Functional Screening of Alzheimer Pathology Genome-wide Association Signals in Drosophila
2011; Elsevier BV; Volume: 88; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2011.01.006
ISSN1537-6605
AutoresJoshua M. Shulman, Portia Chipendo, Lori B. Chibnik, Cristin Aubin, Dong Tran, Brendan T Keenan, Patricia L. Kramer, Julie A. Schneider, David Bennett, Mel Β. Feany, Philip L. De Jager,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoWe have leveraged a Drosophila model relevant to Alzheimer disease (AD) for functional screening of findings from a genome-wide scan for loci associated with a quantitative measure of AD pathology in humans. In six of the 15 genomic regions evaluated, we successfully identified a causal gene for the association, on the basis of in vivo interactions with the neurotoxicity of Tau, which forms neurofibrillary tangles in AD. Among the top results, rs10845990 within SLC2A14, encoding a glucose transporter, showed evidence of replication for association with AD pathology, and gain and loss of function in glut1, the Drosophila ortholog, was associated with suppression and enhancement of Tau toxicity, respectively. Our strategy of coupling genome-wide association in humans with functional screening in a model organism is likely to be a powerful approach for gene discovery in AD and other complex genetic disorders. We have leveraged a Drosophila model relevant to Alzheimer disease (AD) for functional screening of findings from a genome-wide scan for loci associated with a quantitative measure of AD pathology in humans. In six of the 15 genomic regions evaluated, we successfully identified a causal gene for the association, on the basis of in vivo interactions with the neurotoxicity of Tau, which forms neurofibrillary tangles in AD. Among the top results, rs10845990 within SLC2A14, encoding a glucose transporter, showed evidence of replication for association with AD pathology, and gain and loss of function in glut1, the Drosophila ortholog, was associated with suppression and enhancement of Tau toxicity, respectively. Our strategy of coupling genome-wide association in humans with functional screening in a model organism is likely to be a powerful approach for gene discovery in AD and other complex genetic disorders. Genome-wide association studies (GWAS) have emerged as powerful tools for the dissection of complex genetic traits, such as susceptibility to Alzheimer disease (AD, MIM 104300);1Bertram L. Tanzi R.E. Genome-wide association studies in Alzheimer's disease.Hum. Mol. Genet. 2009; 18: R137-R145Crossref PubMed Scopus (157) Google Scholar however, efficient methods are needed to enhance follow-up of association signals in order to accelerate the identification and functional validation of genes affected by causal variants.2Ioannidis J.P.A. Thomas G. Daly M.J. Validating, augmenting and refining genome-wide association signals.Nat. Rev. Genet. 2009; 10: 318-329Crossref PubMed Scopus (304) Google Scholar On the basis of recent analyses, the top of GWAS-results distributions (10−3 < p < 10−7), though falling short of genome-wide significance (p < 5 × 10−8), are likely enriched for true associations, but these signals are obscured by a substantial number of chance observations with comparable statistical evidence.3Purcell S.M. Wray N.R. Stone J.L. Visscher P.M. O'Donovan M.C. Sullivan P.F. Sklar P. International Schizophrenia ConsortiumCommon polygenic variation contributes to risk of schizophrenia and bipolar disorder.Nature. 2009; 460: 748-752Crossref PubMed Scopus (3138) Google Scholar, 4Yang J. Benyamin B. McEvoy B.P. Gordon S. Henders A.K. Nyholt D.R. Madden P.A. Heath A.C. Martin N.G. Montgomery G.W. et al.Common SNPs explain a large proportion of the heritability for human height.Nat. Genet. 2010; 42: 565-569Crossref PubMed Scopus (2492) Google Scholar, 5Bush W.S. Sawcer S.J. de Jager P.L. Oksenberg J.R. McCauley J.L. Pericak-Vance M.A. Haines J.L. International Multiple Sclerosis Genetics ConsortiumEvidence for polygenic susceptibility to multiple sclerosis–the shape of things to come.Am. J. Hum. Genet. 