Development of Purkinje cell degeneration in a knockin mouse model reveals lysosomal involvement in the pathogenesis of SCA6.

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.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca(V)) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca(V)2.1 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 Ca(V)2.1 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 Ca(V)2.1 channels.

Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol.

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.

Efficient In Vivo Delivery of siRNA to the Liver by Conjugation of α-Tocopherol.

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 α-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The α-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 α-tocopherol. The C6 hydroxyl group of α-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.

Efficient regulation of viral replication by siRNA in a non-human primate surrogate model for hepatitis C.

RNA interference (RNAi) represents a new technology which could offer potential applications for the therapeutics of human diseases. RNAi-mediated therapy has recently been shown to be effective toward infectious diseases in in vitro and rodent models, however, it remains unclear whether RNAi therapy with systemic application could be effective in primates. In this study, we examined if RNAi therapy could be effective toward infectious diseases by using a non-human primate surrogate model for hepatitis C. Administration into marmosets of cationic liposome-encapsulated siRNA (CL-siRNA) for GB virus B (GBV-B), which is most closely related to hepatitis C virus, repressed GBV-B replication in a dose-dependent manner. Especially, 5 mg/kg of the CL-siRNA completely inhibited the viral replication. Since the serum interferons (IFNs) were induced by CL-siRNA in vivo, inhibition of viral regulation by anti-GBV-B CL-siRNA may include an antiviral effect of IFN. However, contribution of induced IFN may be partial, since the control CL-siRNA which induced a stronger IFN response than GBV-B CL-siRNA could only delay the viral replication. Our results suggest the feasibility of systemic administration of CL-siRNA as an antiviral strategy.