Biochemical and Biophysical Research Communications
rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse
Section snippets
Materials and methods
HD model mouse. The HD190QG transgenic mouse was used as a HD model in this study. The HD190QG transgenic mouse harbors mutant truncated N-terminal htt containing 190 CAG repeats fused with EGFP in its genome. This animal shows progressive motor abnormality, and neuropathology such as formation of aggregates in brain, and shorter viability [21]. All the experiments with mice were approved by the Animal Experiment Committee of the RIKEN Brain Science Institute.
Construction and production of rAAV.
Gene silencing by rAAV-shRNA
In vitro screening was used to identify the efficiency of mRNA ablation of shRNAs directed to EGFP and EGFP-fused truncated htt-polyQ. EGFP-fused truncated htt-190Q is identical to the pathogenic transgene present in the HD190QG mouse [21]. The gene silencing function of 10 candidate shRNA sequences targeting EGFP was evaluated by the EGFP expression of co-transfected Neuro2A cells with shRNA and EGFP (data not shown). An shRNA targeting EGFP sequence 5′-GCAAGCTGACCCTGAAGTTCAT-3′ (shEGFP)
Discussion
In this study, we demonstrated that neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by RNAi transduction. Following shRNA transduction, the number of neuronal aggregates in the striatum detected by ubiquitin antibody was reduced to 34.1% of that in the sham-treated striatum. Importantly, the number of aggregates in the shEGFP-transduced striatum was less than that in the striatum
Acknowledgments
The authors thank Dr. John A. Chiorini for providing pAAV5-RNL and pAAV5-RepCap (identical to 5RepCapB) and Avigen, Inc. (Alameda, CA) for providing pAAV-LacZ, pHLP19, and pAdeno. We also thank Drs. Nobuhisa Iwata, Takaomi Saido, Mayumi Okada, and Ms. Miyoko Mitsu for their technical supports and Drs. Joanna Doumanis and Hong-Kit Wong for their critical readings. This work was supported in part by grants from Grants-in-Aid for Scientific Research on Priority Areas 17025044 from The Ministry of
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