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Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (CaV) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of CaV2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein.We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6 84Q ) developed progressive motor impairment and aggregation of mutant CaV2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca 2؉ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6 84Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant CaV2.1 channels.cerebullum ͉ polyglutamine ͉ P/Q-type voltage-dependent calcium channel

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the Ca v 2.1 voltage-gated calcium channel. To elucidate how the expanded polyglutamine tract in this plasma membrane protein causes the disease, we created a unique knockin mouse model that modestly overexpressed the mutant transcripts under the control of an endogenous promoter (MPI-118Q). MPI-118Q mice faithfully recapitulated many features of SCA6, including selective Purkinje cell degeneration. Surprisingly, analysis of inclusion formation in the mutant Purkinje cells indicated the lysosomal localization of accumulated mutant Ca v 2.1 channels in the absence of autophagic response. The lack of cathepsin B, a major lysosomal cysteine proteinase, exacerbated the loss of Purkinje cells and was accompanied by an acceleration of inclusion formation in this model. Thus, the pathogenic mechanism of SCA6 involves the endolysosomal degradation pathway, and unique pathological features of this model further illustrate the pivotal role of protein context in the pathogenesis of polyglutamine diseases.

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