Using Drosophila to study the contribution of human kinases to tau toxicity
The major neuropathological feature associated with tauopathies is the aggregation of the microtubule associated protein tau within neurons. Aberrant phosphorylation events on tau contribute to the aggregation during disease. The kinases and the phosphorylation sites on tau responsible for the generation of pathogenic tau forms are still unclear. My thesis proposes to establish a Drosophila model to investigate the human kinases and phosphorylation sites responsible for the generation of human tau toxicity. Expression of human tau in the photoreceptors of transgenic Drosophila leads to a degenerative phenotype proportional to the transcriptional level of the transgene. Since the genomic position of the transgene influences its expression, I used a novel method of transgenesis to insert the human tau transgene in a reproducible position within the fly genome. Using this system, tau isoforms differing for the presence of two N-terminal domains were shown to generate the same level of toxicity in the fly eye. Moreover, in contrast with other studies, the tauopathy-associated mutation R406W on human tau did not cause an enhancement of tau-mediated degeneration of the fly eye. Selected human kinases relevant to tau pathology were tested for their ability to enhance tau toxicity in flies. Only human GSK3β showed a robust enhancement of tau-mediated eye degeneration. For the first time I showed that a human kinase, GSK3β, could specifically phosphorylate human tau in Drosophila on sites not targeted by endogenous fly kinases. In particular, tau phosphorylation of T181, S396 and S404 was found to significantly increase upon GSK3β expression. Site-directed mutagenesis on tau suggested that S404 might play a central role in mediating human tau toxicity in the fly eye. Finally this thesis demonstrates that the Drosophila model of tauopathy here created can be used to assess the importance of human kinases and of single phosphorylated sites in the generation of tau toxicity.
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