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.

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Autosomal-dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. In this study, we performed genetic analysis of a unique form of SCA (SCA36) that is accompanied by motor neuron involvement. Genome-wide linkage analysis and subsequent fine mapping for three unrelated Japanese families in a cohort of SCA cases, in whom molecular diagnosis had never been performed, mapped the disease locus to the region of a 1.8 Mb stretch (LOD score of 4.60) on 20p13 (D20S906-D20S193) harboring 37 genes with definitive open reading frames. We sequenced 33 of these and observed a large expansion of an intronic GGCCTG hexanucleotide repeat in NOP56 and an unregistered missense variant (Phe265Leu) in C20orf194, but we found no mutations in PDYN and TGM6. The expansion showed complete segregation with the SCA phenotype in family studies, whereas Phe265Leu in C20orf194 did not. Screening of the expansions in the SCA cohort cases revealed four additional occurrences, but none were revealed in the cohort of 27 Alzheimer disease cases, 154 amyotrophic lateral sclerosis cases, or 300 controls. In total, nine unrelated cases were found in 251 cohort SCA patients (3.6%). A founder haplotype was confirmed in these cases. RNA foci formation was detected in lymphoblastoid cells from affected subjects by fluorescence in situ hybridization. Double staining and gel-shift assay showed that (GGCCUG)n binds the RNA-binding protein SRSF2 but that (CUG)(6) does not. In addition, transcription of MIR1292, a neighboring miRNA, was significantly decreased in lymphoblastoid cells of SCA patients. Our finding suggests that SCA36 is caused by hexanucleotide repeat expansions through RNA gain of function.

Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUGexp) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAGexp transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUGexp transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUGexp transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUGexp transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUGexp disorders. Moreover, our demonstration that relatively short CUGexp transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.