Advertisement

Morphometric Analysis of Huntington’s Disease Neurodegeneration in Drosophila

  • Wan Song
  • Marianne R. Smith
  • Adeela Syed
  • Tamas Lukacsovich
  • Brett A. Barbaro
  • Judith Purcell
  • Doug J. Bornemann
  • John Burke
  • J. Lawrence Marsh
Part of the Methods in Molecular Biology book series (MIMB, volume 1017)

Abstract

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. The HD gene encodes the huntingtin protein (HTT) that contains polyglutamine tracts of variable length. Expansions of the CAG repeat near the amino terminus to encode 40 or more glutamines (polyQ) lead to disease. At least eight other expanded polyQ diseases have been described [1]. HD can be faithfully modeled in Drosophila with the key features of the disease such as late onset, slowly progressing degeneration, formation of abnormal protein aggregates and the dependence on polyQ length being evident. Such invertebrate model organisms provide powerful platforms to explore neurodegenerative mechanisms and to productively speed the identification of targets and agents that are likely to be effective at treating diseases in humans. Here we describe an optical pseudopupil method that can be readily quantified to provide a fast and sensitive assay for assessing the degree of HD neurodegeneration in vivo. We discuss detailed crossing schemes as well as factors including different drivers, various constructs, the number of UAS sites, genetic background, and temperature that can influence the result of pseudopupil measurements.

Key words

Huntington’s disease Drosophila model Neurodegeneration Polyglutamine disease Pseudopupil assay Ommatidium Photoreceptor cell death 

