RT Journal Article
SR Electronic
T1 B10 The role of n-terminal phosphorylation in huntingtin's oligomerisation, aggregation and toxicity
JF Journal of Neurology, Neurosurgery & Psychiatry
JO J Neurol Neurosurg Psychiatry
FD BMJ Publishing Group Ltd
SP A8
OP A8
DO 10.1136/jnnp-2012-303524.26
VO 83
IS Suppl 1
A1 Herrera, F
A1 dos Santos, J Branco
A1 Outeiro, T Fleming
YR 2012
UL http://jnnp.bmj.com/content/83/Suppl_1/A8.3.abstract
AB Background Aggregation of mutant huntingtin (Htt) is a dynamic process that starts with the association of a few misfolded Htt monomers in small, soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic species and that larger aggregates are rather neuroprotective. In order to prevent Htt aggregation and toxicity it is essential to understand the molecular mechanisms of oligomerisation.Aims Elucidate the role of Htt's N-terminal region in its oligomerisation, aggregation and toxicity, with particular emphasis on the phosphorylatable residues (T3, S13 and S16).Methods/techniques We have recently developed a cellular model for the visualisation of Htt oligomeric species in living cells, based on the bimolecular fluorescence complementation (BiFC) assay. In this model, Htt exon1 is fused to two non-fluorescent halves of the Venus protein (V1 and V2). When Htt dimerises, the two halves get together and reconstitute the functional fluorophore. We used this BiFC assay to create a series of phosphoresistant (T3A, S13A, S16A) and phosphomimic (T3D, S13D, S16D) Htt mutants.Results/outcome When phosphomimic mutations were present in both 103QHtt-V1 and 103QHtt-V2 BiFC constructs, the generation of inclusion bodies was completely abolished. However, the levels of oligomeric species were similar to the non-mutated and the phosphoresistant BiFC pairs. The combinations of a non-mutated construct with a phosphomimic construct produced intermediate phenotypes in terms of aggregation. Phosphoresistant BiFC pairs did not produce overt phenotypes. Mutations in N-terminal residues had varied effects on Htt toxicity, which apparently were not associated with the levels of oligomeric species or inclusion bodies.Conclusions The phosphorylation state of Htt's N-terminal region is a key player in the formation of inclusion bodies and the toxicity of the protein.