2010; 86: 621-625Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar New strategies are therefore needed, not only to validate associations with the best evidence, but also to facilitate identification of true signals of association in circumstances where statistical power is limited and increased sample size is not feasible. One potential solution is to couple the GWAS with a functional screen that evaluates candidate genes for participation in a relevant pathological cascade, a two-stage strategy that might effectively increase overall study power. Here, we leverage a model system relevant to AD in the fruit fly, Drosophila melanogaster, to perform functional testing of 19 genes from 15 distinct genomic regions identified in a GWAS for loci influencing the burden of AD pathology in humans. AD is the most common cause of dementia, and it is characterized at autopsy by widespread neuronal loss in association with extracellular amyloid plaques and intracellular neurofibrillary tangles, predominantly comprising the amyloid-β peptide (Aß) and Tau, respectively.6Querfurth H.W. LaFerla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3292) Google Scholar Both rare mutations and common polymorphisms have been found to influence susceptibility for AD, and GWAS have recently been successful at discovering such loci.1Bertram L. Tanzi R.E. Genome-wide association studies in Alzheimer's disease.Hum. Mol. Genet. 2009; 18: R137-R145Crossref PubMed Scopus (157) Google Scholar, 7Harold D. Abraham R. Hollingworth P. Sims R. Gerrish A. Hamshere M.L. Pahwa J.S. Moskvina V. Dowzell K. Williams A. et al.Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease.Nat. Genet. 2009; 41: 1088-1093Crossref PubMed Scopus (1970) Google Scholar, 8Lambert J.-C. Heath S. Even G. Campion D. Sleegers K. Hiltunen M. Combarros O. Zelenika D. Bullido M.J. Tavernier B. et al.Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease.Nat. Genet. 2009; 41: 1094-1099Crossref PubMed Scopus (1154) Google Scholar, 9Seshadri S. Fitzpatrick A.L. Ikram M.A. DeStefano A.L. Gudnason V. Boada M. Bis J.C. Smith A.V. Carassquillo M.M. Lambert J.C. et al.Genome-wide analysis of genetic loci associated with Alzheimer disease.JAMA. 2010; 303: 1832-1840Crossref PubMed Scopus (894) Google Scholar Most GWAS conducted to date have relied on the dichotomous outcome of AD clinical diagnosis; however, this study design is potentially confounded by genetic heterogeneity of dementia in cases and subclinical disease in controls. In a complementary approach, we have based our analysis on a relevant AD intermediate phenotype: a quantitative measure of global AD pathology from postmortem counts of amyloid plaques and neurofibrillary tangles. Although this approach potentially offers more statistical power than a case-control study of comparable size,10Bennett D.A. De Jager P.L. Leurgans S.E. Schneider J.A. Neuropathologic intermediate phenotypes enhance association to Alzheimer susceptibility alleles.Neurology. 2009; 72: 1495-1503Crossref PubMed Scopus (71) Google Scholar, 11Shulman J.M. Chibnik L.B. Aubin C. Schneider J. De Jager P. Bennett D. Intermediate phenotypes identify divergent pathways to Alzheimer's disease.PLoS ONE. 2010; 5: e1124Crossref Scopus (36) Google Scholar it is limited by the difficulty in obtaining neuropathologic data on large numbers of older individuals. Thus, we anticipated a challenge in meeting the statistical burden of proof for gene discovery, and therefore we coupled our association analysis with a functional screening paradigm in order to validate our results. A GWAS was performed in an autopsy cohort consisting of 227 participants from the Religious Orders Study and the Rush Memory and Aging Project, two longitudinal, epidemiologic studies of aging and AD that include brain donation at death.12Bennett D.A. Schneider J. Arvanitakis Z. Kelly J. Aggarwal N. Shah R. Wilson R. Neuropathology of older persons without cognitive impairment from two community-based studies.Neurology. 2006; 66: 1837-1844Crossref PubMed Scopus (794) Google Scholar, 13Bennett D.A. Wilson R. Schneider J. Evans D. Beckett L. Aggarwal N. Barnes L. Fox J. Bach J. Natural history of mild cognitive impairment in older persons.Neurology. 2002; 59: 198-205Crossref PubMed Scopus (737) Google Scholar, 14Bennett D.A. Schneider J.A. Buchman A.S. Mendes de Leon C. Bienias J.L. Wilson R.S. The Rush Memory and Aging Project: study design and baseline characteristics of the study cohort.Neuroepidemiology. 