References

  1. 1.
    Paulson HL, Bonini NM, Roth KA (2000) Polyglutamine disease and neuronal cell death. Proc Natl Acad Sci USA 97(24):12957–12958. doi: 10.1073/pnas.210395797 210395797 [pii] PubMedCrossRefGoogle Scholar
  2. 2.
    Gatchel JR, Zoghbi HY (2005) Diseases of unstable repeat expansion: mechanisms and common principles. Nat Rev Genet 6(10):743–755. doi: nrg1691 [pii] 10.1038/nrg1691 PubMedCrossRefGoogle Scholar
  3. 3.
    Macdonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, Barnes G, Taylor SA, James M, Groot N, Macfarlane H, Jenkins B, Anderson MA, Wexler NS, Gusella JF, Bates GP, Baxendale S, Hummerich H, Kirby S, North M, Youngman S, Mott R, Zehetner G, Sedlacek Z, Poustka A, Frischauf AM, Lehrach H, Buckler AJ, Church D, Doucettestamm L, Odonovan MC, Ribaramirez L, Shah M, Stanton VP, Strobel SA, Draths KM, Wales JL, Dervan P, Housman DE, Altherr M, Shiang R, Thompson L, Fielder T, Wasmuth JJ, Tagle D, Valdes J, Elmer L, Allard M, Castilla L, Swaroop M, Blanchard K, Collins FS, Snell R, Holloway T, Gillespie K, Datson N, Shaw D, Harper PS (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntingtons-disease chromosomes. Cell 72(6):971–983CrossRefGoogle Scholar
  4. 4.
    Gusella JF, Macdonald ME (1995) Huntingtons-disease. Semin Cell Biol 6(1):21–28PubMedCrossRefGoogle Scholar
  5. 5.
    Kremer B, Goldberg P, Andrew SE, Theilmann J, Telenius H, Zeisler J, Squitieri F, Lin BY, Bassett A, Almqvist E, Bird TD, Hayden MR (1994) A worldwide study of the Huntingtons-disease mutation—the sensitivity and specificity of measuring Cag repeats. N Engl J Med 330(20):1401–1406PubMedCrossRefGoogle Scholar
  6. 6.
    Nagai Y, Fujikake N, Ohno K, Higashiyama H, Popiel HA, Rahadian J, Yamaguchi M, Strittmatter WJ, Burke JR, Toda T (2003) Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila. Hum Mol Genet 12(11):1253–1259PubMedCrossRefGoogle Scholar
  7. 7.
    Jackson GR, Salecker I, Dong XZ, Yao X, Arnheim N, Faber PW, MacDonald ME, Zipursky SL (1998) Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron 21(3):633–642PubMedCrossRefGoogle Scholar
  8. 8.
    Marsh JL, Walker H, Theisen H, Zhu YZ, Fielder T, Purcell J, Thompson LM (2000) Expanded polyglutamine peptides alone are intrinsically cytotoxic and cause neurodegeneration in Drosophila. Hum Mol Genet 9(1):13–25PubMedCrossRefGoogle Scholar
  9. 9.
    Warrick JM, Paulson HL, Gray-Board GL, Bui QT, Fischbeck KH, Pittman RN, Bonini NM (1998) Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell 93(6):939–949PubMedCrossRefGoogle Scholar
  10. 10.
    Kazemi-Esfarjani P, Benzer S (2000) Genetic suppression of polyglutamine toxicity in Drosophila. Science 287(5459):1837–1840. doi: 8327 [pii] PubMedCrossRefGoogle Scholar
  11. 11.
    Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, Kazantsev A, Schmidt E, Zhu YZ, Greenwald M, Kurokawa R, Housman DE, Jackson GR, Marsh JL, Thompson LM (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413(6857):739–743PubMedCrossRefGoogle Scholar
  12. 12.
    Lee WCM, Yoshihara M, Littleton JT (2004) Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington’s disease. Proc Natl Acad Sci USA 101(9):3224–3229PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang S, Binari R, Zhou R, Perrimon N (2010) A genomewide RNA interference screen for modifiers of aggregates formation by mutant Huntingtin in Drosophila. Genetics 184(4):1165–1179. doi: genetics.109.112516 [pii] 10.1534/genetics.109.112516 PubMedCrossRefGoogle Scholar
  14. 14.
    Romero E, Cha GH, Verstreken P, Ly CV, Hughes RE, Bellen HJ, Botas J (2008) Suppression of neurodegeneration and increased neurotransmission caused by expanded full-length huntingtin accumulating in the cytoplasm. Neuron 57(1):27–40. doi: S0896-6273(07)00985-3 [pii] 10.1016/j.neuron.2007.11.025 PubMedCrossRefGoogle Scholar
  15. 15.
    Mugat B, Parmentier ML, Bonneaud N, Chan HY, Maschat F (2008) Protective role of Engrailed in a Drosophila model of Huntington’s disease. Hum Mol Genet 17(22):3601–3616. doi: ddn255 [pii] 10.1093/hmg/ddn255 PubMedCrossRefGoogle Scholar
  16. 16.
    Franceschini N (1972) Information processing in the visual systems of arthropods. Springer, BerlinGoogle Scholar
  17. 17.
    Agrawal N, Pallos J, Slepko N, Apostol BL, Bodai L, Chang LW, Chiang AS, Thompson LM, Marsh JL (2005) Identification of combinatorial drug regimens for treatment of Huntington’s disease using Drosophila. Proc Natl Acad Sci USA 102(10):3777–3781. doi: 10.1073/Pnas.0500055102 PubMedCrossRefGoogle Scholar
  18. 18.
    Brand AH, Perrimon N (1993) Targeted gene-expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415PubMedGoogle Scholar
  19. 19.
    Marsh JL, Thompson LM (2004) Can flies help humans treat neurodegenerative diseases? Bioessays 26(5):485–496. doi: 10.1002/Bies.20029 PubMedCrossRefGoogle Scholar
  20. 20.
    Lin DM, Goodman CS (1994) Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13(3):507–523. doi: 0896-6273(94)90022-1 [pii] PubMedCrossRefGoogle Scholar
  21. 21.
    Fernandez-Funez P, Nino-Rosales ML, de Gouyon B, She WC, Luchak JM, Martinez P, Turiegano E, Benito J, Capovilla M, Skinner PJ, McCall A, Canal I, Orr HT, Zoghbi HY, Botas J (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature 408(6808):101–106PubMedCrossRefGoogle Scholar
  22. 22.
    Karpilow JM, Pimentel AC, Shamloula HK, Venkatesh TR (1996) Neuronal development in the Drosophila compound eye: photoreceptor cells R1, R6, and R7 fail to differentiate in the Retina aberrant in pattern (rap) mutant. J Neurobiol 31(2):149–165PubMedCrossRefGoogle Scholar
