An advanced model for neurodegeneration studies in the fruit fly Drosophila melanogaster

The senile dementias are the 4th most common cause of death, with 700,000 people in the UK suffering from neurodegenerative disorders, with an associated annual cost to the economy of #17 billion. This will rise precipitously as the age profile of our population increases over the next 35 years. Research into neurodegenerative disorders predominantly uses mice and rats, and these mammals are sacrificed in their thousands in laboratories throughout the world. The fruitfly is an alternative model that can be genetically engineered to develop neurodegeneration, and indeed, it has contributed significantly to understanding how these diseases develop, and how they might be alleviated. However, the timing of the events that lead to neurodegeneration within nerve cells has not received much attention. We propose to develop a robust system in the fly in which two genes can be independently regulated. We shall express the mutant human huntingtin gene (that gives flies a form of Huntington?s Disease, HD) in specific neurons in the fly, and by turning the gene on or off at different times, we shall discover when the neurons are most sensitive to the HD mutation. We shall then use our new system to turn on and off at variable times another gene, that we know can alleviate HD in the fly, to see when it is most effective. There are examples where suppressors of neurodegeneration in the fly can have a beneficial effect at one time (in a young fly), but a detrimental effect at another. By determining when these suppressors of HD act, in relation to the critical period when HD irreversibly ?takes hold? of a neuron, we can design a rational therapy that may act as a prophylactic for HD. Our results will benefit neurodegeneration research in general, but also stimulate a refocusing and refining of experimental work in the mammal. This in turn should lead to a reduction in the number of mammalian experiments that are carried out. The advantage of our method is that it can be applied to almost every character one would wish to study in a fly or a mouse, and is not simply limited to diseases.

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.