Identification of genetic modifiers of mammalian prion disease modelled in Drosophila

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PhD project strategic theme: Biosciences for an integrated understanding of healthThe aim of this project is to identify genetic modifiers of acquired and genetic mammalian prion disease modelled in Drosophila and mice. This will be achieved by RNASeq analysis of actively translated mRNA isolated from prion-infected neurons (both total neurons and specific subsets) in the brains of prion-diseased transgenic Drosophila and mice in order to identify potential genetic modifiers of prion disease. Candidate genetic modifiers will be validated through modulation of their expression (decreased or increased by RNAi or transgene over-expression, respectively) during ongoing prion disease in PrP transgenic Drosophila and analysing any changes in prion-induced pathology. The specific objectives of the project are:1. Generation of PrP / ribo-tag transgenic DrosophilaFly lines will be generated that are transgenic for mammalian PrP and co-express a ribosomal tag (ribo-tag) that allows isolation of mRNA undergoing translation. Ribo-tag Drosophila will be made transgenic for wild type ovine or murine PrP, or murine PrP with a mutation that correlates with genetic prion disease in humans (genetic CJD). Both the ribo-tag and the PrP will either be expressed pan neuronally or in specific neuronal subsets.2. Perform RNASeq analysis on prion-diseased PrP / ribo-tag transgenic DrosophilaAcquired prion disease will be established in wild type ovine or murine PrP transgenic Drosophila exposed to ovine or murine prion inoculum, respectively, that express PrP pan neuronally or in specific neuronal subsets. This will allow comparisons to be made between the neuropathology caused by different species' PrP, as well as comparisons between the neurodegeneration undergone by different neuronal subsets. Genetic prion disease will be established in Drosophila transgenic for murine-CJD PrP to allow differences to be identified between the neuropathology of acquired and genetic prion diseases. mRNA associated with ribosomes which possess the ribo-tag will be extracted from neurones of prion-diseased Drosophila and subjected to RNASeq. Subsequent bio-informatic analysis will identify biochemical pathways, along with important genetic modifiers associated with those pathways, that are involved in acquired and genetic prion disease in the different Drosophila models. 3. Perform RNASeq analysis on prion-diseased PrP / ribo-tag transgenic mouse brainsRibosomal-associated mRNA will be extracted from the brains of prion-diseased mice and subjected to RNASeq, and subsequent bio-informatic analysis. This will identify biochemical pathways and genetic modifiers involved in acquired prion disease in the mouse. 4. Modulated expression of genetic modifiers during prion disease in DrosophilaData from the Drosophila- and mouse-based RNASeq analyses will be compared to identify genetic modifiers of prion disease common between these two hosts, in order to validate the relevance of the Drosophila models. The role of identified candidate genetic modifiers of prion disease will be validated by modulation of their expression, achieved by RNAi or transgene over-expression, during an ongoing prion infection in PrP transgenic Drosophila. Validation of the genetic modifiers will be determined by assessing changes in the accumulation of prion seeding activity and the prion-induced neurotoxic phenotype (in terms of survival and locomotor ability) of prion-infected PrP transgenic Drosophila.