GSK-3 Inhibition Ameliorates Ab Pathology Figure 2. Arctic Ab42 peptide in the adult Drosophila nervous system is mostly in an insoluble fibrillar state. In the absence of the activator mifepristone (RU486; 2RU), a negligible amount of soluble protein is observed at day 15. Following treatment with RU486 (+RU; dark green) the Ab peptide expression is seen in both soluble and insoluble fractions with a significant proportion observed in the insoluble fraction. Data are presented as means 6 SEM and were analysed by ANOVA, P,0.01 when protein levels of soluble and insoluble fractions of Ab42 expressing flies were compared. doi:10.1371/journal.pgen.1001087.g002 As a behavioural measure of neuronal dysfunction in our inducible model, locomotor activity was assessed using a negative geotaxis (climbing) assay that has been used extensively to characterise fly models of neurodegenerative diseases [33,36]. Drosophila display an age-related decline in climbing behaviour, and this was apparent in the non-RU-treated and elavGS;UAS- Ab40 +RU control flies used in the current study (Figure 5). We found that flies expressing Arctic Ab42 displayed a reduced negative geotaxis in comparison to their 2RU control flies and the Ab40 +RU and 2RU flies (Figure 5). The climbing behaviour of the Arctic Ab42 flies had declined to a level by day 15 that was reached by the control flies only by day 28. Finally, we quantified neuronal loss, as measured by the number of cell bodies in one hemisphere, in flies over-expressing Arctic Ab42 peptide in adult neurons compared to non-expressing controls. No neuronal loss was evident in the brains of these flies (Figure S2). Collectively, these data demonstrate that expression of Arctic Ab42 specifically in the neurons of the adult fly leads to early death and progressive neuronal dysfunction, in the apparent absence of neuronal loss. Hence we have successfully developed an inducible Drosophila model of AD that will provide a useful system in which to further investigate the potential mechanisms underlying pathogenesis in Alzheimer’s disease, without any confounding effects on neuronal development. Adult-onset expression of Ab42 increases the activity of Shaggy in the adult nervous system Because of the described role of GSK-3 in Alzheimer’s disease, we investigated the activity of the fly orthologue of GSK-3, Sgg, in the Ab42-expressing flies. Phosphorylation at Ser9 of Sgg is important in suppressing its kinase activity. We found that expression of Arctic Ab42 in the adult nervous system decreased the Ser9 phosphorylation level of Sgg, indicating an up-regulation of the activity of the kinase (see Figure 6). This increase in Sgg activity could have contributed to the toxicity we observed in our Ab42 expressing flies. Lithium is a GSK-3 inhibitor, and we therefore tested its effect on Ser9 phosphorylation in the Ab42 flies and, indeed, we found an increase in Ser9 phosphorylation compared to untreated controls (Figure 6). Inhibition of Shaggy activity in the adult nervous system suppresses the toxicity of Arctic Ab42 We next investigated if the increase in Sgg activity that we observed in the Artic Ab42-expressing flies contributed to Ab42 toxicity. To do this, we co-expressed in adult neurons a dominant negative form of Sgg, the S9E mutant, which mimics an inhibited state of the kinase [37,38] and renders it inactive. Expression of this dominant-negative Sgg increased the median and maximum lifespan of flies expressing Arctic Ab42. Flies co-expressing Arctic Ab42 and the dominant negative mutant S9E lived significantly longer than control flies co-expressing Arctic Ab42 and GFP (Figure 7), to control for any titration effect of GAL4 in the presence of a second UAS-transgene. Furthermore, inactivation of Sgg, either by expressing the dominant negative mutant S9E or feeding the flies lithium in adulthood, significantly suppressed the climbing deficit of the Arctic Ab42-expressing flies (Figure 8). Two different doses of lithium (30mM and 100mM) both rescued the climbing deficit of the Ab42-expressing flies. These data demonstrate that inhibiting the activity of Sgg in neurons in adults suppresses the adult onset Arctic Ab42 induced toxicity, and demonstrate experimentally a functional role of GSK-3 in mediating Ab42 toxicity. PLoS Genetics | www.plosgenetics.org 4 September 2010 | Volume 6 | Issue 9 | e1001087