2005; 25: 163-175Crossref PubMed Scopus (274) Google Scholar Written informed consent was given and an Anatomic Gift Act signed by all study participants after the procedures were fully explained, and both studies were approved by the institutional review board of Rush University Medical Center. Subjects were nondemented at recruitment and were followed prospectively with annual clinical evaluations. Proximate to death, 40% of subjects had normal cognition, 22% had mild cognitive impairment, and 38% met clinical criteria for AD (Table S1 available online). After quality control, 334,575 SNP genotypes were available for analysis (Figure S1). The outcome was a continuous measure of global AD pathology, based on averaged counts of neuritic plaques, diffuse plaques, and neurofibrillary tangles on silver-stained tissue sections from five brain regions (midfrontal, middle temporal, inferior parietal, and entorhinal cortices and the hippocampal CA1 sector).15Bennett D.A. Schneider J.A. Tang Y. Arnold S.E. Wilson R.S. The effect of social networks on the relation between Alzheimer's disease pathology and level of cognitive function in old people: a longitudinal cohort study.Lancet Neurol. 2006; 5: 406-412Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 16Bennett D.A. Wilson R.S. Schneider J.A. Evans D.A. Aggarwal N.T. Arnold S.E. Cochran E.J. Berry-Kravis E. Bienias J.L. Apolipoprotein E epsilon4 allele, AD pathology, and the clinical expression of Alzheimer's disease.Neurology. 2003; 60: 246-252Crossref PubMed Scopus (202) Google Scholar Linear regression was used to evaluate SNP associations with the continuous AD pathological trait, adjusting for both age at death and APOE ɛ4 (MIM 107741) genotype. The top independently associated regions (p < 1 × 10−3) containing candidate genes are presented in Table 1 (for full results, see Table S2). Of note, the subjects in the study cohort were also part of a larger autopsy collection used for a recent candidate-based analysis of associations with AD pathology intermediate phenotypes;11Shulman J.M. Chibnik L.B. Aubin C. Schneider J. De Jager P. Bennett D. Intermediate phenotypes identify divergent pathways to Alzheimer's disease.PLoS ONE. 2010; 5: e1124Crossref Scopus (36) Google Scholar however, none of the loci examined in that study exceeded the significance threshold applied here, and many of those SNPs were not captured by the Illumina genotyping platform used in this genome scan.Table 1GWAS Results and Functional ScreeningSNPLocusAllelesMAFBeta (95% CI)p ValueHuman Gene(s)Functional ScreenFly OrthologLOFGOFrs39356919q13C/T0.490.15 (0.09 to 0.21)1.64 × 10−6SPTBN4B-specEnh-SHKBP1CG9467-N/ALTBP4rs194152618q12A/G0.280.15 (0.09 to 0.22)6.46 × 10−6PIK3C3Pi3K59F-N/Ars174680719p21C/T0.110.22 (0.12 to 0.31)7.87 × 10−6ELAVL2fneSupEnhrs22808618p21C/T0.25−0.16 (−0.23 to −0.09)1.40 × 10−5ENTPD4NTPase--SLC25A37mfrn--rs100652605q14C/A0.490.13 (0.07 to 0.19)2.38 × 10−5SCAMP1Scamp--LHFPL2CG3770-N/Ars193550210p12A/G0.300.15 (0.08 to 0.21)2.66 × 10−5SLC39A12CG10006-N/Ars382498211p14T/C0.220.15 (0.08 to 0.22)3.22 × 10−5MPPED2CG16717-N/Ars123786479q33G/A0.350.14 (0.08 to 0.21)3.44 × 10−5DBC1rs168985q14T/C0.31−0.13 (−0.19 to −0.07)4.64 × 10−5HAPLN1rs21087207p14T/C0.22−0.16 (−0.23 to −0.08)5.23 × 10−5POU6F2pdm3-N/Ars52734612p13G/A0.45−0.12 (−0.18 to −0.06)5.72 × 10−5TSPAN9tsp5D-N/Ars1084599012p13T/G0.390.13 (0.06 to 0.19)6.93 × 10−5SLC2A14Glut1EnhSupNANOGbsh--rs951312213q32G/A0.43−0.12 (−0.18 to −0.06)1.70 × 10−4HS6ST3hs6stEnh-rs75917082p15T/C0.350.12 (0.06 to 0.18)1.93 × 10−4EHBP1CG15609-N/Ars712806311q14A/G0.25−0.13 (−0.20 to −0.06)5.93 × 10−4DLG2dlgEnh-rs126346903p12T/C0.33−0.11 (−0.17 to −0.04)1.32 × 10−3ROBO2robo--rs2978085q35G/A0.360.09 (0.03 to 0.15)2.60 × 10−3SLIT3slitEnhSupAlleles are denoted as minor/major. Beta is calculated per copy of minor allele under the additive genetic model with adjustment for age at death and APOE ɛ4 genotype. CI, confidence interval. Functional Screen shows screening results based on testing of gain or loss of function (GOF and LOF, respectively) in orthologous fly genes for enhancement (Enh) or suppression (Sup) of Tau toxicity. MAF, minor allele frequency; -, no interaction observed; N/A, genetic reagent not available. Fly orthologs were identified on the basis of implementation of the tBLASTn algorithm50Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (56937) Google Scholar within the annotated Drosophila genome. All orthologs had highly significant BLAST results: E value < 10−10 and mean score = 398 (range: 67–1462). Fly genes with evidence of functional interactions with Tau toxicity are shown in boldface type. Open table in a new tab Alleles are denoted as minor/major. Beta is calculated per copy of minor allele under the additive genetic model with adjustment for age at death and APOE ɛ4 genotype. CI, confidence interval. Functional Screen shows screening results based on testing of gain or loss of