Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, PC21
An in vivo model to investigate tau hyperphosphorylation in AlzheimerG
G. Povellato1, D. Hanger2, G. Tear1
1. MRC Centre for Developmental Neurobiology, King's College London, London, United Kingdom. 2. MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, London, United Kingdom.
Hyperphosphorylation of tau plays a critical role in Alzheimer’s disease (AD) pathology. Many kinases phosphorylate tau in vitro, but it is not certain which ones are responsible for the neurodegenerative process in AD. Drosophila provides a model system for studying tau toxicity in vivo. Expression of human tau in Drosophila photoreceptor neurons results in neurodegeneration that bears many of the hallmarks of AD. We are utilising Drosophila to identify the human kinases and their phosphorylation sites responsible for the generation of toxic forms of tau in vivo. Expression of human tau in the photoreceptors of several transgenic Drosophila lines revealed varying levels of degeneration by scanning electron microscopy (SEM). We investigated the transcript levels of human tau expressed by qPCR. The levels of Sarcosyl-soluble and insoluble tau produced were quantified by western blot (WB). Data were analyzed by t-test or one-way ANOVA followed by the post-hoc Tukey's multiple comparison test. Significance level was set to P < 0.05. We found that the level of tau transcript and insoluble but not soluble human tau expression correlates with the degenerative phenotype in the eye. Human kinases were expressed together with human tau in the fly eye to see whether the tau toxicity was enhanced. Glycogen synthase 3β (GSK3β) is one of the major kinases involved in tau hyperphosphorylation in AD brains. When human GSK3β was co-expressed with human tau the eye degenerative phenotype increased compared to that seen with tau alone. Western blot analysis showed that the degenerative enhancement is coincident with specific phosphorylation of human tau by GSK3β on sites recognised by PHF-1, a phospho-dependent tau antibody for Ser396/404. This demonstrates the phosphorylation sites on tau responsible for increased toxicity in vivo can be identified using Drosophila. In the same way we are investigating additional kinases potentially responsible for tau hyperphosphorylation in AD including the dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A). DYRK1A expression also enhances the human tau phenotype in the fly eye. We are currently mapping the phosphorylation sites responsible by western blot. We find increased photoreceptor degeneration when expressing both human tau and human kinases in the Drosophila eye. This allows us to evaluate the contribution made by individual human kinases and specific sites to generate toxic forms of tau in vivo.
Where applicable, experiments conform with Society ethical requirements