GSK-3 Inhibition Ameliorates Ab Pathology Figure 3. Expression of Arctic Ab42 specifically in the adult nervous system shortens lifespan. Lifespans were determined as described in materials & methods. Survival curves are depicted and data were compared using the log-rank test. P,0.01 comparing median lifespan of UAS- ArcAb42/+;elavGS/+ +RU flies to their 2RU controls. P,0.01 comparing UAS-ArcAb42/+;elavGS/+ +RU flies to UAS-Ab40/+;elavGS/+ +RU controls. Induction of Ab40 in the adult nervous system did not alter lifespan in comparison to non-RU486-treated controls. doi:10.1371/journal.pgen.1001087.g003 Arctic Ab42 toxicity, and protection by Shaggy inhibition, is not mediated predominantly through alterations in tau phosphorylation Since GSK-3 is a well-established tau kinase, and tau is abnormally phosphorylated in AD, we next investigated whether the protective effect of Sgg inhibition on Ab42 toxicity in our fly model is mediated via alterations in tau phosphorylation. Hence, we examined the phosphorylation of Drosophila tau in flies over- expressing Arctic Ab42 peptide in the absence or presence of lithium-treatment (Figure 9). We analysed tau phosphorylation using Phos-tag acrylamide gels, a technique for separating phosphorylated protein isoforms [39], which has been employed previously to investigate the phosphorylation of fly proteins [40]. Phos-tag is a phosphate-binding compound which, when incorporated into polyacrylamide gels, can result in an exaggerated mobility shift for phosphorylated proteins, dependent on the degree of phosphorylation. When heat-stable fly head homogenates were run on Phos-tag gels, several prominent tau bands were detected, implying that endogenous tau is phosphorylated at multiple sites in WT tissue (Figure 9A). De-phosphorylation using l-protein phosphatase confirmed that the high molecular weight bands were due to tau phosphorylation, and that non-phosphorylated fly tau runs as a doublet (Figure 9A). This method is thus capable of detecting at least some phosphorylation changes on the endogenous fly tau. Using this method, no difference in the level of tau phosphorylation was observed in flies over-expressing Arctic Ab42 compared to non-expressing controls or to Arctic Ab42 flies treated with lithium chloride (Figure 9A and Figure S3A). The phosphorylation sites on fly tau have not yet been extensively characterized. Drosophila-specific phosphorylation-de- pendent antibodies are hence not available for the examination of specific sites. However, several GSK-3 specific sites [41,42], and sites reported to be altered by Ab42 peptide [43–45], on human tau appear to be conserved in the Drosophila tau sequence (Figure S4). Of these, Ser262, and Ser356 phosphorylation-dependent human tau antibodies were found to detect fly tau protein specifically (Figure 9B and Figure S3B). We therefore used these antibodies to examine the effects on tau phosphorylation at these sites. Arctic Ab42 over-expression, or treatment of Ab42- expressing flies with lithium, did not modify phosphorylation at the Ser262 or Ser356 homologous tau epitopes (Figure 9B). This suggests that neither Ab42 nor GSK-3 are predominant in vivo regulators of the phosphorylation of these sites on fly tau. Loss of Tau reduces adult-onset Ab42 toxicity Although we could not uncover a role for tau phosphorylation in Ab42 toxicity, we investigated whether the presence of tau modulates Ab42 pathology. We found that loss of tau reduced the Arctic Ab42 climbing dysfunction. UAS-ArcAb42/+;elavGS tau EP3203/tau deficiency (dfc) flies, which express Arctic Ab42 in a genetic background homozygous mutant for tau (see Figure 10A for tau expression levels; tau antibody has previously been described [46]), had improved locomotor ability compared to UAS-ArcAb42/+;elavGS tau EP2303/TM6 flies, which express a much greater level of tau (P,0.0001, two-way ANOVA; Figure 10A) and UAS-ArcAb42/+;elavGS/+ flies, which express PLoS Genetics | www.plosgenetics.org 5 September 2010 | Volume 6 | Issue 9 | e1001087