function (GOF and LOF, respectively) in orthologous fly genes for enhancement (Enh) or suppression (Sup) of Tau toxicity. MAF, minor allele frequency; -, no interaction observed; N/A, genetic reagent not available. Fly orthologs were identified on the basis of implementation of the tBLASTn algorithm50Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (56937) Google Scholar within the annotated Drosophila genome. All orthologs had highly significant BLAST results: E value < 10−10 and mean score = 398 (range: 67–1462). Fly genes with evidence of functional interactions with Tau toxicity are shown in boldface type. As expected for our small study, no variant achieved genome-wide significance, and we therefore implemented our functional screening strategy. Candidate genes in the vicinity of top-scoring SNPs were identified on the basis of linkage disequilibrium criteria (Table 1 and Table S2), and in each case, all such genes were included for further evaluation in an unbiased fashion. In nine out of 24 cases, no candidate genes were identified in the target genomic region around an index SNP, and these association signals were not pursued further. We additionally chose to evaluate two genomic regions that were identified by SNP associations of more modest significance but contained genes (SLIT3 [MIM 603745] and ROBO2 [MIM 602431]) that function as ligand and receptor, respectively, in a common neuronal signaling pathway. Nineteen out of the 22 candidate genes had conserved orthologs in Drosophila and were promoted to functional testing. A variety of Drosophila experimental models relevant to AD have been developed, including transgenic systems based on the neurotoxicity of both Aß and Tau.17Finelli A. Kelkar A. Song H.J. Yang H. Konsolaki M. A model for studying Alzheimer's Abeta42-induced toxicity in Drosophila melanogaster.Mol. Cell. Neurosci. 2004; 26: 365-375Crossref PubMed Scopus (209) Google Scholar, 18Moloney A. Sattelle D.B. Lomas D.A. Crowther D.C. Alzheimer's disease: insights from Drosophila melanogaster models.Trends Biochem. Sci. 2010; 35: 228-235Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 19Wittmann C.W. Wszolek M. Shulman J. Salvaterra P. Lewis J. Hutton M. Feany M. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.Science. 2001; 293: 711-714Crossref PubMed Scopus (692) Google Scholar For functional screening of GWAS results, we selected the Tau transgenic model because (1) it has previously been successfully employed for rapid genetic screening20Shulman J.M. Feany M. Genetic modifiers of tauopathy in Drosophila.Genetics. 2003; 165: 1233-1242Crossref PubMed Google Scholar and (2) there is growing consensus that Tau is a downstream mediator of Aß toxicity in AD.6Querfurth H.W. LaFerla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3292) Google Scholar, 21Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wölfing H. Chieng B.C. Christie M.J. Napier I.A. et al.Dendritic Function of Tau Mediates Amyloid-beta Toxicity in Alzheimer's Disease Mouse Models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1194) Google Scholar, 22Roberson E.D. Scearce-Levie K. Palop J. Yan F. Cheng I. Wu T. Gerstein H. Yu G. Mucke L. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model.Science. 2007; 316: 750-754Crossref PubMed Scopus (1373) Google Scholar, 23Vossel K.A. Zhang K. Brodbeck J. Daub A.C. Sharma P. Finkbeiner S. Cui B. Mucke L. Tau Reduction Prevents Abeta-Induced Defects in Axonal Transport.Science. 2010; 330: 198Crossref PubMed Scopus (358) Google Scholar Expression of human Tau (MAPT [MIM 157140]) in the Drosophila nervous system recapitulates several features of AD, including age-dependent neurodegeneration, decreased lifespan, and abnormally phosphorylated and misfolded Tau.19Wittmann C.W. Wszolek M. Shulman J. Salvaterra P. Lewis J. Hutton M. Feany M. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.Science. 2001; 293: 711-714Crossref PubMed Scopus (692) Google Scholar We used transgenic animals, allowing tissue-specific expression of TauV337M, a mutant form of Tau associated with familial frontotemporal dementia (FTD [MIM 600274]). Importantly, wild-type and mutant forms of human Tau demonstrate similar mechanisms of toxicity when expressed in the Drosophila nervous system and show consistent interactions with known genetic modifiers.19Wittmann C.W. Wszolek M. Shulman J. Salvaterra P. Lewis J. Hutton M. Feany M. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.Science. 