Development of a Reporter System for In Vivo Monitoring of γ-Secretase Activity in Drosophila

  • Hong, Young Gi (Department of Molecular Biology, Chonbuk National University) ;
  • Roh, Seyun (Department of Molecular Biology, Chonbuk National University) ;
  • Paik, Donggi (Department of Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School) ;
  • Jeong, Sangyun (Department of Molecular Biology, Chonbuk National University)
  • Received : 2016.12.02
  • Accepted : 2016.12.28
  • Published : 2017.01.31


The ${\gamma}$-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the ${\beta}$-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the ${\gamma}$-secretase catalytic component, presenilin, which lead to increased amyloid ${\beta}$-peptide production, are responsible for early-onset familial Alzheimer's disease. ${\beta}$-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of ${\gamma}$-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate ${\gamma}$-secretase activity and/or APPL metabolism.


${\gamma}$-secretase;Alzheimer's disease;APPL;Notch;presenilin


  1. Weiss, J.B., Suyama, K.L., Lee, H.H., and Scott, M.P. (2001). Jelly belly: a Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation. Cell 107, 387-398.
  2. Wharton, K.A., Johansen, K.M., Xu, T., and Artavanis-Tsakonas, S. (1985). Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43, 567-581.
  3. Zhang, Z., Nadeau, P., Song, W., Donoviel, D., Yuan, M., Bernstein, A., and Yankner, B.A. (2000). Presenilins are required for $\gamma$-secretase cleavage of $\beta$-APP and transmembrane cleavage of Notch-1. Nat. Cell Biol. 2, 463-465.
  4. Zhou, S., Zhou, H., Walian, P.J., and Jap, B.K. (2005). CD147 is a regulatory subunit of the $\gamma$-secretase complex in Alzheimer's disease amyloid $\beta$-peptide production. Proc. Natl. Acad. Sci. USA 102, 7499-7504.
  5. Lu, B., and Vogel, H. (2009). Drosophila models of neurodegenerative diseases. Annu. Rev. Pathol. 4, 315-342.
  6. Luo, L., Tully, T., and White, K. (1992). Human amyloid precursor protein ameliorates behavioral deficit of flies deleted for appl gene. Neuron 9, 595-605.
  7. Martin-Morris, L.E., and White, K. (1990). The Drosophila transcript encoded by the $\beta$-amyloid protein precursor-like gene is restricted to the nervous system. Development 110, 185-195.
  8. McCarthy, J.V., Twomey, C., and Wujek, P. (2009). Presenilin-dependent regulated intramembrane proteolysis and $\gamma$-secretase activity. Cell. Mol. Life Sci. 66, 1534-1555.
  9. Nicolas, M., and Hassan, B.A. (2014). Amyloid precursor protein and neural development. Development 141, 2543-2548.
  10. Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563-564.
  11. Selkoe, D.J. (1998). The cell biology of $\beta$-amyloid precursor protein and presenilin in Alzheimer's disease. Trends Cell Biol. 8, 447-453.
  12. Selkoe, D.J., and Wolfe, M.S. (2007). Presenilin: running with scissors in the membrane. Cell 131, 215-221.
  13. Stempfle, D., Kanwar, R., Loewer, A., Fortini, M.E., and Merdes, G. (2010). In vivo reconstitution of $\gamma$-secretase in Drosophila results in substrate specificity. Mol. Cell. Biol. 30, 3165-3175.
  14. Tanzi, R.E. (2012). The genetics of Alzheimer disease. Cold Spring Harb. Perspect. Med. 2, a006296.
  15. Teranishi, Y., Inoue, M., Yamamoto, N.G., Kihara, T., Wiehager, B., Ishikawa, T., Winblad, B., Schedin-Weiss, S., Frykman, S., et al. (2015). Proton myo-inositol cotransporter is a novel $\gamma$-secretase associated protein that regulates $A{\beta}$ production without affecting Notch cleavage. FEBS J. 282, 3438-3451.
  16. Wakabayashi, T., Craessaerts, K., Bammens, L., Bentahir, M., Borgions, F., Herdewijn, P., Staes, A., Timmerman, E., Vandekerckhove, J., Rubinstein, E., et al. (2009). Analysis of the $\gamma$-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat. Cell Biol. 11, 1340-1346.
  17. Wang, X., Wang, Z., Chen, Y., Huang, X., Hu, Y., Zhang, R., Ho, M.S., and Xue, L. (2014). FoxO mediates APP-induced AICD-dependent cell death. Cell Death Dis. 5, e1233.
  18. Goedert, M. (2015). NEURODEGENERATION. Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled $A{\beta}$, tau, and $\alpha$-synuclein. Science 349, 1255555.
  19. Greeve, I., Kretzschmar, D., Tschape, J.A., Beyn, A., Brellinger, C., Schweizer, M., Nitsch, R.M., and Reifegerste, R. (2004). Age-dependent neurodegeneration and Alzheimer-amyloid plaque formation in transgenic Drosophila. J. Neurosci. 24, 3899-3906.
