Background Aggregation of mutant huntingtin is a dynamic process that starts with the association of a few misfolded huntingtin monomers in small, soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic species of mutant huntingtin and that the largest aggregates are rather neuroprotective. In order to prevent huntingtin aggregation and toxicity it is essential to understand the molecular mechanisms of oligomerisation. However, existing experimental models of Huntington's disease do not enable the direct visualisation of the smaller intermediary species in the aggregation process, only of the larger huntingtin aggregates.
Aims To develop a cellular model for the visualisation and study of dimers and oligomers of mutant huntingtin in living cells.
Methods We generated two different constructs that carried complementary portions of the Venus fluorescent reporter protein fused to the exon 1 of mutant huntingtin (103Q glutamine tract). When the exon-1 of mutant huntingtin dimerises inside the cells, both complementary halves of Venus reconstitute the functional fluorophore and emit green fluorescence, which can be easily visualised and measured.
Results Oligomer generation and toxicity was evaluated over time and confirmed by different methods. We used similar fusion constructs with wild-type huntingtin exon 1 (25Q glutamine tract) as a negative control for oligomerisation and toxicity. Additional controls included, among others, the transfection of cells with only one of the constructs or with the two halves of the Venus protein without huntingtin. The robustness of our model is being tested by analysing the effect of known modifiers of aggregation, such as heat shock proteins, on huntingtin oligomerisation.
Conclusions Our preliminary results indicate that this model can be a powerful tool for the identification of new molecular targets for the treatment of Huntington's disease.
- bifluorescent complementation assay
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