Identification of genetic modifiers of mammalian prion disease modelled in Drosophila
Lead Research Organisation:
University of Cambridge
Department Name: Veterinary Medicine
Abstract
PhD project strategic theme: Biosciences for an integrated understanding of health
The 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 Drosophila
Fly 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 Drosophila
Acquired 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 brains
Ribosomal-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 Drosophila
Data 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.
The 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 Drosophila
Fly 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 Drosophila
Acquired 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 brains
Ribosomal-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 Drosophila
Data 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.
Organisations
| Description | The work funded by this award has identified key gene expression changes in cellular and biochemical pathways in transgenic Drosophila models of different prion diseases, which are a type of transmissible neurodegenerative disease. To make Drosophila susceptible to prion disease, Drosophila were previously generated that were transgenic for the prion protein (PrP). These PrP transgenic Drosophila were exposed to different prion inocula to model different types of acquired prion disease. A type of genetic prion disease was also previously modelled in Drosophila that were transgenic for PrP with a mutation associated with this disease. In this study, data generated from RNA sequencing (RNAseq) was analysed to identify gene expression changes during preclinical and clinical disease stages in the different prion models, including cellular and biochemical pathways that were perturbed. The RNAseq analysis compared results between Drosophila models of genetic and acquired models of prion disease to identify similarities and differences in the neurotoxic mechanism between prion diseases initiated by different mechanisms. The RNAseq analysis also compared the results between two different types of acquired prion disease to determine the similarity in neurotoxicity caused by prion diseases initiated by the same mechanism but originating from different species. |
| Exploitation Route | The current outcomes of the award can be used to enhance the current understanding of prion-induced neurotoxicity. In particular, the results of the RNAseq analyses in prion inoculated transgenic Drosophila can be compared to future analyses in this type of prion model, as well as other types of animal model for prion disease. This will help to identify fundamental processes that occur during prion disease, which can then be investigated further by functional studies, including testing potential anti-prion compounds. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |