GSK-3 Inhibition Ameliorates Ab Pathology difference was seen in the 2RU controls. (B). Expression of Shaggy S9E alone did not affect control lifespan. No significant difference was observed comparing elavGS/UAS-SggS9E +RU to either elavGS/UAS-SggS9E 2RU, or UAS-gfp/elavGS/+ +RU control lifespans. doi:10.1371/journal.pgen.1001087.g007 in the brains of these flies. This finding contrasts with previous reports that flies over-expressing Arctic Ab42, using a constitutive neuronal driver, develop vacuoles [28,33], These contrasting results could be reconciled if neuronal loss upon Ab42 over- expression is a consequence of developmental abnormalities in the Drosophila brain or if neuronal loss represents an end-stage event in response to Ab42 toxicity, since vacuolation has been reported only under the most extreme conditions of expression, age and temperature, while neuronal toxicity in these models is already apparent under less stringent conditions [33]. Importantly, our findings agree with those of other studies demonstrating that neuronal loss is generally not evident in murine models of amyloidosis, such as in mice transgenic for the amyloid precursor protein [53]. Moreover, our study has provided direct evidence of neuronal dysfunction in response to Ab42, by electrophysiological methods, at a timepoint more suitable to understanding the early events that lead to neuronal decline in AD. GSK-3 activity was increased in our flies upon Arctic Ab42 over-expression, as measured by reductions in phosphorylation of endogenous Sgg at the inhibitiory Ser9 site. This is consistent with previous observations showing that Ab42 alters GSK-3 phosphor- ylation in cells [54] and in mice [14]. Contrary to these findings, Figure 8. Inhibition of Shaggy, either by expression of SggS9E in the adult nervous system or treatment with lithium, suppresses the locomotor dysfunction phenotype of Arctic Ab42 flies. (A) Climbing ability of UAS-ArcAb42/UAS-gfp;elavGS/+ and UAS-ArcAb42/ +;elavGS/UAS-SggS9E flies on +RU486 SY medium was assessed at the indicated time-points (see Materials and Methods). Data are presented as the percentage climbing performance of flies 6 SD. P,0.001 when UAS-ArcAb42/UAS-gfp;elavGS/+ and UAS-ArcAb42/+;elavGS/UAS-SggS9E flies are compared at day 21 (one-way ANOVA, number of independent tests (n) = 3). Graph shows one representative data of repeated experiments. (B) Expression of Shaggy S9E alone does not reduce climbing ability of control flies. elavGS/UAS-SggS9E +RU flies display a similar locomotor function compared to both elavGS/UAS-SggS9E 2RU and UAS-gfp/+;elavGS/+ +RU control flies. (C) Climbing ability of UAS-ArcAb42/+;elavGS/+ and UAS- ArcAb42/UAS-gfp;elavGS/+ on +RU486 SY medium was assessed in the presence and absence of lithium chloride (30mM and 100mM). P,0.001 when UAS-ArcAb42 flies were fed lithium and compared to flies not fed lithium at day 18 (one-way ANOVA, n = 3). Graph shows one representative data of repeated experiments. (D) Lithium had no effect on negative geotaxis of control flies. UAS-gfp/+;elavGS/+ +RU and UAS-gfp/+;elavGS/+ +RU in the presence of lithium had similar locomotor function. The crosses were performed at 27uC. doi:10.1371/journal.pgen.1001087.g008 PLoS Genetics | www.plosgenetics.org 10 September 2010 | Volume 6 | Issue 9 | e1001087