In vivo molecular research in late-onset neurodegenerative diseases necessarily uses animal models, but symptoms in transgenic mice often take weeks to develop, and numbers of individuals required to obtain phenotypic statistical significance can be high. Invertebrate models such as the fruitfly play a significant role in replacing, refining and reducing the emphasis on mice, and have provided considerable molecular insight into these diseases. However, the current fly disease models are constitutive and the relevant transgenes are usually expressed pan-neurally or in specific organs such as the eye. Our aim is to use the unsurpassed molecular genetic ‘toolbox' of Drosophila, to refine the study of neurodegeneration in the fly. Specifically, we shall, 1. Develop a system whereby we can systematically control the expression levels of a disease bearing transgene such as mutant human huntingtin (mHtt) 2. Refine the system so we can examine the onset of neurodegeneration in a specific cell group rather than pan-neuronally or in particular organs. 3. Further refine the system so that the temporal dynamics of the onset of neurodegenerative disease can be studied with the use of inducible transgenes 4. Validate our approach and show proof-of-principle by extending this controlled methodology to studying the temporal dynamics of suppressors of the Huntington's phenotype that we have previously identified in our laboratories. The experimental design involves modifying the Ga4/UAS inducible Gene Switch system to make it responsive to LexA rather than Gal4, while incorporating promoters to drive expression of mutant Huntingtin (mHtt) in the Pigment Dispersing Factor (PDF) circadian neurons in the brain. We shall induce mHtt at different ages during adult life and quantify neurodegeneration in PDF clock neurons by assessing circadian behaviour and by microscopy, thereby identifying the critical period when the clock neurons pass the point of ‘no return'. We shall then use suppressors of mHtt induced degeneration under Pdf-Gal4_Gene Switch spatio/temporal control, and investigate whether these suppressors can act as prophylactics to prevent subsequent neuronal damage. The impact of this new model shall be in its contribution to the refocusing of efforts on the timing of the critical cellular (and possibly reversible) events that lead to neurodegeneration. This refinement will consequently lead to a reduction in the numbers used, and, hopefully, to a paradigm change in the types of experiments that are done in mice. Our novel method can be generalised to almost any phenotype, and provides significant added value in the 3Rs.
Also received an NC3Rs Public Engagement Award in 2014:
An advanced model for neurodegeneration studies in the fruit fly Drosophila melanogaster. Dr Özge Özkaya, University of Leicester, £550.
Dissel S, Hansen CN, Özkaya Ö, Hemsley M, Kyriacou CP, Rosato E (2014). The logic of circadian organization in Drosophila. Curr Biol. 24(19): 2257-66 doi: 10.1016/j.cub.2014.08.023
Ozkaya O, Rosato E (2012). The circadian clock of the fly: a neurogenetics journey through time. Adv Genet. 77: 79-123 doi: 10.1016/B978-0-12-387687-4.00004-0
Green EW, Giorgini F (2012) Choosing and using Drosophila models to characterize modifiers of Huntington's disease. Biochemical Society Transactions 40(4): 739-45 doi:10.1042/BST20120072
Green EW, Campesan S, Breda C, Sathyasaikumar KV, Muchowski PJ, Schwarcz R, Kyriacou CP, Giorgini F (2012) Drosophila eye color mutants as therapeutic tools for Huntington disease. Fly 6(2): 117-20 doi:10.4161/fly.19999
Principal investigatorDr Ezio Rosato
InstitutionUniversity of Leicester
Co-InvestigatorDr Flaviano Giorgini
Professor Charalambos Kyriacou