  23. 23.
    Spradling AC, Rubin GM (1983) The effect of chromosomal position on the expression of the Drosophila xanthine dehydrogenase gene. Cell 34(1):47–57PubMedCrossRefGoogle Scholar
  24. 24.
    Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phi C31. Genetics 166(4):1775–1782PubMedCrossRefGoogle Scholar
  25. 25.
    Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34(1–2):1–15. doi: 10.1002/Gene.1015 PubMedCrossRefGoogle Scholar
  26. 26.
    Matsumoto K, Tohe A, Oshima Y (1978) Genetic-control of galactokinase synthesis in Saccharomyces cerevisiae—evidence for constitutive expression of positive regulatory gene Gal4. J Bacteriol 134(2):446–457PubMedGoogle Scholar
  27. 27.
    Elliott DA, Brand AH (2008) The GAL4 system: a versatile system for the expression of genes. Methods Mol Biol 420:79–95. doi: 10.1007/978-1-59745-583-1_5 PubMedCrossRefGoogle Scholar
  28. 28.
    McGuire SE, Le PT, Osborn AJ, Matsumoto K, Davis RL (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302(5651):1765–1768PubMedCrossRefGoogle Scholar
  29. 29.
    Ito K, Awano W, Suzuki K, Hiromi Y, Yamamoto D (1997) The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124(4):761–771PubMedGoogle Scholar
  30. 30.
    Pignoni F, Zipursky SL (1997) Induction of Drosophila eye development by Decapentaplegic. Development 124(2):271–278PubMedGoogle Scholar
  31. 31.
    Osterwalder T, Yoon KS, White BH, Keshishian H (2001) A conditional tissue-specific transgene expression system using inducible GAL4. Proc Natl Acad Sci USA 98(22):12596–12601PubMedCrossRefGoogle Scholar
  32. 32.
    Roman G, Endo K, Zong L, Davis RL (2001) P{Switch}, a system for spatial and temporal control of gene expression in Drosophila melanogaster. Proc Natl Acad Sci USA 98(22):12602–12607PubMedCrossRefGoogle Scholar
  33. 33.
    Rorth P, Szabo K, Bailey A, Laverty T, Rehm J, Rubin GM, Weigmann K, Milan M, Benes V, Ansorge W, Cohen SM (1998) Systematic gain-of-function genetics in Drosophila. Development 125(6):1049–1057PubMedGoogle Scholar
  34. 34.
    Rorth P (1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci USA 93(22):12418–12422PubMedCrossRefGoogle Scholar
  35. 35.
    Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci USA 99(20):12651–12656. doi: 10.1073/Pnas.202320599 PubMedCrossRefGoogle Scholar
  36. 36.
    Ni JQ, Liu LP, Binari R, Hardy R, Shim HS, Cavallaro A, Booker M, Pfeiffer BD, Markstein M, Wang H, Villalta C, Laverty TR, Perkins LA, Perrimon N (2009) A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182(4):1089–1100. doi: 10.1534/Genetics.109.103630 PubMedCrossRefGoogle Scholar
  37. 37.
    Pfeiffer BD, Ngo TTB, Hibbard KL, Murphy C, Jenett A, Truman JW, Rubin GM (2010) Refinement of tools for targeted gene expression in Drosophila. Genetics 186(2):735–755. doi: 10.1534/Genetics.110.119917 PubMedCrossRefGoogle Scholar
  38. 38.
    Robinow S, White K (1988) The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev Biol 126(2):294–303PubMedCrossRefGoogle Scholar
  39. 39.
    Lee WC, Yoshihara M, Littleton JT (2004) Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington’s disease. Proc Natl Acad Sci USA 101(9):3224–3229. doi: 10.1534/Genetics.110.119917 PubMedCrossRefGoogle Scholar
  40. 40.
    Bonini NM, Fortini ME (2003) Human neurodegenerative disease modeling using Drosophila. Annu Rev Neurosci 26:627–656. doi: 10.1146/annurev.neuro.26.041002.131425 041002.131425 [pii] PubMedCrossRefGoogle Scholar
  41. 41.
    Ellis MC, Oneill EM, Rubin GM (1993) Expression of Drosophila glass protein and evidence for negative regulation of its activity in nonneuronal cells by another DNA-binding protein. Development 119(3):855–865PubMedGoogle Scholar
  42. 42.
    Sang TK, Li C, Liu W, Rodriguez A, Abrams JM, Zipursky SL, Jackson GR (2005) Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis. Hum Mol Genet 14(3):357–372. doi: ddi032 [pii] 10.1093/hmg/ddi032 PubMedCrossRefGoogle Scholar
  43. 43.
    Kirschfeld K, Feiler R, Franceschini N (1978) Photo-stable pigment within rhabdomere of fly photoreceptors No 7. J Comp Physiol 125(3):275–284CrossRefGoogle Scholar
  44. 44.
    Chyb S, Hevers W, Forte M, Wolfgang WJ, Selinger Z, Hardie RC (1999) Modulation of the light response by cAMP in Drosophila photoreceptors. J Neurosci 19(20):8799–8807PubMedGoogle Scholar
  45. 45.
    Slepko N, Bhattacharyya AM, Jackson GR, Steffan JS, Marsh JL, Thompson LM, Wetzel R (2006) Normal-repeat-length polyglutamine peptides accelerate aggregation nucleation and cytotoxicity of expanded polyglutamine proteins. Proc Natl Acad Sci USA 103(39):14367–14372. doi: 10.1073/Pnas.0602348103 PubMedCrossRefGoogle Scholar
  46. 46.
    Branco J, Al-Ramahi I, Ukani L, Perez AM, Fernandez-Funez P, Rincon-Limas D, Botas J (2008) Comparative analysis of genetic modifiers in Drosophila points to common and distinct mechanisms of pathogenesis among polyglutamine diseases. Hum Mol Genet 17(3):376–390. doi: ddm315 [pii] 10.1093/hmg/ddm315 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Wan Song
    • 1
  • Marianne R. Smith
    • 1
  • Adeela Syed
    • 1
  • Tamas Lukacsovich
    • 1
  • Brett A. Barbaro
    • 1
  • Judith Purcell
    • 1
  • Doug J. Bornemann
    • 1
  • John Burke
    • 1
  • J. Lawrence Marsh
    • 1
  1. 1.Department of Developmental and Cell BiologyUniversity of California, IrvineIrvineUSA

Personalised recommendations