Award details

Deciphering the cellular mechanism of seeded prion aggregation in neuronal cells

Reference	BB/V001310/1
Principal Investigator / Supervisor	Dr Peter Kloehn
Co-Investigators / Co-Supervisors	Professor Sharon Tooze
Institution	University College London
Department	MRC PRION Unit at UCL
Funding type	Research
Value (£)	413,375
Status	Current
Type	Research Grant
Start date	01/06/2021
End date	31/05/2024
Duration	36 months

Abstract

Prions, the infectious pathogens of prion diseases are thought to arise by template-assisted conversion of the cellular prion protein, but the underpinning cellular mechanism of this pathogenic process remains unknown. While a growing body of data suggests that self-templating assemblies of protein aggregates are the basis of many, if not all neurodegenerative diseases, such "prion-like" mechanisms are ill-defined, which underscores the importance to better define common pathogenic mechanisms. We provide first evidence of how prions replicate in neuronal cells and propose, in collaboration with Sharon Tooze, an expert in protein secretion from the Francis Crick Institute, a comprehensive work plan to gain further evidence in support of our preliminary data. Our results suggest that aggregates of disease-associated PrP (PrPd) segregate into the protein secretory pathway and reach the plasma membrane, where they convert PrPc to full-length (FL-) PrPd. Formation of long rod-like FL-PrPd fibrils, detected by anti-PrP antibodies against FL-PrP, can be blocked by lowering cellular cholesterol and stimulated by dissipation of the vesicular pH gradient, a treatment that concomitantly led to an increase in prion release. Owing to evidence that neuropeptides and prohormones are sorted into the regulated secretory pathway by virtue of protein aggregation, we will address the important questions whether (i) prions are sorted into the secretory pathway by default and (ii) whether the cellular environment of vesicle biogenesis favours misfolding of aggregation-prone proteins. This project contributes to a better characterisation of seeded aggregation and may provide guiding principles to characterise prion-like mechanisms.

Summary

Prion diseases, transmissible diseases of the brain, affect animals and humans alike and include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. Although prions, the infectious pathogens of prion diseases have been thoroughly characterised over recent decades, we still have not found the cellular mechanism by which they propagate in neuronal cells. While prions are thought to replicate by conversion of the cellular prion protein (PrPc), a protein expressed on the surface of neurons, to disease-associated conformers of itself, the cellular site and the molecular mechanism of replication remain enigmatic. Our preliminary data present the first molecular clues on how new aggregates of disease-associated PrP (PrPd) arise at the plasma membrane of neuronal cells. A breakthrough in the detection of PrPd variants enabled us to distinguish intracellular from extracellular PrPd aggregates. This helped us to infer that the assembly and growth of fibrillar PrPd aggregates at the plasma membrane is driven by aggregation seeds from inside the cell. Importantly, inhibition and stimulation of the transport of these seeds to the plasma membrane prevented and accelerated fibril growth, respectively. Together with Prof Sharon Tooze, an expert in protein secretion from the Francis Crick Institute, we plan to investigate the molecular mechanism on how the intracellular pool of PrPd reaches the plasma membrane. This is of great importance, since our preliminary data suggest inhibiting the transport of PrPd on its way to the plasma membrane may block the generation of prions.

Impact Summary

The work outlined in this proposal will have a range of important impacts on stake holders and beneficiaries of BBSRC-funded research as elaborated below. Dementia research: Historically, different types of dementia, like Alzheimer's, Parkinson's and prion diseases were considered phenotypically distinct, but dementia research has been converging in recent years, most arguably due to the finding that "prion-like" phenomena, including seeded aggregation, self-templating protein assemblies and the dissemination of pathogenic protein aggregates, appear to be a common denominator of disease pathogenesis. Our ongoing work to characterise the molecular mechanism of prion replication will not only help to further elucidate the cellular mechanism of prion propagation, but may also provide guiding principles for the molecular underpinning of "prion-like" mechanisms identified in other types of dementia. Our experimental evidence that seeded aggregation of rogue prion proteins at the plasma membrane can be stimulated by dissipating the vesicular pH gradient may prompt other scientists in dementia research to revisit the role of protein secretion in the dissemination of amyloidogenic proteins. Furthermore, our planned studies to investigate whether the cellular environment in secretory vesicles may favour aggregation of rogue prion conformers may inform common pathogenic mechanisms in Alzheimer's and prion diseases. In summary, provision of insights into cellular mechanisms of seeded prion aggregation may further elucidate the phenomenon of "prion-like" mechanisms. Basic research: The collaborative and cross-disciplinary nature of this application may benefit basic research on unsolved mechanisms of protein sorting pathways. It has long been acknowledged that prohormones and neuropeptides form amyloid-like aggregation states during their transport through secretory pathways, but the molecular mechanisms remains unknown. Based on a wealth of novel data on amyloidogenic proteins in dementia, a growing number of research groups in the exocytosis field are scrutinising the significance of amyloid-like assemblies and molecular crowding in protein secretory pathways. This is of central importance to better understand the biology of neurotransmitters. Our aim to scrutinise how rogue prion proteins enter secretory pathways may not only explain the rapid dissemination of prions in the brain, but may also help to identify critical control mechanisms for entry into secretory pathways. Our collaboration with Sharon Tooze, an eminent expert in the exocytosis field will help to excel knowledge transfer and exchange with basic research. Prion disease research: Our aim to identify the cellular mechanism of seeded prion aggregation will greatly benefit the prion field. While mechanistic insight into the sequential proteolytic cleavage of the amyloid precursor protein has been of central importance to understand how amyloidogenic proteins are formed, the molecular underpinning of protein misfolding in prion diseases is unknown. Our finding that antibodies against full-length PrP recognise rod-like assemblies of disease-associated PrP (PrPd) at the plasma membrane, but not intracellularly, points to a critical role of proteolytic cleavage, hence identification of the cellular protease will be pivotal to decipher the cellular mechanism of prion propagation.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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