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B06 Exploring The Role Of Phosphorylation In Huntingtin Aggregation Dynamics
  1. J Branco dos Santos1,
  2. F Herrera1,4,
  3. F Giorgini2,
  4. TF Outeiro1,3
  1. 1Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa, 1649-028 Portugal
  2. 2Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK
  3. 3Department of Neurodegeneration and Restorative Research, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany
  4. 4Cellular Neurobiology Laboratory, Instituto de Tecnologia Quimica e Biologica (ITQB), Av. da República, Estação Agronómica Nacional, Oeiras, 2780-157 Portugal


Background Huntington’s disease (HD) is characterised by misfolding and aggregation of mutant huntingtin (HTT), a dynamic process that starts with the association of misfolded HTT monomers into soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic HTT species while large aggregates may in fact be neuroprotective. To better understand HTT toxicity it is essential to understand the molecular mechanisms of aggregation.

Aims Our goal was to elucidate the role of HTT N-terminal phosphorylation in the aggregation and toxicity of mutant HTT.

Methods/techniques Bimolecular fluorescence complementation (BiFC) was employed to visualise soluble and insoluble HTT species. We used this model to study the behaviour of HTT phosphomimic and phosphoresistant mutants in living cells. Moreover, we tested several compounds that modulate phosphorylation in Drosophila expressing HTT exon 1 (HTT93Q) under the control of the GAL4/UAS system. Pan-neuronal expression of HTT93Q by elavGAL4 gives a variety of disease-relevant phenotypes, including degeneration of photoreceptor neurons. We compared the neurodegeneration of treated or non-treated HD flies using the light microscopy-based pseudopupil assay.

Results/outcome When phosphomimic mutations were present in HTT103Q BiFC constructs, the generation of inclusion bodies was completely abolished. Phosphomimic and Phosphoresistant mutants had varied effects on HTT toxicity, which were not associated with the levels of oligomers or inclusion bodies. A CDC25 phosphatase inhibitor decreased aggregation in cells expressing HTT103Q and increased neurodegeneration in HTT93Q flies. Notably, a specific protein phosphatase 2A (PP2A) inhibitor completely reversed HTT93Q neurotoxicity in Drosophila.

Conclusions The phosphorylation state of HTT N-terminal region plays a role in aggregation and toxicity of the protein, and this can be pharmacologically modulated by targeting cellular phosphatases.

  • phosphorylation
  • N-terminal
  • phosphatase
  • bimolecular fluorescence complementation assay

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