2001; 293: 711-714Crossref PubMed Scopus (692) Google Scholar, 24Fulga T.A. Elson-Schwab I. Khurana V. Steinhilb M.L. Spires T.L. Hyman B.T. Feany M.B. Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.Nat. Cell Biol. 2007; 9: 139-148Crossref PubMed Scopus (336) Google Scholar, 25Khurana V. Lu Y. Steinhilb M. Oldham S. Shulman J. Feany M. TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model.Curr. Biol. 2006; 16: 230-241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar Therefore, similar to transgenic mouse models based on FTD mutant Tau,26Gotz J. Chen F. van Dorpe J. Nitsch R. Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils.Science. 2001; 293: 1491-1495Crossref PubMed Scopus (1190) Google Scholar, 27Lewis J. McGowan E. Rockwood J. Melrose H. Nacharaju P. Van Slegtenhorst M. Gwinn-Hardy K. Paul Murphy M. Baker M. Yu X. et al.Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.Nat. Genet. 2000; 25: 402-405Crossref PubMed Scopus (1048) Google Scholar the fly model selected for our study is relevant to understanding the mechanisms of Tau toxicity in AD.28Götz J. Ittner L.M. Animal models of Alzheimer's disease and frontotemporal dementia.Nat. Rev. Neurosci. 2008; 9: 532-544Crossref PubMed Scopus (531) Google Scholar TauV337M expression in the fly eye causes a moderately reduced eye size and roughened surface (Figure 1B), a phenotype that is amenable to rapid screening for second-site genetic modifiers.20Shulman J.M. Feany M. Genetic modifiers of tauopathy in Drosophila.Genetics. 2003; 165: 1233-1242Crossref PubMed Google Scholar Specifically, by scoring for lines that either exacerbate or rescue the eye phenotype, genes can be characterized as enhancers or suppressors of Tau toxicity, respectively. For loss-of-function analysis, transgenic RNA-interference (RNAi) lines were tested for all 19 target genes,29Dietzl G. Chen D. Schnorrer F. Su K.C. Barinova Y. Fellner M. Gasser B. Kinsey K. Oppel S. Scheiblauer S. et al.A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.Nature. 2007; 448: 151-156Crossref PubMed Scopus (1846) Google Scholar, 30Ni J.Q. Markstein M. Binari R. Pfeiffer B. Liu L.P. Villalta C. Booker M. Perkins L. Perrimon N. Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster.Nat. Methods. 2008; 5: 49-51Crossref PubMed Scopus (195) Google Scholar and classical Drosophila mutant alleles were also available in most cases.31Matthews K.A. Kaufman T.C. Gelbart W.M. Research resources for Drosophila: the expanding universe.Nat. Rev. Genet. 2005; 6: 179-193Crossref PubMed Scopus (101) Google Scholar, 32Tweedie S. Ashburner M. Falls K. Leyland P. McQuilton P. Marygold S. Millburn G. Osumi-Sutherland D. Schroeder A. Seal R. et al.FlyBase: enhancing Drosophila Gene Ontology annotations.Nucleic Acids Res. 2009; 37: D555-D559Crossref PubMed Scopus (599) Google Scholar In addition, we evaluated lines known or predicted to activate gene expression, allowing assessment for gain-of-function interactions for many loci.33Bellen H.J. Levis R.W. Liao G. He Y. Carlson J.W. Tsang G. Evans-Holm M. Hiesinger P.R. Schulze K.L. Rubin G.M. et al.The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.Genetics. 2004; 167: 761-781Crossref PubMed Scopus (649) Google Scholar Genetic modifier effects were scored with the use of a semiquantitative rating scale of rough-eye severity, allowing statistical comparison with Tau transgenic controls (Figure S4). Out of the 19 genes evaluated in the fly model, six genes show interactions with Tau toxicity in vivo (Table 1, Figure 1, and Figure S2), providing functional evidence that strengthens the validity of the GWAS results. In three notable cases, both loss- and gain-of-function experiments demonstrate reciprocal interactions. Specifically, SLC2A14 (MIM 611039) was selected for evaluation on the basis of an associated intronic SNP (rs10845990), and a single ortholog (glut1) is present in the Drosophila genome.34Escher S.A. Rasmuson-Lestander A. The Drosophila glucose transporter gene: cDNA sequence, phylogenetic comparisons, analysis of functional sites and secondary structures.Hereditas. 