Use of PrP transgenic Drosophila to measure mammalian prion infectivity
Lead Research Organisation:
University of Cambridge
Department Name: Veterinary Medicine
Abstract
Prion diseases are invariably fatal neurodegenerative diseases of the brain that affect humans and other vertebrate species. These diseases include CJD of humans, BSE of cattle and scrapie of sheep. Prion diseases occur when a protein termed PrP undergoes a change in shape, and aggregates in the brain where it damages neurons. While other neurodegenerative diseases also occur through their own specific protein misfolding event, prion diseases are unique because they are transmissible, within individuals of the same species and also between different species. During prion disease, the transmissible infectious agent associated with these conditions, which is thought to be the misfolded form of PrP, accumulates in the brain of affected individuals. This infectious agent is termed a prion. Prions are a significant risk to public health through their potential to spread from animals to humans, seen by the appearance of BSE in UK cattle and subsequent emergence of variant CJD in humans. The most common route of transmission of prion diseases is by oral inoculation with prion-infected material i.e. by inadvertently eating prions. Individuals that become infected in such a manner accumulate prion infectivity in lymphoid tissue and blood.
It is important to determine how much prion infectivity is present in the brain and different tissues, and blood from prion-infected individuals. This is necessary to understand the biology of infectious prions, to ensure the safety of animal products destined for human consumption, and to help to establish a blood test for these diseases. The prion is an unconventional infectious agent and the only reliable method to detect its presence is to inoculate experimental animals with prion-infected samples and see if the recipients develop prion disease. Inevitably, this has led to a large number of experimental animals, in particular mice and sheep, being used to assess mammalian prion infectivity, and as a consequence, dying from terminal prion disease. This proposal aims to reduce and replace, where possible, the use of mice and sheep to measure prion infectivity with an invertebrate host, Drosophila melanogaster. This in turn, will help to refine prion infectivity experiments by using less sentient animals that are not known to have a pain system or a comparable level of awareness as mammalian hosts.
We will model scrapie of sheep in Drosophila. We have already generated Drosophila transgenic for sheep PrP and have shown these flies are susceptible to the toxic effects of sheep prions. Adult flies, fed prion infectivity as larvae, show a decreased ability to move and show the presence of the misfolded form of PrP in their brains. In the experiments of this proposal, we intend to validate the sensitivity of our new prion fly model to determine the extent to which our new animal model can replace mice and sheep in measuring prion infectivity. Our use of PrP transgenic Drosophila will produce humane, high-quality, reproducible prion-based science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using PrP transgenic Drosophila. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates can be tested at the same time using a financially viable model. Our previous experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using PrP transgenic Drosophila so we can sensibly inform other scientists and encourage less dependency on mouse and sheep prion bioassays. We will use previously generated prion-infected samples to test in our Drosophila model so that no other experimental animals need to be injected with prion material in order to obtain samples for use within this study. We have preliminary test results from many of these samples so they can be directly compared with the results we generate with our PrP transgenic Drosophila.
It is important to determine how much prion infectivity is present in the brain and different tissues, and blood from prion-infected individuals. This is necessary to understand the biology of infectious prions, to ensure the safety of animal products destined for human consumption, and to help to establish a blood test for these diseases. The prion is an unconventional infectious agent and the only reliable method to detect its presence is to inoculate experimental animals with prion-infected samples and see if the recipients develop prion disease. Inevitably, this has led to a large number of experimental animals, in particular mice and sheep, being used to assess mammalian prion infectivity, and as a consequence, dying from terminal prion disease. This proposal aims to reduce and replace, where possible, the use of mice and sheep to measure prion infectivity with an invertebrate host, Drosophila melanogaster. This in turn, will help to refine prion infectivity experiments by using less sentient animals that are not known to have a pain system or a comparable level of awareness as mammalian hosts.
We will model scrapie of sheep in Drosophila. We have already generated Drosophila transgenic for sheep PrP and have shown these flies are susceptible to the toxic effects of sheep prions. Adult flies, fed prion infectivity as larvae, show a decreased ability to move and show the presence of the misfolded form of PrP in their brains. In the experiments of this proposal, we intend to validate the sensitivity of our new prion fly model to determine the extent to which our new animal model can replace mice and sheep in measuring prion infectivity. Our use of PrP transgenic Drosophila will produce humane, high-quality, reproducible prion-based science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using PrP transgenic Drosophila. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates can be tested at the same time using a financially viable model. Our previous experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using PrP transgenic Drosophila so we can sensibly inform other scientists and encourage less dependency on mouse and sheep prion bioassays. We will use previously generated prion-infected samples to test in our Drosophila model so that no other experimental animals need to be injected with prion material in order to obtain samples for use within this study. We have preliminary test results from many of these samples so they can be directly compared with the results we generate with our PrP transgenic Drosophila.
