GSK-3 Inhibition Ameliorates Ab Pathology Figure 1. Adult-onset induction of Arctic Ab42 peptide in the Drosophila nervous system. (A) A schematic representation of the GeneSwitch-UAS expression system (based on [30]). Driver lines expressing the transcriptional activator GeneSwitch under control of the nervous system-specific elav promoter (elavGS) are crossed to flies expressing an Ab transgene fused to a GAL4-binding upstream activation sequence (UAS- Ab). In the absence of the activator mifepristone (RU486; 2RU), the GeneSwitch protein is expressed in neurons but remains transcriptionally silent, so that Ab is not expressed. Following treatment with RU486 (+RU; green) the GeneSwitch protein is transcriptionally activated, binds to UAS and thus mediates expression of Ab peptide specifically in the fly nervous system. Ab42 RNA (B) and protein (C) levels were quantified at four days and 21 days post-RU486 treatment (see Materials and Methods). Data are presented as means 6 SEM and were analysed by two-way ANOVA and Tukey’s honestly significant difference (HSD) post-hoc comparisons. P,0.05 comparing Ab RNA expression in RU486-treated UAS-ArcAb42/+;elavGS/+ flies to their 2RU486 controls at both time-points (Tukey’s HSD). P,0.01 comparing Ab42 protein levels in RU486-treated UAS-ArcAb42/+;elavGS/+ flies to untreated controls at both time-points. doi:10.1371/journal.pgen.1001087.g001 phenotypes, we examined survival, neuronal and behavioural dysfunction in our inducible Drosophila model of AD. First, we measured the effects of Arctic Ab42 expression on lifespan, by treating elavGS;UAS-Arctic Ab42 and elavGS;UAS- Ab40 flies with RU from two days post-eclosion and recording their survival. Expression of Arctic Ab42 in adult neurons shortened median lifespan by about 50% and maximum lifespan by about 45% in comparison to non-RU-treated flies, and to Ab40 +RU and 2RU control flies (Figure 3), demonstrating a specific lifespan-shortening effect of Arctic Ab42 compared to the Ab40 form of the peptide. Next, we determined whether adult-onset expression of Arctic Ab42 peptide in fly neurons caused neuronal toxicity, by analysing neuronal function. As a direct measure of physiological activity, we examined the electrophysiological responses of the Drosophila giant fibre system (GFS; Figure 4A). Adult elavGS;UAS-Arctic Ab42 flies were fed + or 2 RU486 media from two days post-eclosion, and GFS activity measured at day 16 and day 28 into treatment. Giant fibres (GF) were stimulated via electrodes inserted inside the compound eye, and post-synaptic potentials recorded in the tergotrochanteral muscle (TTM) and the dorsal longitudinal flight muscle (DLM) (Figure 4A); parameters measured were the latency from GF stimulation to muscle response and the stability of the response to high frequency stimulation. At day 16, response latencies in the TTM, DLM and the TTM to high frequency stimulation were comparable between elavGS;UAS-Arctic Ab42 flies on + and 2 RU486 food (Figure 4B and Figure S1). However, at day 28, expression of Arctic Ab42 peptide significantly in- creased the response latency measured in both the TTM and DLM, and inhibited the stability of the TTM response to high frequency stimulation (at 100, 200 and 250 Hz) in comparison to untreated control flies (Figure 4C and Figure S1). This indicates a progressive neuronal dysfunction following adult-onset induction of Arctic Ab42, with young flies exhibiting no dysfunction in the GFS, while older flies showed obvious defects in response to both a single stimulus and to high frequency stimuli. PLoS Genetics | www.plosgenetics.org 3 September 2010 | Volume 6 | Issue 9 | e1001087