1999; 130: 95-103Crossref PubMed Scopus (36) Google Scholar A line predicted to increase glut1 expression was a potent Tau suppressor, restoring the eye to nearly wild-type appearance (Figure 1C), and a glut1 RNAi line had the opposite effect, enhancing Tau toxicity and leading to a worsened eye phenotype (Figure 1F). Similarly, SLIT3 was selected for testing on the basis of an intronic SNP, rs297808. Increasing expression of the orthologous fly gene, slit, rescues the Tau-induced eye phenotype (Figure 1D), whereas slit RNAi increases Tau toxicity (Figure 1G). In addition, we find evidence to support functional validation of ELAVL2 (MIM 601673), a gene found in the vicinity of rs17468071. Transgene-mediated expression of found in neurons (fne), an ortholog of ELAVL2, strongly increased Tau toxicity in the fly eye (Figure 1H), and at higher levels, fne caused pupal lethality when coexpressed with Tau. Reciprocally, an fne RNAi line attenuated Tau toxicity (Figure 1E). The Drosophila genome contains two other ELAVL2 orthologs, including the founding family member, elav, and Rbp9; however, manipulating the expression of these genes in the absence of Tau was associated with substantial toxicity, limiting further evaluation using our screening strategy. Finally, RNAi directed against three other fly genes, β-spectrin, heparan sulfate 6-O-sulfotransferase, and discs large 1, each enhance Tau toxicity, supporting functional validation of the orthologous loci implicated by our GWAS (Table 1 and Figure S2). For the six loci highlighted by the Drosophila functional screen, we genotyped the index SNP in an additional 305 deceased study participants with completed neuropathological evaluation (Table S3). rs10845990, within the SLC2A14 locus, showed suggestive evidence of replication (p = 0.03), and the association was improved in a pooled analysis of 532 subjects, including both the discovery and the replication cohorts (pDISC = 6.9 × 10−5, pJOINT = 8.1 × 10−6). SLC2A14, encoding a glucose transporter (GLUT14), is an attractive biological candidate given the well-known dysregulation of glucose metabolism in the AD brain and likely pathogenic role of oxidative stress.6Querfurth H.W. LaFerla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3292) Google Scholar Although predominantly expressed in the testes,35Wu X. Freeze H.H. GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms.Genomics. 2002; 80: 553-557Crossref PubMed Scopus (124) Google Scholar less abundant SLC2A14 transcripts are also detected in the central nervous system, on the basis of publically available transcriptome data (see Web Resources).36Rhead B. Karolchik D. Kuhn R.M. Hinrichs A.S. Zweig A.S. Fujita P.A. Diekhans M. Smith K.E. Rosenbloom K.R. Raney B.J. et al.The UCSC Genome Browser database: update 2010.Nucleic Acids Res. 2010; 38: D613-D619Crossref PubMed Scopus (493) Google Scholar, 37Su A.I. Cooke M.P. Ching K.A. Hakak Y. Walker J.R. Wiltshire T. Orth A.P. Vega R.G. Sapinoso L.M. Moqrich A. et al.Large-scale analysis of the human and mouse transcriptomes.Proc. Natl. Acad. Sci. USA. 2002; 99: 4465-4470Crossref PubMed Scopus (1217) Google Scholar, 38Wu C. Orozco C. Boyer J. Leglise M. Goodale J. Batalov S. Hodge C.L. Haase J. Janes J. Huss J.W. et al.BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.Genome Biol. 2009; 10: R130Crossref PubMed Scopus (1023) Google Scholar Glucose transporter expression has been reported to be reduced in brains affected by AD, correlated with both Tau phosphorylation and neurofibrillary tangle burden.39Liu Y. Liu F. Iqbal K. Grundke-Iqbal I. Gong C.-X. Decreased glucose transporters correlate to abnormal hyperphosphorylation of tau in Alzheimer disease.FEBS Lett. 2008; 582: 359-364Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar Interestingly, genetic and pharmacological manipulation of oxidative stress has previously been shown to modulate Tau-induced toxicity in flies,40Dias-Santagata D. Fulga T.A. Duttaroy A. Feany M.B. Oxidative stress mediates tau-induced neurodegeneration in Drosophila.J. Clin. Invest. 2007; 117: 236-245Crossref PubMed Scopus (200) Google Scholar potentially consistent with this mechanism of action for the observed interaction with glut1. In summary, on the basis of genetic association in humans and functional screening in a pertinent model organism, we have identified six candidate loci that influence the accumulation of AD neuropathology. Our strategy of integrating human GWAS with a Drosophila genetic screen builds on similar successful cross-species studies in which fly models of neurodegenerative disease enabled secondary screens to reinforce findings from mammalian systems, including transcriptome analysis41Desai U.A. Pallos J. Ma A.A.K. Stockwell B.R. Thompson L.M. Marsh J.L. Diamond M.I. Biologically active molecules that reduce polyglutamine aggregation and toxicity.Hum. Mol. Genet. 2006; 15: 2114-2124Crossref PubMed Scopus (61) Google Scholar and drug discovery.42Karsten S.L. Sang T.