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

National Institute Of Allergy And Infectious Diseases

Investigators

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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, a Recipher analyser, 10x and Fluent single cell platforms, 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. As the organoid model closely mirrors the propagation of human prion in disease, we have also been able to test putative anti-prion therapeutics in this model. Our research published this year has identified ferroptosis as a putative pathway by which cells may die during disease. In a large collaborative study with Joel Schick at Helmholtz Zentrum Munich, our organoid model showed significant changes in proteins involved in the ferroptosis pathway that mirrored equivalent changes occurring in human brain tissue at terminal disease. Further experiments revealed that the diseased organoids were more vulnerable to chemical induction of ferroptosis than healthy organoids and that the prion protein may itself have a role in this death pathway as PrP knock-out organoids were comparatively resistant. An ongoing complication of working with prions is the difficulty of decontamination. Prions are resistant to decontamination by many methods that are effective for viruses or bacteria. Historically many of the protocols used for human prion decontamination have been based on studies using guinea pig passaged human CJD prions. Although, these passaged prions are assumed to be a close representation of the original human prion, they may display differing resistance to decontamination. In the absence of information on decontamination of prions from human brain, we undertook a study to characterise the minimum hypochlorite concentration and time that are required for effective removal of seeding activity, which is a highly sensitive read out of propagation capable prions. In this study we also characterised human prions from our organoid infections as this new model may be used by other groups in the future. In addition to our human prion work we have also investigated prion pathogenesis in vivo using mouse models of infection. Clinical manifestations of human prion disease include cognitive decline and mood-related behavioral changes. Cognition and mood are linked to the neurophysiology of the limbic system. Little is known about how prion disease affects the synaptic activity in brain parts associated with this system. In this research, we studied how prion infection in mice disrupts the synaptic function in three limbic regions, the hippocampus, hypothalamus, and amygdala, at a pre-clinical stage (mid-incubation period) and early clinical onset. Our findings showed calcium flux dysregulation associated with lesser spontaneous synchronous neuronal firing and slowing neural oscillation at the pre-clinical stage in the hippocampal CA1, ventral medial hypothalamus, and basolateral amygdala (BLA). At clinical onset, synaptic transmission and synaptic plasticity became significantly disrupted. This correlated with a substantial depletion of the soluble prion protein, loss of total synapses, abnormal neurotransmitter levels and synaptic release, decline in synaptic vesicle recycling, and cytoskeletal damage. Further, the amygdala exhibited distinct disease-related changes in synaptic morphology and physiology compared with the other regions, but generally to a lesser degree, demonstrating how different rates of damage in the limbic system influence the evolution of clinical disease.