Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8


We previously reported that a (CTG)n expansion causes spinocerebellar ataxia type 8 (SCA8), a slowly progressive ataxia with reduced penetrance. We now report a transgenic mouse model in which the full-length human SCA8 mutation is transcribed using its endogenous promoter. (CTG)116 expansion, but not (CTG)11 control lines, develop a progressive neurological phenotype with in vivo imaging showing reduced cerebellar-cortical inhibition. 1C2-positive intranuclear inclusions in cerebellar Purkinje and brainstem neurons in SCA8 expansion mice and human SCA8 autopsy tissue result from translation of a polyglutamine protein, encoded on a previously unidentified antiparallel transcript (ataxin 8, ATXN8 ) spanning the repeat in the CAG direction. The neurological phenotype in SCA8 BAC expansion but not BAC control lines demonstrates the pathogenicity of the (CTG-CAG)n expansion. Moreover, the expression of noncoding (CUG)n expansion transcripts (ataxin 8 opposite strand, ATXN8OS ) and the discovery of intranuclear polyglutamine inclusions suggests SCA8 pathogenesis involves toxic gain-of-function mechanisms at both the protein and RNA levels.

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Figure 1: BAC expansion and control constructs.
Figure 2: Progressive neurological phenotype in BAC expansion mice.
Figure 3: Responses to parallel fiber stimulation in FVB, BAC-Exp and BAC-Ctl mice.
Figure 4: BAC-Exp animals and SCA8 autopsy brain have 1C2- and ubiquitin-positive intranuclear inclusions.
Figure 5: Full-length and truncated (exon A only) ATXN8OS cDNAs are capable of producing 1C2 protein, with size dependant upon repeat length.
Figure 6: 1C2 protein made from the ATXN8 cDNA results from translation of a polyglutamine ORF.
Figure 7: Endogenous promoter activity, translation of polyglutamine protein and detection of CAG-containing ATXN8 transcripts in BAC transgenic mice and humans.

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We thank SCA8 family members for their participation; M.S. Swanson for critically reviewing our manuscript; H.T. Orr, M.T. Su, H.M. Hsieh-Li, G.W. Lee-Chen for helpful discussions and D. Norton, A. Rose and A. Koeppen for providing control autopsy tissues. Grant support from the National Ataxia Foundation and the US National Institutes of Health (RO1 NS40389) is gratefully acknowledged.

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Correspondence to Laura P W Ranum.

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Supplementary information

Supplementary Fig. 1

Immunohistochemistry of TATA binding protein. (PDF 72 kb)

Supplementary Fig. 2

Immunohistochemistry with polyglutamine-specific MW5 antibody. (PDF 98 kb)

Supplementary Fig. 3

Ataxin 8 (ATXN8) ORF and flanking sequence. (PDF 25 kb)

Supplementary Fig. 4

Genomic organization of SCA8 region and Klhl1 expression in BAC transgenic mice. (PDF 70 kb)

Supplementary Table 1

Primer sequences. (PDF 10 kb)

Supplementary Video 1

SCA8 BAC-Exp2 mouse. A high-copy SCA8 BAC-expansion animal (BAC-Exp2 at 20 weeks) displays severe motor deficits including stiffness and posturing with hindlimbs fully extended and unable to relax. (MOV 2276 kb)

Supplementary Video 2

SCA8 BAC-Exp5 mouse. A single-copy SCA8 BAC-expansion animal (BAC-Exp5 at 19 months) displays milder motor deficits including abnormal gait, low stance and hindlimb dragging. (MOV 691 kb)

Supplementary Video 3

SCA8 BAC-Control mouse. A single-copy SCA8 BAC-control animal (BAC-Ctl1 at 18 months) shows no motor phenotype. (MOV 1528 kb)

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Moseley, M., Zu, T., Ikeda, Y. et al. Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8. Nat Genet 38, 758–769 (2006).

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