Ataxin-2 Regulates RGS8 Translation in a New BAC-SCA2 Transgenic Mouse Model

2015-04-22 00:02:38

PLOS Genetics; 22 April 2015; DOI: 10.1371/journal.pgen.1005182

Warunee Dansithong, Sharan Paul, Karla P. Figueroa, Marc D. Rinehart, Shaina Wiest, Lance T. Pflieger, Daniel R. Scoles,
Stefan M. Pulst


Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling.


Spinocerebellar ataxia type 2 (SCA2) is an inherited neurodegenerative disorder leading to predominant loss of Purkinje cells in the cerebellum and impairment of motor coordination. The mutation is expansion of a protein domain consisting of a stretch of glutamine amino acids. We generated a mouse model of SCA2 containing the entire human normal or mutant ATXN2 gene using bacterial artificial chromosome (BAC) technology. Mice expressing a BAC with 72 glutamines (BAC-Q72) developed a progressive cerebellar degeneration and motor impairment in contrast to mice carrying the normal human gene (BAC-Q22). We found that even prior to behavioral onset of disease, the abundance of specific messenger RNAs changed using deep RNA-sequencing. One of the mRNAs with early and significant changes was Rgs8. Levels of Rgs8 protein were even further reduced than mRNA levels in BAC-Q72 cerebella suggesting to us that mutant ATXN2 might have a role in mRNA stability and translation. Using a cellular model, we showed that the ATXN2 protein interacted with RGS8 mRNA and that this interaction differed between normal and mutant ATXN2. Presence of mutant ATXN2 resulted in reduced RGS8 protein translation in a cellular model. Our studies describe a mouse model of SCA2 expressing the entire human ATXN2 gene and emphasize the role of ATXN2 in mRNA metabolism.


Spinocerebellar ataxia type 2 (SCA2) belongs to the group of neurodegenerative diseases caused by polyglutamine (polyQ) expansion. This group includes SCA1, Machado-Joseph disease (SCA3 or MJD), SCA6, SCA7, SCA17, Huntington's disease, spinal bulbar muscular atrophy (SBMA) and dentatorubral-pallidoluysian atrophy (DRPLA). SCA2 is an autosomal dominant disorder leading to motor incoordination which is caused by progressive degeneration of cerebellar Purkinje cells, and selective loss of neurons within the brainstem and spinal cord. As with most autosomal dominant ataxias, symptoms are characterized by a progressive loss of motor coordination, neuropathies, slurred speech, cognitive impairment and loss of other functional abilities arising from Purkinje cells and deep cerebellar nuclei.

In SCA2, expansion of a CAG repeat in exon 1 of the Ataxin-2 (ATXN2) gene causes expansion of a polyQ domain in the ATXN2 protein. As in the other polyQ diseases, the length of the polyQ repeat is inversely correlated with age of onset (AO) in SCA2. In contrast to other polyQ diseases, mutant ATXN2 does not enter the nucleus in appreciable amounts in early stages of disease. This is also confirmed by protein interaction studies that have identified ATXN2 interactors with cytoplasmic localization. Polyglutamine disorders show their pathology through a toxic gain of function of the protein and larger polyQ expansions have been associated with greater pathology.

ATXN2 is widely expressed in the mammalian nervous system. It is involved in regulation of the EGF receptor, and the inositol 1,4,5-triphosphate receptor (IP3R) whereby increased cytosolic Ca2+ occurs with CAG repeat expansion. ATXN2 functions are also associated with the endoplasmic reticulum, and the Golgi complex. Studies in Caenorhabditis elegans support a role for ATXN2 in translational regulation as well as embryonic development. ATXN2 is also important in energy metabolism and weight regulation, as mice lacking Atxn2, developed obesity and insulin resistance. Furthermore, ATXN2 interacts with multiple RNA binding proteins, including polyA binding protein 1 (PABP1), the RNA splicing factor A2BP1/Fox1, DDX6, TDP-43, and has been localized in polyribosomes and stress granules demonstrating its unique role in RNA metabolism.

Several SCA2 mouse models have been generated. We have reported two transgenic mouse models in which expression of full-length ATXN2 with 58 or 127 CAG repeats (ATXN2-[Q58] or ATXN2-[Q127]) is targeted to Purkinje cells (PCs) using the Purkinje cell protein-2 (Pcp2) promoter. These lines show progressive motor phenotypes accompanied by the formation of insoluble cytoplasmic aggregates, loss of PCs, and shrinkage of the molecular layer associated with the reduction of calbindin staining in PC bodies and dendrites. Onset of the motor phenotype of Pcp2-ATXN2[Q127] mice is associated with reduced PC firing that is progressive with age. Another Atxn2-CAG42 knock-in mouse model demonstrated very late-onset motor incoordination associated, but this was seen only in homozygous knock-in animals. This was associated with Pabpc1 deficiency, and upregulation of Fbxw8, but without loss of calbindin staining or downregulation of Calb1 mRNA.

In order to model human diseases using cis-regulatory elements, recent mouse and rat models have been created by transgenesis using human bacterial artificial chromosomes (BACs). In the BAC approach, an entire human gene including introns and regulatory regions is introduced into the mouse genome. BAC models often have lower genomic copy numbers than conventional cDNA transgenic models resulting in more physiological expression levels and a potentially more faithful late onset of disease.

We developed new BAC-SCA2 transgenic mouse lines expressing full-length human wild-type or mutant ATXN2 genes including upstream and downstream regulatory sequences. BAC mice with mutant ATXN2 exhibited progressive neurological symptoms and morphological changes in cerebellum. We used this mouse model to confirm changes in key PC-genes identified in a cDNA transgenic model, in particular the effects of mutant ATXN2 on Rgs8 steady state protein levels.

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