GSK-3 Inhibition Ameliorates Ab Pathology Figure 4. Arctic Ab42 peptides induce progressive, adult-onset neuronal defects in Drosophila. (A) A schematic illustration of the Drosophila giant fibre system (GFS; adapted from [74]). Giant fibres (GFs; blue) relay signals from the brain to the thoracic musculature via mixed electrochemical synapses with the motorneurons (TTMn, red) of the tergotrancheral muscle (TTM; left), and the peripherally synapsing interneuron (PSI; green), which subsequently forms chemical synapses with the motorneurons (DLMn; orange) of the dorsal longitudinal muscles (DLM; right). Note only one of the TTMn axons is shown exiting the nervous system and contacting the muscle on the left hand side and one set of the DLMns and corresponding neuromuscular junctions are depicted on the right hand side. GFS activity was measured in UAS-ArcAb42;elavGS flies at (B) 16 days and (C) 28 days post-RU486 treatment (see Materials and Methods); parameters measured were the latencies from GF stimulation to muscle response (response latency DLM and TTM) and the stability of the response to high frequency stimulation at 100, 200 and 250 Hz (high frequency stimulation TTM). Data are presented as the mean response 6 SEM and were analysed by student’s t-test, at each time point, on log-derived data. *P,0.05, **P,0.01 comparing response latency or response to high frequency stimulation of UAS-ArcAb42/+;elavGS/+ +RU flies to 2RU controls at 28 days post-induction. doi:10.1371/journal.pgen.1001087.g004 wild-type tau levels (P = 0.0002, two-way ANOVA; Figure 10B), on +RU food. It is important to note, however, that tau loss of function flies themselves displayed some locomotor dysfunction compared to controls (Figure 10B; P,0.0001 comparing UAS- ArcAb42/+;elavGS/+ to UAS-ArcAb42/+;elavGS tau EP3203/ tau dfc on 2RU486, two-way ANOVA), thus potentially reducing the apparent protective effect of removing tau on Ab42 pathology. These data parallel observations in mammals, where loss of murine Tau rescued Ab-induced behavioural deficits in a mouse AD model [8]. We further investigated interactions between Drosophila tau and GSK-3 in the protection against Ab42 pathology, to determine if they might act in the same biochemical pathway (Figure 10B). Lithium treatment alone had a greater protective effect against Ab42 toxicity than did loss of tau function alone, although this may have been confounded by the reduced climbing ability of flies with reduced tau. Moreover, lithium treatment rescued Ab42- induced climbing dysfunction to the same extent in the presence or absence of tau, (P = 0.692 comparing UAS-ArcAb42/+;elavGS/+ and UAS-ArcAb42/+;elavGS tau EP3203/tau dfc flies on +RU, + lithium food, two-way ANOVA) suggesting that endogenous tau is not required for the lithium effect. This suggests that a large proportion of the protective effect of GSK-3 inhibition on Ab42 toxicity is mediated via non-tau-dependent mechanisms. We also show that tau is required for the manifestation of Ab42 effects. Our experimental design does not address, neither excludes, the possibility that a direct interaction of GSK-3 and tau may also affect Ab42 toxicity. Inhibition of Shaggy in the adult nervous system reduces Ab load in Arctic Ab42-expressing flies Because the amelioration of Ab42 toxicity by reduced GSK-3 activity did not appear to be mediated mainly through tau, we next examined the direct effect of GSK-3 inhibition on Ab42 levels. Interestingly, we found by ELISA analysis that Ab peptide was significantly reduced in flies expressing Arctic Ab42 when Sgg activity was reduced. Adult flies that co-expressed Arctic Ab42 and the inactive dominant negative mutant Sgg S9E, or adult flies that expressed Arctic Ab42 and were fed lithium, showed a major reduction in total Ab42 levels in comparison to flies expressing Arctic Ab42 alone and reared on food without lithium (Figure 11A). The transcript levels of Ab42 in the presence of functional or inhibited Sgg activity were not significantly different Figure 5. Expression of Arctic Ab42 peptides in the adult fly nervous system causes locomotor dysfunction. Climbing ability of UAS- ArcAb42/+;elavGS/+ and UAS-Ab40/+;elavGS/+ flies on + and 2 RU486 SY medium was assessed at the indicated time-points (see Materials and Methods). Data are presented as the average performance index (PI) 6 SEM and were compared using two-way ANOVA and Tukey’s honestly significant difference (HSD) post-hoc analyses (number of independent tests (n) = 3, number of flies per group (nt) = 39–45). *P,0.05 comparing PI of UAS-ArcAb42/+;elavGS/+ +RU486 flies to that of untreated and Ab40 over-expressing controls at the indicated time points (Tukey’s HSD). doi:10.1371/journal.pgen.1001087.g005 PLoS Genetics | www.plosgenetics.org 7 September 2010 | Volume 6 | Issue 9 | e1001087