Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Markerless modification of trinucleotide repeat loci in BACs.

Transcription and splicing of human genes are regulated by nucleotide sequences encoded across large segments of our genome, and trinucleotide repeat expansion mutations can have both profound and subtle effects on these processes. In the course of our work to understand the impact of the Spinocerebellar Ataxia type 8 (SCA8) CTG repeat expansion on the transcription and splicing of the RNAs encoded near the SCA8 locus, we have developed a set of reagents and protocols for modifying large genomic BAC clones of this region. We describe the two-step procedure that allows us to precisely replace unexpanded trinucleotide repeats with expanded variants of these repeat sequences without leaving any exogenous sequences in the final constructs, and we discuss how this approach can be adapted to make other desired sequence changes to these genomic clones.

Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits.

Spinocerebellar ataxia type 8 (SCA8) patients typically have a slowly progressive, adult-onset ataxia. SCA8 is dominantly inherited and is caused by large CTG repeat expansions in the untranslated antisense RNA of the Kelch-like 1 gene (KLHL1), but the molecular mechanism through which this expansion leads to disease is still unknown. To more fully characterize the underlying molecular mechanisms involved in SCA8, we developed a mouse model in which Klhl1 is deleted in either all tissues or is deleted specifically in Purkinje cells only. We found that mice that are either homozygous or heterozygous for the Klhl1 deletion have significant gait abnormalities at an early age and develop a significant loss of motor coordination by 24 weeks of age. This loss progresses more rapidly in homozygous knock-outs. Mice with Klhl1 specifically deleted in only Purkinje cells had a loss of motor coordination that was almost identical to the total-tissue deletion mice. Finally, we found significant Purkinje cell dendritic deficits, as measured by the thickness of the molecular layer, in all mice in which Klhl1 was deleted (both total and Purkinje cell-specific deletions) and an intermediate reduction in molecular layer thickness in mice with reduced levels of Klhl1 expression (heterozygous deletions). The results from this mouse model show that even a partial loss of Klhl1 function leads to degeneration of Purkinje cell function and indicates that loss of KLHL1 activity is likely to play a significant part in the underlying pathophysiology of SCA8.

The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila.

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.

Instability of CAG-trinucleotide repeats in chronic lymphocytic leukemia.

Anticipation--earlier onset and more severe disease in the offspring generation--is a well documented feature of familial chronic lymphocytic leukaemia (CLL). In a number of Mendelian diseases, anticipation is caused by expansion of contiguous triplets of nucleotides. The severity of disease expression and penetrance is related to the extent of the triplet expansion. To investigate whether repeat nucleotide repeat expansion is a feature of CLL, the repeat expansion detection (RED) technique was applied to samples from 17 patients with familial disease and 32 patients with early-onset CLL disease. No potentially pathological CAG expansions were detected. We conclude that unstable CAG repeat expansion is not a feature of CLL and that other processes are likely to be involved in generating anticipation in familial forms of the disease.

The KLHL1-antisense transcript ( KLHL1AS) is evolutionarily conserved.

Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 ( KLHL1) gene. The normal function of this transcript is currently unknown. We have now identified the promoter region for the KLHL1-antisense ( KLHL1AS) RNA and report that a Klhl1as transcript is present in the mouse as well. Human and mouse KLHL1AS are transcribed from homologous promoter regions in the first intron of KLHL1 and extend through the transcription and translation start sites as well as the first splice donor sequence of KLHL1. We found that the mouse Klhl1as RNA is not spliced and terminates in a polyadenylation site in the Klhl1 promoter region, whereas both the present and previous work show that human KLHL1AS is highly variably spliced into processed transcripts that contain up to six exons. Mouse Klhl1as transcript was detected in RNA isolated from the cerebellum and from total adult brain and total fetal tissue, and at a low level in testis and ovary. Similarly, human KLHL1AS is expressed in various brain tissues, including the cerebellum, the tissue most affected by SCA8, and was detected at low levels in testis and kidney. The evolutionary conservation of this antisense/sense transcriptional organization strongly indicates that KLHL1AS transcripts play a significant biological role in both human and mouse, presumably as a regulator of KLHL1 expression.