  20. Gunawardena, S., and Goldstein, L.S. (2001). Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila. Neuron 32, 389-401.
  21. Han, K., and Manley, J.L. (1993). Transcriptional repression by the Drosophila even-skipped protein: definition of a minimal repression domain. Genes Dev. 7, 491-503.
  22. Hay, B.A., Wolff, T., and Rubin, G.M. (1994). Expression of baculovirus P35 prevents cell death in Drosophila. Development 120, 2121-2129.
  23. He, G., Luo, W., Li, P., Remmers, C., Netzer, W.J., Hendrick, J., Bettayeb, K., Flajolet, M., Gorelick, F., Wennogle, L.P., et al. (2010). Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease. Nature 467, 95-98.
  24. Herreman, A., Serneels, L., Annaert, W., Collen, D., Schoonjans, L., and De Strooper, B. (2000). Total inactivation of $\gamma$-secretase activity in presenilin-deficient embryonic stem cells. Nat. Cell Biol. 2, 461-462.
  25. Jeong, S., Juhaszova, K., and Kolodkin, A.L. (2012). The control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in Drosophila. Neuron 76, 721-734.
  26. Johnson, R.L., Grenier, J.K., and Scott, M.P. (1995). patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121, 4161-4170.
  27. Ju, B.G., Jeong, S., Bae, E., Hyun, S., Carroll, S.B., Yim, J., and Kim, J. (2000). Fringe forms a complex with Notch. Nature 405, 191-195.
  28. Kim, J., Irvine, K.D., and Carroll, S.B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82, 795-802.
  29. Louvi, A., and Artavanis-Tsakonas, S. (2006). Notch signalling in vertebrate neural development. Nat. Rev. Neurosci. 7, 93-102.
  30. Brand, A.H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
  31. Brown, M.S., Ye, J., Rawson, R.B., and Goldstein, J.L. (2000). Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100, 391-398.
  32. Cao, X., and Sudhof, T.C. (2001). A transcriptionally active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 293, 115-120.
  33. Carmine-Simmen, K., Proctor, T., Tschape, J., Poeck, B., Triphan, T., Strauss, R., and Kretzschmar, D. (2009). Neurotoxic effects induced by the Drosophila amyloid-$\beta$ peptide suggest a conserved toxic function. Neurobiol Dis. 33, 274-281.
  34. Chen, F., Hasegawa, H., Schmitt-Ulms, G., Kawarai, T., Bohm, C., Katayama, T., Gu, Y., Sanjo, N., Glista, M., Rogaeva, E., et al. (2006). TMP21 is a presenilin complex component that modulates $\gamma$-secretase but not $\varepsilon$-secretase activity. Nature 440, 1208-1212.
  35. de Celis, J.F., and Garcia-Bellido, A. (1994a). Roles of the Notch gene in Drosophila wing morphogenesis. Mech. Dev. 46, 109-122.
  36. de Celis, J.F., and Garcia-Bellido, A. (1994b). Modifications of the notch function by Abruptex mutations in Drosophila melanogaster. Genetics 136, 183-194.
  37. de Celis, J.F. (1998). Positioning and differentiation of veins in the Drosophila wing. Int. J. Dev. Biol. 42, 335-343.
  38. De Strooper, B. (2003). Aph-1, Pen-2, and Nicastrin with Presenilin generate an active $\gamma$-Secretase complex. Neuron 38, 9-12.
  39. De Strooper, B., Vassar, R., and Golde, T. (2010). The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat. Rev. Neurol. 6, 99-107.
  40. Esler, W.P., and Wolfe, M.S. (2001). A portrait of Alzheimer secretases--new features and familiar faces. Science 293, 1449-1454.
  41. Fernandez-Funez, P., de Mena, L., and Rincon-Limas, D.E. (2015). Modeling the complex pathology of Alzheimer's disease in Drosophila. Exp. Neurol. 274, 58-71.
  42. Fortini, M.E. (2009). Notch signaling: the core pathway and its posttranslational regulation. Dev. Cell 16, 633-647.
  43. Bai, X.C., Yan, C., Yang, G., Lu, P., Ma, D., Sun, L., Zhou, R., Scheres, S.H., and Shi, Y. (2015). An atomic structure of human $\gamma$-secretase. Nature 525, 212-217.
  44. Bailey, A.M., and Posakony, J.W. (1995). Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9, 2609-2622.
  45. Bertet, C., Li, X., Erclik, T., Cavey, M., Wells, B., and Desplan, C. (2014). Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper. Cell 158, 1173-1186.
  46. Bier, E. (2005). Drosophila, the golden bug, emerges as a tool for human genetics. Nat. Rev. Genet. 6, 9-23.
  47. Brachmann, C.B., and Cagan, R.L. (2003). Patterning the fly eye: the role of apoptosis. Trends Genet. 19, 91-96.