 1998, Perlmutter et al., 1998) and investigations of neurotransmitter levels (Augood et al., 2002, Pisani et al., 2006, Torres et al., 2004) suggest that DYT1 dystonia result from a dysfunction of the dopamine system in the basal ganglia. Homozygous DYT1 knock-in and knock-out mouse models point to a loss of function mutation as animals die shortly after birth, suggesting that torsinA is indispensable for specific developmental processes in mammalian brain (Goodchild et al., 2005). By contrast, the GAG deletion is reported to be deleterious in a toxic gain of function manner, causing redistribution of torsinA from the ER to the NE (Goodchild and Dauer, 2004, Misbahuddin et al., 2005), abnormal protein interaction (Goodchild and Dauer, 2004, Goodchild and Dauer, 2005) and formation of inclusion bodies in cell culture (Hewett et al., 2000).

In the present paper we show that the pathology of DYT1 dystonia is not restricted to a dopaminergic dysfunction of the basal ganglia but involves different neurotransmitters and brain regions, than previously thought. We further demonstrate for the first time that overexpression of human wildtype torsinA alters normal function of neurons and might cause behavioral and pathological abnormalities in a transgenic mouse model when expressed at high levels. We hypothesize that overexpression or prolonged exposure to the human wildtype torsinA protein might therefore be a detrimental factor for the neuronal cell.