GSK-3 Inhibition Ameliorates Ab Pathology Figure 6. Flies expressing Ab42 in adult neurons show a decrease in Shaggy inhibitory Ser9 phosphorylation. (A) Western blot analyses for pan-Sgg and phospho Ser9 Sgg revealed a decrease in Ser9 phosphorylation in flies expressing Ab42 by RU induction for 15 days (UAS-ArcAb42/ +;elavGS/+ or UAS-ArcAb42/GFP;elavGS/+) in comparison to their 2RU controls, while an increase in Ser9 phosphorylation was observed when the Ab42 expressing flies were fed lithium (+RU +Li). Flies over expressing Sgg were used as positive control. (B) Quantification of the western blot analysis in (A), n = 3, is depicted in the bar chart, with significant differences seen between ArcAb42/+;elavGS/+ +RU flies to 2RU controls (P,0.01), and between ArcAb42/+;elavGS/+ +RU +Li flies to +RU controls (P,0.001). doi:10.1371/journal.pgen.1001087.g006 (Figure 11B), suggesting that Sgg does not affect transgene expression, but rather acts directly or indirectly on Ab degrada- tion/sequestration. These data demonstrate for the first time a role of GSK-3 in determining the level of Ab42 peptide, in the absence of effects on APP processing. Furthermore, this reduction in the levels of Ab42 peptide by GSK-3 inhibition is most likely not mediated by tau, since loss of tau function partially rescued Ab toxicity, but did not affect Ab42 levels (Figure 10A and Figure S5). These data again suggest that GSK-3 can modify Ab42 toxicity via tau-independent mechanisms. Discussion Glycogen synthase kinase-3 is increasingly thought to play a pivotal role in the pathogenesis of Alzheimer’s disease, both as a regulator of the accumulation of Ab peptide [12,17,47] and through its well-established role as a tau kinase [48–52]. Although previous studies in mice have suggested that GSK-3 alters Ab levels via modulation of APP processing [12,17], the direct effects of the enzyme on Ab toxicity, and in the adult nervous system, have not been examined. We therefore performed a more direct analysis of the specific role of GSK-3 in regulating Ab42 toxicity in adult neurons in vivo, by modulating its activity in an adult-onset Drosophila model of Alzheimer’s disease. Our study shows for the first time that GSK-3 inhibition ameliorates Ab42 toxicity in adult flies, and also highlights a novel mechanism of protection by which GSK-3 directly regulates Ab42 levels in the absence of any effects on APP processing. We have generated an inducible Drosophila model of Alzheimer’s disease. Over-expression of the Arctic Ab42 peptide in adult fly neurons led to shortened lifespan, neuronal dysfunction and behavioural impairments. However, no neuronal loss was evident PLoS Genetics | www.plosgenetics.org 8 September 2010 | Volume 6 | Issue 9 | e1001087