Technical Summary
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.
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.
Planned Impact
Prion infection and the development of prion disease has a profound impact on the relatively large number of experimental mice (54,643 in 2010, with 5,877 of those experiencing direct injection into the brain), and sheep (21 during 2010) used to assess prion strains and prion infectivity in the UK (Home Office Figures as of 13.01.2012). Therefore it is appropriate, and timely, to consider developing new animal models to assess mammalian prion infectivity with the focus on the 3Rs policy.
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 specific objectives 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
These studies will contribute to a reduction and replacement, where possible, of experimental rodents or sheep currently used to measure scrapie prion infectivity by the use of Drosophila, a less sentient host. Our studies will also contribute to the development of an invertebrate model of mammalian prion disease, an important goal in prion biology, whilst adhering to the 3Rs strategy with respect to the use of animals in research. In this context we aim to: (a) reduce the numbers of mice and sheep kept in laboratory conditions generally; (b) reduce the number of mice and sheep that experience poor welfare as a consequence of experimental prion inoculation. Poor welfare arises as a result of (i) CNS-diseased animals lacking control of their interactions with each other and/or with their environment; (ii) the experience of fear and frustration as a consequence of lack of control; and (iii) the experience of some degree of pain; (c) replace mice and sheep, where possible, with the use of animals not known to have a pain system or a comparable level of awareness.
Drosophila melanogaster is an appropriate species to consider for this role and has already been used to model a number of human neurodegenerative diseases. A major strength of Drosophila model systems is the ability to rapidly generate transgenic flies that express heterologous proteins in a tissue-specific manner.
This proposal aims to reduce and replace mice with Drosophila for testing prion-infected samples, which in turn will help to refine experiments by using less sentient animals that exhibit less pain sensation. Based on our initial studies, the use of Drosophila will enable us to produce humane, high-quality, reproducible science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using Drosophila in place of rodents or large animal models. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates tested at the same time using a financially viable model. Our past experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using Drosophila, so we can sensibly inform other scientists and encourage less dependency on rodents and sheep for this purpose. Our proposed fly model will give value for money in a timely fashion, realistically address all the 3Rs and impact on all the criteria listed by the NC3Rs.
This project will have a very high impact on our research locally as we will move away, where possible, from experimental rodent and sheep models for initial prion screens and concentrate on the fly model. We will be able to assess prion infectivity much faster than in the mouse model and use large numbers of flies to ensure statistically significant data. Once the fly model is validated within our lab, we will encourage other researchers to take up the model following publication of our results to substantiate and further our findings.
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 specific objectives 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
These studies will contribute to a reduction and replacement, where possible, of experimental rodents or sheep currently used to measure scrapie prion infectivity by the use of Drosophila, a less sentient host. Our studies will also contribute to the development of an invertebrate model of mammalian prion disease, an important goal in prion biology, whilst adhering to the 3Rs strategy with respect to the use of animals in research. In this context we aim to: (a) reduce the numbers of mice and sheep kept in laboratory conditions generally; (b) reduce the number of mice and sheep that experience poor welfare as a consequence of experimental prion inoculation. Poor welfare arises as a result of (i) CNS-diseased animals lacking control of their interactions with each other and/or with their environment; (ii) the experience of fear and frustration as a consequence of lack of control; and (iii) the experience of some degree of pain; (c) replace mice and sheep, where possible, with the use of animals not known to have a pain system or a comparable level of awareness.
Drosophila melanogaster is an appropriate species to consider for this role and has already been used to model a number of human neurodegenerative diseases. A major strength of Drosophila model systems is the ability to rapidly generate transgenic flies that express heterologous proteins in a tissue-specific manner.
This proposal aims to reduce and replace mice with Drosophila for testing prion-infected samples, which in turn will help to refine experiments by using less sentient animals that exhibit less pain sensation. Based on our initial studies, the use of Drosophila will enable us to produce humane, high-quality, reproducible science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using Drosophila in place of rodents or large animal models. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates tested at the same time using a financially viable model. Our past experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using Drosophila, so we can sensibly inform other scientists and encourage less dependency on rodents and sheep for this purpose. Our proposed fly model will give value for money in a timely fashion, realistically address all the 3Rs and impact on all the criteria listed by the NC3Rs.
This project will have a very high impact on our research locally as we will move away, where possible, from experimental rodent and sheep models for initial prion screens and concentrate on the fly model. We will be able to assess prion infectivity much faster than in the mouse model and use large numbers of flies to ensure statistically significant data. Once the fly model is validated within our lab, we will encourage other researchers to take up the model following publication of our results to substantiate and further our findings.