Cell-type-specific alternative splicing in spinocerebellar ataxia type 6.

The alpha1A voltage-dependent calcium-channel (Ca(v)2.1) gene, the causative gene for spinocerebellar ataxia type 6 (SCA6), is transcribed into two major mRNA isoforms by alternative splicing at the intron 46-exon 47 boundary. One isoform has a stop codon upstream of the CAG repeat. The other "toxic isoform" has an alternatively spliced 5-nucleotide (GGCAG) insertion at the beginning of exon 47. This insertion leads to disruption of the following stop codon and transcription of a polyglutamine-encoding Ca(v)2.1 mRNA. The aim of our study is to investigate whether the expanded CAG repeat of exon 47 in Ca(v)2.1 gene increases the relative amount of the toxic isoform in Purkinje cells. Purkinje and granule cells were independently isolated in brain from subjects with SCA6 and quantified the amount of the toxic isoform mRNA by using real-time reverse transcription (RT)-PCR. We designed two sets of probe and primers: Set A for assessing total Ca(v)2.1 mRNA, and Set B for assessing the toxic isoform mRNA. The ratio of total Ca(v)2.1 mRNA to G3PDH mRNA was similar between Purkinje and granule cells in brain from both normal controls and patients with SCA6, and the ratio of toxic isoform mRNA to total Ca(v)2.1 mRNA did not differ between Purkinje and granule cells in control brains. However, this ratio was increased in Purkinje cells but not in granule cells in SCA6 brains. Our results suggest that toxic isoform mRNA is increased in a Purkinje cell-specific manner, which may result in SCA6-associated selective neurodegeneration.