P3‐123: Which attributes of tau mediate toxicity: Phosphorylation or aggregation?

We have found that Drosophila which express wild-type human 3-repeat tau (h-3RTau) provide an interesting model of tauopathies such as Alzheimer's disease, and have provided insights into the mechanisms by which tau can cause neuronal dysfunction (Mudher et al., 2004; Chee et al., 2005; Cowan et al., 2010). Flies expressing h-3RTau in their motor neurons have a readily-measurable loco motor behavioral phenotype. This reflects neuronal dysfunction which results from pre-synaptic dysfunction (at the NMJ synapse of motor neuron onto muscle, in this case); which in turn results from defective fast axonal transport in the affected neurons. The defective axonal transport in h-3RTau-expressing animals results from the breakdown of microtubules, which we have measured by transmission electron microscopy. H-3RTau is highly phosphorylated at GSK-3b sites in the fly, and is only weakly able to bind microtubules, as measured by an ex vivo biochemical assay. Remarkably, the presence of h-3RTau also significantly diminishes the ability of the endogenous Drosophila tau (d-Tau) to bind and stabilize microtubules, despite the fact that it fails to form any insoluble tau (such as filaments or tangles) that we have been able to detect. It does this not by sequestering free cytoplasmic unphosphorylated tau into filaments, but by binding it in asoluble complex. This indicates that formation of insoluble tau is not necessary for tau toxicity. Electron microscopy, locomotion assays, Western blotting. By treating the h-3RT-expressing flies with LiCl (which has well-known GSK-3b inhibitory activity, as well as other actions) we are able to reduceh-3RTau phosphorylation levels; reduce binding of h-3RTau to d-Tau; improve binding of both h-3RTau and d-Tau to microtubules; restore microtubule integrity; restore axonal transport; and improve locomotion (Mudher et al.,2004; Cowan et al., 2010). Here we show that LiCl also has the remarkable unexpected effects of increasing total levels of h-3RTau protein, producing insoluble tau, and producing small granular electron-dense structures which might represent granular tau oligomers. We are currently undertaking experiments to determine which of the 3 effects of LiCl - the reduced tauphosphorylation, the increased tau levels, or the tau aggregate formation - are necessary or sufficient for the observed rescue of tau phenotype. A central question in this set of studies is whether these effects are lithium-specific, and whether they are due to actions of lithium in addition to its GSK-3B inhibition. This is important from amechanistic standpoint, and also because lithium is not a well-tolerated long-term treatment in humans. We explored this using the more specific GSK-3b inhibitor AR-A01448. When we rear Drosophila on media containing 20 μM AR-A01448, we reduce phosphorylation of h-3RTau andimprove locomotion just as with LiCl treatment (Mudher et al., 2004). We now show here that AR-A01448 treatment, like lithium treatment, achieves this rescue by restoring microtubule integrity. Additionally AR-A01448, like lithium, increases levels of h-3RTau protein, and produces ∼20 nmelectron-dense intra-axonal structures which might be insoluble tau. These results indicate that both the expected effects of lithium (in terms of rescuing the tau phenotype) and the unexpected effects of lithium (i.e. increasing the levels of tau protein and producing insoluble tau), are due to GSK-3B inhibition. Our working interpretation of the remarkable fact that two treatments which rescue the tau phenotype also act to increase levels of tau and produce insoluble tau, is that the phosphorylation of tau at key GSK-3b sites is more important in mediating tau toxicity than are tau levels or solubility.

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