Author Summary Alzheimer’s disease (AD) is the leading cause of dementia in the ageing population. Symptoms include memory loss and decline in understanding and reasoning. Alois Alzheimer, who reported the first case of AD, observed plaques and tangles in the brains of patients. The plaques are made up of amyloid protein, while the tangles are of tau protein. One of the main scientific ideas about AD is that it starts with build-up of amyloid, which then alters tau protein, causing the disease. Another protein, called GSK-3, also seems to play a part. Simple invertebrates such as flies are useful for understanding human diseases. We have created an AD model in the fruit fly Drosophila where amyloid protein is present in the nerve cells of the adult fly; this caused the flies to be impaired in their survival, nerve function, and behavior. We found that amyloid increased the activity of GSK-3, and so we experimentally turned down its activity and found that this improved the survival and behavior of the flies. Importantly, turning down the activity of GSK-3 in flies that did not have amyloid did not seem to harm them. GSK-3 could therefore be a good target for drugs against AD. pathogenesis [11]. GSK-3a has been implicated in the amyloido- genic processing of APP to yield Ab peptides [12], while GSK-3b has been implicated in the tau-related pathogenesis of AD, by colocalizing with tau tangles and phosphorylating tau [9]. As yet, the exact role of GSK-3 in the generation of Ab peptides is not known. GSK-3 itself is also regulated by phosphorylation. Phosphorylation at Ser9 of GSK-3b and the equivalent Ser21 of GSK-3a inhibits activity, while phosphorylation at Tyr216/ Tyr279 of GSK-3b and GSK-3a, respectively, is thought to increase activity [13]. Remarkable similarities are seen between double transgenic mice expressing tau either with APP or with GSK-3b. This finding is consistent with the hypothesis that amyloid acts via activation of GSK-3 to modulate tau function [5,9,14]. Lithium chloride, which is used as a mood stabilizing agent in patients with bipolar disorders, inhibits GSK-3 activity, either by competing with magnesium ions [15] or by increasing Ser9 phosphorylation [16]. Lithium reduces amyloid production by altering the role of GSK-3a in APP processing/cleavage; selective inhibition of GSK-3 by siRNA or expressing a dominant negative form of GSK-3 also decreases Ab production in cultured cells and mice [12,17]. Furthermore, lithium reduces both tau phosphorylation at several GSK-3 epitopes and tauopathy in a mouse model expressing mutant human tau [12,18]. However, in another study, lithium was seen to reduce tau phosphorylation but not to affect Ab load in a triple mutant mouse expressing human APPswe, human tauP301L and with mutant presenilin 1 PS1M146V knock-in. The differing observations might be due to variations in the age at which the mice were treated with lithium, as suggested by the authors [19]. Fruit flies, Drosophila melanogaster, can provide useful invertebrate models of neurodegeneration because of their complex brains, short lifespans and relative ease of genetic manipulation. Several fly models of aspects of AD biology have been made, including ones that over-express either Drosophila or human tau, and show neuronal dysfunction phenotypes [20–22]. Co-expression of human tau protein with Shaggy (Sgg), the Drosophila homologue of GSK-3 [23], exacerbates these neurotoxic phenotypes and leads to the appearance of neurofibrillary tangles [22,24,25]. Fly models expressing Ab peptides have also been generated, and show neurodegeneration and amyloid deposits [26,28]. Although an GSK-3 Inhibition Ameliorates Ab Pathology APP orthologue exists in flies, the Ab sequence is not conserved, and Drosophila models directly expressing Ab allow study of Ab toxicity in the absence of any endogenous amyloid production [29]. In this study we have generated a fly model that expresses Arctic mutant Ab42 peptide in the nervous system of adult flies, using an inducible system for gene expression, because we wished to understand the underlying mechanism of disease progression of AD in adults, without complications from developmental effects. We first characterised this model, and then used it to investigate the amyloid cascade hypothesis, by modulating the levels of endogenous fly tau and examining the effects on phenotypes consequent upon expression of Ab. We also investigated the requirement for GSK-3 in Ab pathology and its role in direct regulation of Ab peptides. Results Arctic Ab42 expression can be induced in adult Drosophila neurons To generate an adult-onset fly model of Alzheimer’s disease, we expressed Arctic mutant Ab42 peptides using an inducible pan- neuronal driver. An elav GeneSwitch (elavGS) driver line [30–31] that has been used previously to develop an adult-onset Drosophila model of spinocerebellar ataxia (SCA) [32] was used to direct expression of a UAS-Arctic Ab42 transgene [33] both spatially and temporally, to neurons of the adult fly (Figure 1A). A UAS-Ab40 line [33] was used as a control for over-expression of non-toxic forms of Ab in fly neurons, since this form of the peptide has previously been shown to have no detrimental effect in flies [26–28]. We measured expression of Ab peptides in adult neurons when we treated elavGS;UAS-Arctic Ab42 flies with the activator mifepristone (RU486;RU) from two days post-eclosion, by measur- ing RNA and protein levels at 4 and 21 days into treatment (Figure 1B and 1C). Ab transcripts were clearly elevated in RU- treated elavGS;UAS-Arctic Ab42 flies in comparison with untreat- ed (2RU) flies at both time-points (Figure 1B). Moreover, an Ab42- specific ELISA confirmed that Ab42 protein was elevated in elavGS;UAS-Arctic Ab42 (+RU) flies compared to untreated (2RU) flies and that the level of protein increased with age (Figure 1C). Since RNA transcript level decreased with age, this age- dependent accumulation of Ab42 protein is most likely to be attributable to an increased rate of translation of the protein relative to the rate of protein degradation. Aggregation of Ab has been shown to be of critical importance for its pathogenicity [34]. Therefore, we assessed the state of aggregation of Ab42 in the mutant flies by separating soluble and insoluble protein fractions from fly brain extracts. At day 15, when the first signs of pathology were observed in the Arctic Ab42 flies (see below), we found that most of the Ab42 protein had accumulated into an insoluble, fibrillar form (Figure 2), consistent with the aggregation-promoting effects of the Arctic mutation [35]. Overall these results confirm that the elavGS-UAS system used in this study is sufficient to induce over-expression of Ab peptides specifically in the adult fly nervous system, and that Arctic mutant Ab42 protein accumulates with age. Over-expression of Arctic Ab42 peptide in adult neurons increases mortality and induces neuronal dysfunction in Drosophila, without evidence of neuronal cell loss Previously published studies have shown that constitutive expression of Arctic Ab42 peptide in fly neurons significantly shortens lifespan, induces behavioural impairments and causes neuronal death [26–28]. To determine whether adult-onset expression of Arctic Ab42 peptide in neurons causes similar PLoS Genetics | www.plosgenetics.org 2 September 2010 | Volume 6 | Issue 9 | e1001087