STRUCTURAL BASIS OF PRION INFECTION: Tracking fluorescent prions to define their pathogenic pathways by cutting-edge light and electron microscopy

Prions, the infectious agents causing mad cow disease (BSE) and CJD in humans are unique in medical research. Unlike all other infectious agents (bacteria and viruses) the infectious particle does not contain genetic information but instead consists of clumps of misshapen, rogue forms of one of the body's own proteins called the prion protein (PrP). Once formed in the body, rogue PrP particles (prions) act as seeds to convert normal PrP into a likeness of themselves setting off a chain reaction leading to progressive accumulation of prions throughout the brain. This accumulation causes nerve cells to die leading to severe brain damage, dementia and ultimately death of the infected individual. Understanding what is special about the structure of prions and how they grow and kill nerve cells is increasingly important as it is thought that similar processes, with the spread of growing misshapen protein seeds, are also involved in more common forms of brain disease such as Alzheimer's and Parkinson's diseases. Currently, in the absence of effective treatments, institutional care for patients with dementia costs the UK NHS tens of billions of pounds each year.

Despite decades of research, it is still not clear how prions grow or how they kill nerve cells. A major reason for these gaps in our knowledge is that prion infection has never been observed in sufficient detail in isolated living nerve cells. However recent advances in technology with light and electron microscopes will now allow us to directly see prions as they infect cells. The research aims of this proposal are 1) to find out which parts of the nerve cell prions bind to during initial phases of infection, 2) to identify which part of the nerve cell prions move to in order to cause their toxic effects, 3) to identify specific structural components of the nerve cell that are involved in each part of the process. To achieve these aims, I will use cells that have been genetically modified to produce PrP with a built-in fluorescent chemical tag which will produce light that can be seen using new, powerful, light microscopes. This will enable tracking of "glowing" prions in real time as infection proceeds in living cells. Once the precise steps of infection are worked out and we know exactly where to find prions within the cell, I will zoom-in on these locations using powerful electron microscopes. High-resolution images from electron microscopes will enable us to pinpoint which structural components of the cell are directly interacting with prions. Through this work I hope to provide the first detailed understanding of the how prions interact with cells to cause their lethal effects. Importantly, knowing the structural components of the cell that prions interact with will identify key targets for drugs that may be able to stop prion infection within the brain.

Prions are infectious fibrillar aggregates of misfolded host-encoded prion protein (PrP), which is expressed on the surface of neurons. Prions cause invariably fatal neurodegenerative diseases, such as the Creutzfeldt-Jakob disease (CJD), and are transmissible between individuals by inoculation or other routes. The misfolding and aggregation of PrP is templated by existing (sporadic or acquired) prion seeds, but the precise mechanism of prion propagation is not known. Nor are the factors that mediate prion toxicity. This is due to the lack of a cell-based model system that could deliver high-resolution spatiotemporal data on prion infection pathways. The aim of the proposed research is to define prion pathogenicity mechanisms by single molecule localisation microscopy (SMLM), correlative light and electron cryo-microscopy (cryo-CLEM) and single particle electron cryo-microscopy (cryo-EM) utilising a novel transgenic cell platform. An established neuroblastoma N2a cell line has been equipped with the unnatural amino acid (UAA) incorporation machinery. Super-resolution fluorescence-competent UAA has been introduced into PrP at the site that is compatible with forming fluorescent prions. Thus, prion propagation upon infection with purified prion seeds will be traceable in real time. Candidate prion receptors will be fused with photo-switchable fluorescent proteins (e.g. rsEGFP2) and their interactions with prions will be studied by multicolour live cell SMLM and cryo-CLEM at defined stages of infection. Finally, cryo-EM of purified prions decorated with purified receptors will reveal near-atomic details of the clinically relevant interactions. This research combining dynamic and cross-scale structural data will inform the design of anti-prion therapeutics in the future. It may also benefit patients suffering from the Alzheimer's disease, proven to share PrP pathways, and costing the UK NHS tens of billions of pounds each year.