Prion protein function in redox homeostasis and associated failure in prion disease

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Scope: Prion diseases are infectious diseases affecting humans and animals. They are caused by prions; misfolded conformers of a cellular protein called the prion protein (PrP) that can recruit and convert more PrP molecules into prions in a self-propagating cycle. Disease symptoms vary but always end with death, and no therapy exists to slow or halt their progression. Our research aims to investigate the processes by which neuronal cells become dysfunctional and die during prion disease and to explore avenues that could be exploited to stop, slow, prevent or reverse disease damage. Within this scope, our interests include determining the causes of cytosolic oxidative damage, which influences cellular energy pathways and cytoskeletal architecture, and the importance of such pathological events. Furthermore, our experiments seek to determine whether such changes are occurring due to loss of PrP function or corruption of function of abnormal PrP or both. We intend for our research to generate a significant understanding of both PrP function at the cellular level and the homeostatic systems that fail during prion disease pathogenesis. Research materials, equipment and methods: Our research predominantly uses cell-based techniques to investigate changes in single cells or in cell networks. We have generated a number of cell models, including mouse and human stem cell models. To investigate specific functional and disease-related pathways we use a combination of protein, RNA, live cell function and microscopy analyses. The equipment we hold for these analyses include a chip/multiwell/in vivo MEA system, a Seahorse analyser, two plate readers of differing functional capacity, an automated fluorescence microscope and chemiluminescence/fluorescence imaging system. Research accomplishments: Using human induced pluripotent stem cells to make cerebral organoid cultures we developed the first human three-dimensional brain tissue model of prion disease. This was achieved by infecting the cerebral organoids with human prions. We further demonstrated that cerebral organoid prion infections can reproduce certain features of prion disease that are found in human brains post-mortem. We have since shown that the cerebral organoid model of prion infection faithfully propagates the infecting prion, producing the same disease when passaged back into mice as is seen when mice are infected with the original brain inoculum. In addition to investigation of infection, we have used the organoid model to examine cellular changes associated with mutations within the prion gene. There are two mutations for which we have organoid models; one is associated with genetic Creutzfeldt Jakob Disease (CJD) and the other with fatal familial insomnia. Neither mutation causes a spontaneous disease with the organoids, and we have found that even when oxidative stress or stress induced by viral infection is applied to the organoids the mutated protein is stable and does not initiate the disease. However, both mutations have a dysfunctional phenotype. The mutation causing fatal familial insomnia is associated with cytosolic oxidative stress and changed metabolism. As a result of their shifted metabolism, these cells demonstrate increased acetylcholine and decreased GABA, which supports neuronal excitotoxicity and the enhanced wakefulness/changed sleep patterns that are associated with this disease. Looking at the CJD associated mutation we also noted that cellular changes are not confined to neuronal tissue with cardiomyocytes demonstrating impaired electrophysiological function with increased oxidative stress and changed mitochondrial function. Using mouse models, our research has also identified a new protective role for the prion protein in preserving neurotransmission following oxidative insults.