Quantitative analysis of prion structure and dynamics by super-resolution microscopy

Aims Two processes lie at the heart of protein misfolding diseases such as Creutzfeldt-Jacob, Parkinson’s, and Alzheimer’s disease: the conversion of protein into misfolded, toxic structures, and their replication by a prion-like mechanism (1-2). A main obstacle in understanding their molecular mechanism is the lack of tools to see and differentiate amyloid structures. This project will utilize and further develop super-resolution fluorescence microscopy techniques to analyze structure, toxicity, and structural dynamics of prion replication.

Methods We have developed a new super-resolution microscopy technique using transient amyloid binding (TAB) of single fluorescent probe molecules to visualize amyloid structures on a nanometer scale (3). Images are reconstructed at a resolution better than 60 nm from the localization of single dye molecules that specifically bind to an amyloid fibril. Dye molecules bind to amyloid with defined orientations and can distinguish between oligomeric and fibrillary amyloid species. The project will furthermore provide training in a wide range of biophysical and biochemical techniques: fluorescence microscopy and spectroscopy, atomic force and electron microscopy, protein expression and purification, chemical modification of proteins, handling of mouse and human prions, SDS-PAGE and Western blotting, mammalian cell culture.

Rotation projectThe imaging methodology will first be established in vitro in a model system using recombinant protein. We will grow fibrils of the prion protein (PrP) in vitro, deposit these fibrils onto the glass surface of imaging chambers and image them by TAB microscopy. Then we will add monomeric recombinant PrP to the solution and follow growth of the prion rods in real time.

PhD project The methodology offers a unique view of the dynamics of single prion fibres in real time, which allows to answer several key questions about the mechanism of amyloid formation and prion disease: a) How do anti-amyloid and anti-prion drugs affect the self-assembly mechanism? (4, 5) b) How do mutations in the prion protein affect self-assembly? c) How do oligomeric PrP assemblies and fibrils interconvert and replicate?

Oligomeric assemblies of the Aβ42 peptide are believed to be the main toxic species in AD. Similarly, data from the laboratory of John Collinge suggest that a toxic assembly of PrP precedes the accumulation of fibrillary PrP in the brain (6). The project will use TAB microscopy to differentiate oligomeric and fibrillary assemblies through the orientation of ThT probe binding. We will analyse PrP aggregates isolated from mouse brain at different time points after inoculation, corresponding to different stages of the disease.

Prion propagation in vitro: elucidating the mechanism of replication. Dr Graham Jackson

Aims
Whilst the majority of native proteins can misfold and aggregate into amyloid structures given the right conditions a subset misfold in vivo resulting in various proteinopathies including Alzheimer’s Disease, Parkinson’s Disease and CJD. In the case of CJD the misfolding of the prion protein into prions results in unique biological properties distinct from the other proteinopathies, notably the ability to infect a new host. The mechanism of prion replication has not been defined and is poorly understood with the simple linear protein polymerisation model clearly not accounting for many of the properties of prion disease. We have developed a modified Protein Misfolding by Cyclic Amplification (PMCA) reaction that converts recombinant prion protein into, authentic, infectious prions with a specific infectivity and protein architecture superficially identical to prions isolated ex vivo.

We are focused on determining the structures of synthetic prions seeded with various rodent strains using cryo-electron microscopy (EM) and atomic force microscopy (AFM) to elucidate the relative roles that structure and post-translational modifications play in prion structure, the propagation of strains and the phenotypic expression of prion infection.

The ability to propagate prion infectivity in vitro is critically dependent upon the presence of a small percentage of brain homogenate, without which only non-infectious amyloid propagates. Therefore, we are trying to identify the essential cofactor or cofactors in brain that allow prion propagation and may also contribute to the expression of strains.

In conjunction with Dr Jan Bieschke we are using our in vitro prion replication reaction to image nascent, growing prions in real-time using TIRF and super-resolution microscopy. This will allow us to study the kinetics and mechanism of prion replication and again contrast this with the elongation of simpler amyloid forms and compare strains.

Rotation projects
Generation and purification of synthetic prions derived from different originator strains for structural and phenotypic characterisation.
Isolating the crucial cofactor or cofactors required for prion replication. 
The kinetics of prion replication in vitro seeded with rodent strains of varying incubation periods and the role of polymorphisms and mutations.
The structural and functional consequences of the protective G127V polymorphism modelled in PMCA. 
The role of the N-terminus of PrP in the replication of synthetic prions in PMCA.
Fluorescent labelling of synthetic prions for their imaging as seeds and de novo elongating prions by TIRF and super-resolution microscopy.
The isolation of PrP fibrils generated by ‘QuIC’ reactions and their structural comparison with prion rods by EM/AFM.
The effect of Ab assemblies on prion propagation in vitro. [The incidence of CJD declines beyond late middle-age and one hypothesis is that Ab assemblies interfere with prion propagation possible by sequestering PrPC].

PhD project
Prions, the causative agents of diseases such as Creutzfeldt-Jakob disease can be generated in vitro using the PMCA reaction, which exploits the ability of prions to template the conversion of normal PrP (PrPC), present in brain and other tissue homogenates leading to amplification of the seed prion. To date, there remains controversy surrounding the additional requirements for the generation of authentic prions by PMCA; rather than non-infectious amyloid by amyloid-seeding assays, and the most reliable substrate for PMCA remains whole brain homogenate. A key aim of my research is to investigate the minimal requirements for generation of infectious prions in vitro, the kinetics and mechanism of replication and the critical determinants of ‘strains’. The major techniques employed are biochemical fractionations, PMCA and analysis of products by western blotting and enzyme-linked immunosorbent assay (ELISA), an automated cell assay for prion infectivity (ASCA), electron microscopy, atomic force microscopy, super-resolution and TIRF microscopy and fluorescence techniques such as FRET.
Extension of one or a combination more of the outlined rotation projects will form the basis of full period of PhD research. For more details or informal discussion please contact Dr Graham Jackson (g.jackson@prion.ucl.ac.uk).