part, to show that tau is acting downstream of Ab pathology. This inducible model of AD will also open the way to understanding the role of events at different ages and of the ageing process itself in the biological pathway leading to this ageing related disease. We have shown the involvement of GSK-3, particularly in adulthood, in AD pathogenesis, and also uncover a novel mechanism by which GSK-3 could be acting directly or indirectly on Ab, by reducing Ab load. These results raise new potential therapeutic benefits of GSK-3 in AD pathology. Materials and Methods Fly stocks and maintenance All fly stocks were maintained at 25uC or 27uC on a 12:12-h light:dark cycle at constant humidity on a standard sugar-yeast (SY) medium (15gl21 agar, 50 gl21 sugar, 100 gl21 autolysed yeast, 100gl21 nipagin and 3ml l21 propionic acid). Adult-onset neuronal-specific expression of Arctic mutant Ab42 peptide or constitutively active Sgg was achieved by using the elav GeneSwitch (elavGS)-UAS system [GAL4-dependant upstream activator sequence; [30]]. ElavGS was derived from the original elavGS 301.2 line [30] and obtained as a generous gift from Dr H. Tricoire (CNRS, France). UAS-ArcAb42 and UAS-SggS9E were obtained from Dr D. Crowther (University of Cambridge, UK) and the Bloomington Drosophila Stock Centre respectively. Tau dfc (9530) and EP line 3203 (17098) were received from Bloomington Drosophila stock centre. The EP line orientation is opposite to tau expression and causes a reduction in tau expression [46]. elavGS and UAS-lines used in all experiments were backcrossed six times into the w1118 genetic background. Male flies expressing UAS-constructs were crossed to female flies expressing elavGS, and adult-onset neuronal expression induced in female progeny by treatment with mifepristone (RU486;200mM) added to the standard SY medium. Lithium treatment protocol Lithium Chloride was made at 1M concentration and added to 200mM RU486 standard SY medium at a final concentration of 30mM or 100mM. Lifespan analyses For all experiments, flies were raised at a standard density on standard SY medium in 200 mL bottles. Two days after eclosion once-mated females were transferred to experimental vials containing SY medium with or without RU486 (200mM) at a density of 10 flies per vial. Deaths were scored almost every other day and flies were transferred to fresh food three times a week. Statistical analyses were performed using JMP (version 7.0) software (SAS Institute, Cary, NC, USA). Data are presented as survival curves and analysis was performed using log-rank tests to compare between groups. Negative Geotaxis Assays To characterise the adult-onset behavioural effects of Arctic Ab42 peptide on neuronal function, climbing assays were initially performed at 25uC according to previously published methods [75]. Climbing ability was analysed every 2–3 days post-RU486 treatment. Briefly, 15 adult flies were placed in a vertical column (25cm long, 1.5cm diameter) with a conic bottom end, tapped to the bottom of the column, and their subsequent climb to the top of the column was analysed. Flies reaching the top and flies remaining at the bottom of the column after a 45 sec period were counted separately, and three trials were performed at 1 min intervals for each experiment. Scores recorded were the mean number of flies at GSK-3 Inhibition Ameliorates Ab Pathology the top (ntop), the mean number of flies at the bottom (nbottom) and the total number of flies assessed (ntot). A performance index (PI) defined as K(ntot+ntop2nbottom)/ntot) was calculated. Data are presented as the mean PI 6 SEM obtained in three independent experiments for each group, and analysis of variances (ANOVA) and post hoc analyses were performed using JMP 7.0 software. To assess various modifiers of the adult-onset neuronal dysfunction in Arctic Ab42 over-expressing flies, a less stringent climbing assay was performed at 27uC. Thirty flies expressing Arctic Ab42 with or without co-expressing modifiers in the neurons (elav- GAL4GS) were used for the climbing assay, adapted from Ganetzky et al. [76]. Climbing performance (ability to climb past a 5cm mark in 18s) was assessed after eclosion post RU486 treatment. Electrophysiology Recordings from the GFS of adult flies were made essentially as described in [77]; a method based on those described by Tanouye and Wyman (1980) [78] and Gorczyca and Hall (1984) [79]. Flies were anaesthetized by cooling on ice and secured in wax, ventral side down, with the wings held outwards in the wax. A tungsten earth wire (ground electrode) was placed into abdomen; tungsten electrodes were pushed through the eyes and into the brain to deliver a 40V pulse for 0.03ms using a Grass S48 stimulator. Recordings were made from the TTM and contralateral DLM muscle using glass microelectrodes (resistance: 40–60 MV). The electrodes were filled with 3M KCl and placed into the muscles through the cuticle. Responses were amplified using Getting 5A amplifiers (Getting Instruments, USA) and the data digitized using an analogue-digital Digidata 1320 and Axoscope 9.0 software (Axon Instruments, USA). For response latency recordings, at least 5 single stimuli were given with a 5s rest period between each stimulus; trains of 10 stimuli, at either 100Hz, 200 Hz or 250Hz, were given a 5s rest interval between each train. Histology For neuronal cell loss, adult heads were fixed, dehydrated and transverse sections were stained with Toluidine blue. Cell bodies were then counted blind for each genotype. Quantitative RT–PCR Total RNA was extracted from 20–25 fly heads using Trizol (GIBCO) according to the manufacturers’ instructions. The concentration of total RNA purified for each sample was measured using an eppendorf biophotometer. 1mg of total RNA was then subjected to DNA digestion using DNAse I (Ambion), immediately followed by reverse transcription using the Super- script II system (Invitrogen) with oligo(dT) primers. Quantitative PCR was performed using the PRISM 7000 sequence-detection system (Applied Biosystems), SYBR Green (Molecular Probes), ROX Reference Dye (Invitrogen), and Hot Star Taq (Qiagen, Valencia, CA) by following manufacturers’ instructions. Each sample was analysed in triplicate with both target gene (Arctic Ab42) and control gene (RP49) primers in parallel. The primers for the Ab transgenes were directed to the 59 end and 39 end of the Ab coding sequence: forward GATCCTTCTCCTGCTAACC; reverse CACCATCAAGCCAATAATCG. The RP49 primers were as follows: forward ATGACCATCCGCCCAGCATCAGG; reverse ATCTCGCCGCAGTAAACG. Quantification of total, soluble, and aggregated Ab42 To extract total Ab42, five fly heads were homogenised in 50 ml GnHCl extraction buffer (5 M Guanidinium HCl, 50 mM Hepes pH 7.3, protease inhibitor cocktail (Sigma, P8340) and 5mM PLoS Genetics | www.plosgenetics.org 15 September 2010 | Volume 6 | Issue 9 | e1001087