-K. Gehman L.T. Chatterjee S. Liu J. Lawless G.M. Sengupta S. Berry R.W. Pomakian J. Oh H.S. et al.A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.Neuron. 2006; 51: 549-560Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar The Drosophila Tau transgenic model selected for our functional screening pipeline has been used in prior successful genetic screens and numerous other investigations,20Shulman J.M. Feany M. Genetic modifiers of tauopathy in Drosophila.Genetics. 2003; 165: 1233-1242Crossref PubMed Google Scholar, 24Fulga T.A. Elson-Schwab I. Khurana V. Steinhilb M.L. Spires T.L. Hyman B.T. Feany M.B. Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.Nat. Cell Biol. 2007; 9: 139-148Crossref PubMed Scopus (336) Google Scholar, 25Khurana V. Lu Y. Steinhilb M. Oldham S. Shulman J. Feany M. TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model.Curr. Biol. 2006; 16: 230-241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 43Blard O. Feuillette S. Bou J. Chaumette B. Frebourg T. Campion D. Lecourtois M. Cytoskeleton proteins are modulators of mutant tau-induced neurodegeneration in Drosophila.Hum. Mol. Genet. 2007; 16: 555-566Crossref PubMed Scopus (79) Google Scholar and many results have been consistent with findings in mouse models and other AD experimental paradigms.28Götz J. Ittner L.M. Animal models of Alzheimer's disease and frontotemporal dementia.Nat. Rev. Neurosci. 2008; 9: 532-544Crossref PubMed Scopus (531) Google Scholar, 44Moloney A. Sattelle D.B. Lomas D.A. Crowther D.C. Alzheimer's disease: insights from Drosophila melanogaster models.Trends Biochem Sci. 2009; 35: 228-235Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar In current hypotheses about the mechanisms of AD pathogenesis, supported by a large body of work, Tau-induced neurotoxicity defines a key pathway mediating the effects of Aß.6Querfurth H.W. LaFerla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3292) Google Scholar, 21Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wölfing H. Chieng B.C. Christie M.J. Napier I.A. et al.Dendritic Function of Tau Mediates Amyloid-beta Toxicity in Alzheimer's Disease Mouse Models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1194) Google Scholar, 22Roberson E.D. Scearce-Levie K. Palop J. Yan F. Cheng I. Wu T. Gerstein H. Yu G. Mucke L. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model.Science. 2007; 316: 750-754Crossref PubMed Scopus (1373) Google Scholar, 23Vossel K.A. Zhang K. Brodbeck J. Daub A.C. Sharma P. Finkbeiner S. Cui B. Mucke L. Tau Reduction Prevents Abeta-Induced Defects in Axonal Transport.Science. 2010; 330: 198Crossref PubMed Scopus (358) Google Scholar Therefore, our functional screen may be relevant to many susceptibility loci that influence downstream mechanisms of Aß toxicity. Nevertheless, our approach would not be expected to detect genes that directly influence the processing of amyloid precursor protein (APP), Aß aggregation, or other proximal events in the pathologic cascade. In the future, such loci might be functionally screened with the use of either APP or Aß transgenic flies or Aß/Tau dual transgenic flies.17Finelli A. Kelkar A. Song H.J. Yang H. Konsolaki M. A model for studying Alzheimer's Abeta42-induced toxicity in Drosophila melanogaster.Mol. Cell. Neurosci. 2004; 26: 365-375Crossref PubMed Scopus (209) Google Scholar, 24Fulga T.A. Elson-Schwab I. Khurana V. Steinhilb M.L. Spires T.L. Hyman B.T. Feany M.B. Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.Nat. Cell Biol. 2007; 9: 139-148Crossref PubMed Scopus (336) Google Scholar, 45Fossgreen A. Bruckner B. Czech C. Masters C. Beyreuther K. Paro R. Transgenic Drosophila expressing human amyloid precursor protein show gamma-secretase activity and a blistered-wing phenotype.Proc. Natl. Acad. Sci. USA. 1998; 95: 13703-13708Crossref PubMed Scopus (125) Google Scholar Additional strengths of our approach include the substantial genomic conservation between flies and mammals46Rubin G.M. Yandell M. Wortman J. Gabor Miklos G. Nelson C. Hariharan I. Fortini M. Li P. Apweiler R. Fleischmann W. et al.Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1307) Google Scholar and the availability of reagents to manipulate the function of nearly all Drosophila genes.31Matthews K.A. Kaufman T.C. Gelbart W.M. Research resources for Drosophila: the expanding universe.Nat. Rev. Genet. 