Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation
Introduction
Spinocerebellar ataxia type 3 (SCA3) or Machado–Joseph disease is an autosomal dominant neurodegenerative disorder caused by CAG trinucleotide repeat expansion within the coding region of SCA3 gene (Kawaguchi et al., 1994, Zoghbi and Orr, 2000, Kobayashi and Kakizuka, 2003). Ataxin-3, the SCA3 gene product, is widely distributed in the central nervous system and peripheral tissues (Kawaguchi et al., 1994, Tait et al., 1998). Normal ataxin-3 contains 12–41 glutamines near the C-terminus, and polyglutamine tract expands to 62–84 glutamines in mutant ataxin-3 (Kawaguchi et al., 1994, Kobayashi and Kakizuka, 2003). Clinical manifestations of SCA3 include cerebellar ataxia, peripheral nerve palsy, pyramidal and extrapyramidal signs (Rosenberg, 1992, Takiyama et al., 1994). In contrast to wide distribution of ataxin-3 in the brain, SCA3 neurodegeneration is mainly found in brainstem, basal ganglia, cerebellum and spinal cord (Durr et al., 1996, Zoghbi and Orr, 2000).
SCA3 belongs to the family of polyglutamine neurodegenerative disorders resulting from an expansion of unstable CAG repeat within the coding region of gene. Up to now, nine polyglutamine diseases, including Huntington's disease (HD), spinobulbar muscular atropy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA) and six forms of spinocerebellar ataxias, have been identified (Zoghbi and Orr, 2000, Everett and Wood, 2004). Accumulating data suggest that nuclear expression of polyglutamine-expanded proteins is required for the pathogenesis of polyglutamine neurodegenerative disorders and that the resulting transcriptional abnormality caused by mutant polyglutamine protein leads to neuronal dysfunction and cell death (Okazawa, 2003, Sugars and Rubinsztein, 2003, Gatchel and Zoghbi, 2005, Duenas et al., 2006). Ataxin-3 contains a putative nuclear localization signal (NLS) close to the glutamine repeat and is transported into the nucleus (Tait et al., 1998). It has been reported that endogenous ataxin-3 interacts with histone as well as transcriptional co-activators and possesses transcriptional repressor activity (Li et al., 2002, Evert et al., 2006), suggesting that one of normal functions of ataxin-3 is regulating gene transcription. Disease-causing polyglutamine ataxin-3 has been shown to be accumulated in ubiquitinated intranuclear inclusions in affected CNS neurons (Paulson et al., 1997). Previous studies also showed that transcription factor TBP and transcription co-factor CBP are incorporated into nuclear inclusions formed by polyglutamine-expanded ataxin-3 (McCampbell et al., 2000). Thus, it is possible that mutant polyglutamine ataxin-3 causes transcriptional dysregulation and resulting neurotoxicity. In accordance with this hypothesis, our recent in vitro study showed that polyglutamine-expanded ataxin-3-Q79 caused neurodegeneration of cultured cerebellar, striatal and substantia nigra neurons by increasing pro-apoptotic Bax mRNA level and downregulating anti-apoptotic Bcl-xL mRNA expression (Chou et al., 2006). It is still unknown whether mutant ataxin-3 induces neurotoxicity in vivo by impairing the normal pattern of transcriptions in affected brain regions.
In the present study, we prepared a SCA3 animal model by generating transgenic mice expressing polyglutamine-expanded ataxin-3-Q79. Ataxin-3-Q79 transgenic mice displayed pronounced ataxic symptoms resulting from cerebellar dysfunction. To test the involvement of transcriptional abnormality in ataxin-3-Q79-induced cerebellar malfunction, microarray analysis was performed to detect altered cerebellar mRNA expressions of SCA3 transgenic mice. Our study provides the evidence that mutant ataxin-3-Q79 causes cerebellar neurotoxicity and dysfunction by altering mRNA expressions of proteins involved in glutamatergic neurotransmission, intracellular calcium signaling/mobilization, MAP kinase pathways or regulating neuronal survival and differentiation, GABAA/B receptor subunits and heat shock proteins.
Section snippets
Generation of transgenic mice
According to our published study (Chou et al., 2006), influenza hemagglutinin epitope (HA, YPYDVPDYA) was added to the N-terminus of human wild-type ataxin-3-Q22 or disease-causing ataxin-3-Q79 by performing PCR amplification using the cDNA of ataxin-3-Q22 or ataxin-3-Q79 (ataxin-3 mjd1a isoform) (Kawaguchi et al., 1994) as the template. The transgene construct was prepared by inserting cDNA of ataxin-3-Q79HA or ataxin-3-Q22HA into mouse prion protein expression vector (MoPrP.Xho) (Borchelt et
Generation of transgenic mice expressing disease-causing or wild-type human ataxin-3
In the present study, transgenic mice expressing disease-causing human ataxin-3 with an expanded polyglutamine tract (79Q; ataxin-3-Q79) were prepared as an in vivo animal model of SCA3. As a control, transgenic mice expressing wild-type human ataxin-3 with a normal polyglutamine tract (22Q; ataxin-3-Q22) was also generated. To characterize wild-type or mutant ataxin-3 protein expressed in brain using immunoblotting assay, influenza hemagglutinin epitope (HA) was added to the N-terminus of
Discussion
In the present study, we prepared an in vivo animal model of SCA3 by generating transgenic mice expressing disease-causing ataxin-3-Q79. Mutant ataxin-3-Q79 protein was expressed in several brain regions including the cerebellum, pontine nucleus and substantia nigra, which are vulnerable to polyglutamine-expanded ataxin-3-induced neurotoxicity in SCA3 patients (Zoghbi and Orr, 2000). Similar to SCA3 human brain, intranuclear inclusions containing mutant ataxin-3-Q79 were also observed in
Acknowledgments
We are grateful to Dr. Akira Kakizuka (Kyoto University) for providing cDNA clones of human ataxin-3-Q22 and ataxin-3-Q79. This work was supported by the National Science Council of ROC (NSC94-2320-B-182-020 and NSC95-2320-B-182-006) and Chang Gung Medical Research Project (CMRP140441 and EMRPD160061).
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