phosphorylation, since Ab increases GSK-3 activity [10]. Fur- thermore, GSK-3 and APP cause similar increases in tau phosphorylation and aggregation in tau over-expressing mice [14]. Because both tau and GSK-3 appeared to play a causal role in Arctic Ab42 toxicity in our flies, we examined whether the protective effect of GSK-3 inhibition on Ab toxicity might be mediated via alterations in phosphorylation of endogenous Drosophila tau. We found that Arctic Ab42 over-expression and lithium treatment of Ab42-expressing flies did not have an observable effect on overall tau phosphorylation, as revealed both by generic measures, and at two specific sites by examination of phosphorylation of fly tau at Ser262 and Ser356 epitopes. However, because the Ser 262 and 356 sites are also predominant in vivo substrates for MARK (microtubule-associated protein (MAP)-microtubule affinity regulating kinase) [58,59], further investigation of more specific GSK-3 sites on Drosophila tau, using mass spectrometric methods, would provide a more definitive analysis of the role of tau phosphorylation by GSK-3 in mediating Ab42 toxicity in the fly. Our data suggest that tau phosphorylation may not be the only mechanism by which Ab42 exerts its toxic effect in Drosophila, since phosphorylation sites thought to be important for mediating Ab42 effects on tau (Thr212/Ser214 [43], Thr231 [45], Ser422, Ser262 [44]) are predominantly not conserved in the fly, or are not altered by Ab42 over-expression in our AD model. These findings seem to contradict previous studies showing that Ab42 increases phos- phorylation of human tau in Drosophila [55,60], and indicating that particular sites, such as Ser262, are important in mediating Ab toxicity in the fly [60]. This disparity could reflect a lack of conservation of the mechanisms through which Ab42 regulates human and Drosophila tau proteins. However, the endogenous fly tau does appear to be an important downstream mediator of Ab42 toxicity, since loss of tau function partially reduced Ab42 pathology in our study. This protective effect of tau against Ab42 toxicity, however, may have been masked by the locomotor dysfunction from reducing levels of tau itself in flies that do not over-express the Ab42 peptide. As a previous study has reported similar tau-dependent neuropathological phenotypes in an APP- overexpressing mouse model of AD [8], our findings suggest that the role of tau in amyloid toxicity is conserved over large evolutionary distances. We further examined the epistatic interaction between GSK-3 inhibition and tau loss of function in protecting against Ab42 toxicity in our fly model. Lithium alone had a greater protective effect against Ab42 toxicity than loss of tau function alone, and lithium could prevent Ab42-induced dysfunction in the presence or absence of tau. This suggests that a large proportion of the protective effect of GSK-3 inhibition on Ab42 toxicity is mediated via non-tau-dependent mechanisms and that tau is not required for this effect. We observed no additive effect of combining lithium treatment and tau loss of function in protection against Ab42- induced pathology; however, this could have been a consequence either of toxicity of loss of tau in the absence of Ab42 or of the level of protection afforded by lithium, which could have produced a ceiling effect. Our findings agree with other recently published studies, showing that loss of tau only partially protects against GSK-3-induced neuronal degeneration in adult mice [61], suggesting that other non-tau-dependent mechanisms of GSK-3 neuro-toxicity exist. Previous studies have shown a reduction in Ab load in mice as a result of GSK-3 inhibition, but this has been explained mainly by dysregulation of APP processing, either by increasing c-secretase activity [12] or by increasing the phosphorylation of APP and therefore directing its subcellular location to sites of secretase GSK-3 Inhibition Ameliorates Ab Pathology activity [17]. Our inducible Drosophila model, however, expresses the Arctic Ab42 peptide directly, thus circumventing the requirement for APP processing. We found that inhibition of GSK-3 caused a reduction in the level of Ab42 peptide, but not in RNA transcript levels. Hence, although previous studies have indicated that GSK-3 does not affect Ab degradation [17], our findings demonstrate a novel effect of GSK-3 in Ab metabolism, irrespective of APP processing, in the adult nervous system. Furthermore, this observation may, partially, explain the non-tau-dependent effect of GSK-3 in protecting against Ab toxicity in our flies. Our study, therefore, implies that GSK-3 may increase Ab degradation or clearance. Candidate in vivo Ab degrading enzymes include neprilysin (NEP) [62], insulin degrading enzyme (IDE) [63–64] and to a lesser extent endothelin converting enzymes (ECE-1,2) [65] and plasmin [66]. Mice deficient in IDE [63,64] or NEP [62] display increased levels of Ab peptides in the brain. Conversely, increasing expression and activity of NEP or IDE reduces the cerebral amyloid plaque burden observed in APP over-expressing mice [67], further implying that these are predominant in vivo Ab degrading enzymes. Direct interactions between GSK-3 and IDE or neprilysin activities in relation to Ab degradation, however, have not been extensively investigated. Studies examining the effects of reduced insulin signalling on amyloid toxicity in APP over-expressing mice have reported either increased GSK-3 activity, reduced IDE activity and increased amyloidosis [68] or decreased GSK-3 activity, increased IDE expression and reduced amyloidosis [69] in the brain, suggesting an inverse correlation between GSK-3 and IDE activities in relation to Ab metabolism. Other studies have reported no correlation between inhibition of GSK-3 activity and neprilysin levels in relation to reduced Ab load in mice [17], but NEP activity was not measured and may provide a more accurate indication of the role of this enzyme in Ab metabolism downstream of GSK-3. As most information from mouse models is correlative, however, further work is required to determine whether these degrading enzymes are direct mediators of Ab degradation in response to GSK-3 inhibition, by modulating their activities in our inducible Drosophila model. Drosophila homologues of both IDE and NEP exist, and over-expression of both NEP [26,70] and IDE [71] have been shown to reduce Ab induced neurotoxicity in flies. This suggests that these degradation mechanisms may be generally conserved, and that the fly is a valuable model for the direct analysis of these genetic interactions with respect to the role of GSK-3 in AD. The more general proteosome degradation pathway could also play a role in regulating Ab degradation or sequestration. Heat shock protein 90 (Hsp90), a protein chaperone involved in the proteosome degradation pathway, is thought to phosphorylate GSK-3 and regulate its activity [13]. In addition, an increase in levels of GSK-3 down-regulates the transcriptional activity of Heat shock factor-1 (HSF-1) and Hsp70 [72]; thus a decrease in GSK-3 activity could lead to an increase in levels/activity of these chaperone molecules and augment the levels of Ab. In a Caenorhabdits elegans worm model of AD, the aggregation-mediated Ab42 toxicity was regulated by modulating the levels of hsf-1; a reduction in hsf-1 increased paralysis in these worms, suggesting a role of hsf-1 in the dis-aggregation of Ab toxic oligomers [73]. Thus, any of these pathways/molecules could play a role in affecting Ab load in our fly model and will require detailed exploration in the future. Our data highlight that this fly model is suitable for the study of AD pathology, since we observe neuronal dysfunction and toxicity that are particular to the expression of Ab42 peptide. Further- more, we have been able to test the amyloid cascade hypothesis in PLoS Genetics | www.plosgenetics.org 14 September 2010 | Volume 6 | Issue 9 | e1001087