2005; 6: 179-193Crossref PubMed Scopus (101) Google Scholar The success rate of our strategy exceeds the returns of unbiased Drosophila genetic screens using the same transgenic model,20Shulman J.M. Feany M. Genetic modifiers of tauopathy in Drosophila.Genetics. 2003; 165: 1233-1242Crossref PubMed Google Scholar suggesting that the list of 19 loci tested was enriched for genes influencing the development of AD pathology. Although a negative result in our screen does not exclude a gene as potentially associated with AD, the six validated loci highlight pathways of potential relevance to disease pathogenesis. Future functional investigation in Drosophila, and in other experimental systems, may reveal the mechanisms by which these genes modulate Tau-induced neurodegeneration, and these loci are also excellent targets for further replication analysis in human cohorts. Importantly, our functional screening strategy highlights genes that are likely responsible for association signals, and in two cases, rs393569 and rs10845990, we are able to nominate causal genes (SPTBN4 and SLC2A14, respectively) for which more than one candidate was initially found on the basis of linkage disequilibrium with the index SNP, a commonly encountered problem in following up GWAS results. The association signals uncovered in our GWAS are comparable to that of numerous published reports in larger case-control cohorts that have identified candidate risk loci with suggestive but not definitive statistical evidence of association to AD or other relevant intermediate traits.1Bertram L. Tanzi R.E. Genome-wide association studies in Alzheimer's disease.Hum. Mol. Genet. 2009; 18: R137-R145Crossref PubMed Scopus (157) Google Scholar Evidence is emerging in support of a polygenic model of inheritance for complex genetic disorders, particularly neuropsychiatric diseases, in which hundreds or even thousands of common variants collectively contribute to disease risk.3Purcell S.M. Wray N.R. Stone J.L. Visscher P.M. O'Donovan M.C. Sullivan P.F. Sklar P. International Schizophrenia ConsortiumCommon polygenic variation contributes to risk of schizophrenia and bipolar disorder.Nature. 2009; 460: 748-752Crossref PubMed Scopus (3138) Google Scholar, 4Yang J. Benyamin B. McEvoy B.P. Gordon S. Henders A.K. Nyholt D.R. Madden P.A. Heath A.C. Martin N.G. Montgomery G.W. et al.Common SNPs explain a large proportion of the heritability for human height.Nat. Genet. 2010; 42: 565-569Crossref PubMed Scopus (2492) Google Scholar, 5Bush W.S. Sawcer S.J. de Jager P.L. Oksenberg J.R. McCauley J.L. Pericak-Vance M.A. Haines J.L. International Multiple Sclerosis Genetics ConsortiumEvidence for polygenic susceptibility to multiple sclerosis–the shape of things to come.Am. J. Hum. Genet. 2010; 86: 621-625Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Given the very small effect sizes, it is unrealistic that the majority of such loci can be validated individually by statistical evidence alone. Our strategy of coupling GWAS in humans to functional genetic screening in a model organism will therefore likely be a powerful strategy for follow-up of such signals in the future for the prioritization of genes and pathways for further investigation. We are grateful to our colleagues, Lei Yu and Sue Leurgans, for assistance with statistical analyses. We thank Christian Klambt, Jimena Sierralta, and Hiroshi Nakato for generously providing Drosophila stocks. We are grateful Dr. Bradley Hyman and Chris Cotsapas for comments on the manuscript and valuable discussion. We also thank the Bloomington Drosophila stock center, the Vienna Drosophila RNAi Center (VDRC), and the Harvard Transgenic RNAi Project (TRiP, NIH/NIGMS R01GM084947) for providing fly stocks. J.M.S. is supported by NIH grant K08AG034290 and by the Clinical Investigator Training Program: Beth Israel Deaconess Medical Center – Harvard/MIT Health Sciences and Technology, in collaboration with Pfizer Inc. and Merck & Co. M.B.F. is supported by the Ellison Medical Foundation. The authors also thank the participants of the Religious Orders Study and the Rush Memory and Aging Project, which were supported by NIH grants P30AG10161, R01AG15819, and R01AG17917. Download .pdf (.23 MB) Help with pdf files Document S1. Four Figures and Three Tables The URLs for data presented herein are as follows:BioGPS, http://biogps.gnf.orgFlyBase, http://flybase.org/Harvard Transgenic RNAi Project (TRiP), http://www.flyrnai.org/TRiP-HOME.htmlOnline Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGatewayVienna Drosophila RNAi Center (VDRC), http://stockcenter.vdrc.at/control/main
Referência(s)