Abstract
Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.
Highlights
Huntington’s disease (HD) is caused by the misfolding of the N-terminal exon1 fragment of the protein product of a mutant gene encoding an expanded (.36 copies) CAG trinucleotide repeat (Hatters, 2008; Imarisio et al, 2008)
Nuclear vs cytoplasmic counting To quantify the subcellular differences in the expression of Httex1-72Q-eGFP when co-transfected with the intrabodies, the extent to which the GFP signal was found in cytoplasm was counted in ST14A cells cotransfected with Httex1-72Q-EGFP, RFP-nuclear localization signal (NLS) and either C4 single-chain Fv (scFv) or VL12.3
We have noted over several years of cellular and in vivo experiments that the localization of Httex1-72Q-eGFP differs between C4 scFv and VL12.3 transductions, with the former clearly much more cytoplasmic
Summary
Huntington’s disease (HD) is caused by the misfolding of the N-terminal exon1 fragment of the protein product of a mutant gene encoding an expanded (.36 copies) CAG trinucleotide repeat (Hatters, 2008; Imarisio et al, 2008). Nuclear vs cytoplasmic counting To quantify the subcellular differences in the expression of Httex1-72Q-eGFP when co-transfected with the intrabodies, the extent to which the GFP signal was found in cytoplasm was counted in ST14A cells cotransfected with Httex1-72Q-EGFP, RFP-NLS and either C4 scFv or VL12.3. We have noted over several years of cellular and in vivo experiments that the localization of Httex1-72Q-eGFP differs between C4 scFv and VL12.3 transductions, with the former clearly much more cytoplasmic.
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