TM6 flies flies on +RU486 SY medium, were measured by ELISA at 21 days post-induction. Data are presented as the average protein concentration, 6 standard error of mean, data were compared using one-way ANOVA and student t-test, number of independent tests (n) = 3. No significant difference was seen in the levels of Ab42. Found at: doi:10.1371/journal.pgen.1001087.s005 (0.08 MB DOC) Acknowledgments We thank Dr. D. Crowther (University of Cambridge) for helpful discussions and kind donation of UAS-Ab42 fly stocks, Dr. H. Tricoire References 1. Spires TL, Hyman BT (2005) Transgenic models of Alzheimer’s disease: learning from animals. NeuroRx 2: 423–437. 2. Selkoe DJ, Schenk D (2003) Alzheimer’s disease: molecular understanding predicts amyloid-based therapeutics. Annu Rev Pharmacol Toxicol 43: 545–584. 3. Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, et al. (1998) Association of missense and 59-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393: 702–705. 4. Rizzu P, Van Swieten JC, Joosse M, Hasegawa M, Stevens M, et al. (1999) High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands. Am J Hum Genet 64: 414–421. 5. Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, et al. (2001) Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293: 1487–1491. 6. Oddo S, Billings L, Kesslak JP, Cribbs DH, LaFerla FM (2004) Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome. Neuron 43: 321–332. 7. Oddo S, Caccamo A, Cheng D, Jouleh B, Torp R, et al. (2007) Genetically augmenting tau levels does not modulate the onset or progression of Abeta pathology in transgenic mice. J Neurochem 102: 1053–1063. 8. Roberson ED, Scearce-Levie K, Palop JJ, Yan F, Cheng IH, et al. (2007) Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer’s disease mouse model. Science 316: 750–754. 9. Muyllaert D, Kremer A, Jaworski T, Borghgraef P, Devijver H, et al. (2008) Glycogen synthase kinase-3beta, or a link between amyloid and tau pathology? Genes Brain Behav 7 Suppl 1: 57–66. 10. Wang ZF, Li HL, Li XC, Zhang Q, Tian Q, et al. (2006) Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture. J Neurochem 98: 1167–1175. 11. Forde JE, Dale TC (2007) Glycogen synthase kinase 3: a key regulator of cellular fate. Cell Mol Life Sci 64: 1930–1944. 12. Phiel CJ, Wilson CA, Lee VM, Klein PS (2003) GSK-3alpha regulates production of Alzheimer’s disease amyloid-beta peptides. Nature 423: 435–439. 13. Lochhead PA, Kinstrie R, Sibbet G, Rawjee T, Morrice N, et al. (2006) A chaperone-dependent GSK-3beta transitional intermediate mediates activation- loop autophosphorylation. Mol Cell 24: 627–633. 14. Terwel D, Muyllaert D, Dewachter I, Borghgraef P, Croes S, et al. (2008) Amyloid activates GSK-3beta to aggravate neuronal tauopathy in bigenic mice. Am J Pathol 172: 786–798. 15. Ryves WJ, Harwood AJ (2001) Lithium inhibits glycogen synthase kinase-3 by competition for magnesium. Biochem Biophys Res Commun 280: 720–725. 16. Chalecka-Franaszek E, Chuang DM (1999) Lithium activates the serine/ threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc Natl Acad Sci U S A 96: 8745–8750. 17. Rockenstein E, Torrance M, Adame A, Mante M, Bar-on P, et al. (2007) Neuroprotective effects of regulators of the glycogen synthase kinase-3beta signaling pathway in a transgenic model of Alzheimer’s disease are associated with reduced amyloid precursor protein phosphorylation. J Neurosci 27: 1981–1991. 18. Noble W, Planel E, Zehr C, Olm V, Meyerson J, et al. (2005) Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc Natl Acad Sci U S A 102: 6990–6995. 19. Caccamo A, Oddo S, Tran LX, LaFerla FM (2007) Lithium reduces tau phosphorylation but not A beta or working memory deficits in a transgenic model with both plaques and tangles. Am J Pathol 170: 1669–1675. 20. Mershin A, Pavlopoulos E, Fitch O, Braden BC, Nanopoulos DV, et al. (2004) Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons. Learn Mem 11: 277–287. 21. Ubhi KK, Shaibah H, Newman TA, Shepherd D, Mudher A (2007) A comparison of the neuronal dysfunction caused by Drosophila tau and human tau in a Drosophila model of tauopathies. Invert Neurosci 7: 165–171. 22. Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J, et al. (2001) Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293: 711–714. GSK-3 Inhibition Ameliorates Ab Pathology (CNRS, France) for kind donation of elavGS fly stocks, Dr. N. Lowe (University of Cambridge) for the Drosophila tau antibody, Dr. M. Bourouis (CNRS, France) for the pan-Sgg antibody, and Dr. J. Fitzgerald (Institute of Neurology, UCL) for advice on using the phos-tag methods. Author Contributions Conceived and designed the experiments: OS FK IR SL LP. Performed the experiments: OS FK IR RK HA CG MJA. Analyzed the data: OS FK IR RK HA CG MJA. Contributed reagents/materials/analysis tools: OS FK MJA. Wrote the paper: OS FK IR JH SL LP. 23. Ruel L, Bourouis M, Heitzler P, Pantesco V, Simpson P (1993) Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch. Nature 362: 557–560. 24. Jackson GR, Wiedau-Pazos M, Sang TK, Wagle N, Brown CA, et al. (2002) Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34: 509–519. 25. Mudher A, Shepherd D, Newman TA, Mildren P, Jukes JP, et al. (2004) GSK- 3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila. Mol Psychiatry 9: 522–530. 26. Finelli A, Kelkar A, Song HJ, Yang H, Konsolaki M (2004) A model for studying Alzheimer’s Abeta42-induced toxicity in Drosophila melanogaster. Mol Cell Neurosci 26: 365–375. 27. Crowther DC, Page R, Chandraratna D, Lomas DA (2006) A Drosophila model of Alzheimer’s disease. Methods Enzymol 412: 234–255. 28. Iijima K, Liu HP, Chiang AS, Hearn SA, Konsolaki M, et al. (2004) Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer’s disease. Proc Natl Acad Sci U S A 101: 6623–6628. 29. Rosen DR, Martin-Morris L, Luo LQ, White K (1989) A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor. Proc Natl Acad Sci U S A 86: 2478–2482. 30. Osterwalder T, Yoon KS, White BH, Keshishian H (2001) A conditional tissue- specific transgene expression system using inducible GAL4. Proc Natl Acad Sci U S A 98: 12596–12601. 31. McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci STKE 2004: pl6. 32. Latouche M, Lasbleiz C, Martin E, Monnier V, Debeir T, et al. (2007) A conditional pan-neuronal Drosophila model of spinocerebellar ataxia 7 with a reversible adult phenotype suitable for identifying modifier genes. J Neurosci 27: 2483–2492. 33. Crowther DC, Kinghorn KJ, Miranda E, Page R, Curry JA, et al. (2005) Intraneuronal Abeta, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer’s disease. Neuroscience 132: 123–135. 34. Bharadwaj PR, Dubey AK, Masters CL, Martins RN, Macreadie IG (2009) Abeta aggregation and possible implications in Alzheimer’s disease pathogenesis. J Cell Mol Med 13: 412–421. 35. Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, et al. (2001) The ‘Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced Abeta protofibril formation. Nat Neurosci 4: 887–893. 36. Feany MB, Bender WW (2000) A Drosophila model of Parkinson’s disease. Nature 404: 394–398. 37. Papadopoulou D, Bianchi MW, Bourouis M (2004) Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster. Mol Cell Biol 24: 4909–4919. 38. Bourouis M (2002) Targeted increase in shaggy activity levels blocks wingless signaling. Genesis 34: 99–102. 39. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T (2006) Phosphate- binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5: 749–757. 40. Oh H, Irvine KD (2008) In vivo regulation of Yorkie phosphorylation and localization. Development 135: 1081–1088. 41. Hanger DP, Byers HL, Wray S, Leung KY, Saxton MJ, et al. (2007) Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis. J Biol Chem 282: 23645–23654. 42. Hanger DP, Anderton BH, Noble W (2009) Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends Mol Med 15: 112–119. 43. Gotz J, Chen F, van Dorpe J, Nitsch RM (2001) Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science 293: 1491–1495. 44. Perez M, Ribe E, Rubio A, Lim F, Moran MA, et al. (2005) Characterization of a double (amyloid precursor protein-tau) transgenic: tau phosphorylation and aggregation. Neuroscience 130: 339–347. PLoS Genetics | www.plosgenetics.org 17 September 2010 | Volume 6 | Issue 9 | e1001087