The aim of this proposal is to test the hypothesis that PrP transgenic Drosophila are a suitable invertebrate host to measure mammalian prion infectivity. The prion disease we will study is scrapie of sheep. We have already generated ovine PrP transgenic Drosophila and have shown exogenous exposure to ovine prions induces a toxic phenotype in these flies.
The specific objectives of this proposal are 1. Determine the sensitivity of ovine PrP transgenic Drosophila to ovine prion infectivity 2. Use ovine PrP transgenic Drosophila to measure prion infectivity in brain, lymphoid tissue and blood from scrapie-infected sheep 3. Strain type ovine prions in ovine PrP transgenic Drosophila.
To achieve these aims, ovine PrP transgenic Drosophila, at the larval stage, will be orally fed ovine prions by the addition of scrapie-infected inocula to Drosophila feed. The prion inocula will include: brain homogenate from classical (ARQ/ARQ and VRQ/VRQ) or atypical (AHQ/AHQ) scrapie-infected sheep brain material; lymphoid tissue homogenate and time course blood plasma samples from classical scrapie-infected sheep; homogenates of serially passaged, defined prion strain-infected ovine PrP transgenic mouse brains. The prion-induced phenotype of ovine PrP transgenic Drosophila will be assessed in post eclosion flies by measurement of locomotor ability using a negative geotaxis climbing assay, together with analysis of disease-associated PrP deposition by IHC and molecular profile by western blot; confocal analysis of PrP in fly brains (+/- Proteinase K) and quantitation of PrP by conformational dependent immunoassay on fly head homogenates.
All of the ovine PrP transgenic flies have already been generated for use in this proposal. All of the prion inocula have already been acquired from, and characterised within, previous prion projects within our laboratory. All of the experimental protocols to be used in this proposal are fully established in our laboratory.
Thackray AM et al. (2020). Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease. Biochemical Journey 477(4): 833–852. doi: 10.1042/BCJ20190872
Thackray AM et al. (2018). The use of PrP transgenic Drosophila to replace and reduce vertebrate hosts in the bioassay of mammalian prion infectivity [version 1; peer review: 2 approved]. F1000Research 7:595. doi: 10.12688/f1000research.14753.1
Thackray AM et al. (2018). Mammalian prion propagation in PrP transgenic Drosophila. Brain 141(9):2700-10. doi: 10.1093/brain/awy183
Thackray AM et al. (2017). Genetic human prion disease modelled in PrP transgenic. The Biochemical Journal 474(19):3523-3267. doi: 10.1042/BCJ20170462
Thackray AM et al. (2016). Bioassay of prion-infected blood plasma in PrP transgenic Drosophila. The Biochemical Journal 473(23):4399-4412. doi: 10.1042/BCJ20160417
Bujdoso R et al. (2015). Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response. World Journal of Virology 4(3):188-97. doi: 10.5501/wjv.v4.i3.188
Thackray AM et al. (2014). Cytosolic PrP can participate in prion-mediated toxicity. Journal of Virology 88(14):8129-38 doi: 10.1128/JVI.00732-14
Thackray AM et al. (2014). Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila. Biochem J. 463(1):31-40. doi: 10.1042/BJ20140129
Thackray AM et al. (2014). The emergence of a Drosophila model to measure ovine prion infectivity. CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources 8(58). doi: 10.1079/PAVSNNR20130058
Thackray AM et al. (2012). Prion-induced toxicity in PrP transgenic Drosophila. Experimental and Molecular Pathology 92(2):194-201. doi: 10.1016/j.yexmp.2012.01.005
Thackray AM et al. (2012). Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival. Biochemical Journal 444(3):487-95. doi: 10.1042/BJ20112141
- 2019 Research Review: Fly infectivity assay for prion disease
- News: New bioassay uses fruit fly to test for mammalian prion disease
- Further Funding: NC3Rs Skills & Knowledge Transfer Grant, The use of PrP transgenic Drosophila to replace and reduce mice in the bioassay of mammalian prions, August 2017, £75,000