Background: Huntington’s disease is an autosomal dominant disease where the huntingtin protein is expanded by polyglutamines, increasing its capacity to aggregate. This results in a toxic gain of function of the protein 1. One of the major ways soluble monomer, oligomers and aggregations can be degraded is by macroautophagy (from here after referred to as autophagy). Furthermore, defective autophagy is involved in the pathology of neurodegenerative diseases 2. LC3 is a protein incorporated uniquely into the membrane of an autophagosome, which is the apparatus of autophagy, and is widely accepted as a marker of autophagy. A relatively new assay has been developed by Kimura where the different stages and activity of autophagy can be more closely studied. This system is based on the production of a fusion protein of LC3 tagged with two fluorescent markers, monomeric red fluorescent protein (mRFP) and a green fluorescent protein (EGFP) 3, and has already been used in mammalian cells.
Aims & Objectives: The aim of this project is to use this system in vivo targeting the mRFP-EGFP-rLC3 construct into the Drosophila genome by site specific integration.
Methods/Study Design: We attempted this by placing the LC3 marker into a Drosophila vector ?C31 pUAST attB. After inserting the marker into the vector the construct was then tested using Drosophila embryonic cell cultures and analysis by fluorescence microscopy.
Results/Findings: When the construct was tested in Drosophila embryonic cells, expression of the two colours red and green were seen, showing that the construct was being expressed correctly. These were the phenotypes of the mRFP and EGFP. The intensity of these colours changed when the levels of autophagy were varied using bafilomycin and rapamycin. However, only a small qualitative difference was seen.
Conclusion: For this construct to be used as a useful marker for autophagy, a robust quantitative analysis still needs to be undertaken. Dose response curves would also produce important data in trying to ascertain what level of calibration is needed to effectively use this transgenic marker.
Simon Rodney, S. Imarisio