Prion-induced neurotoxicity.
Background: Prion diseases are fatal proteinopathies initiated upon recruitment of the cellular prion protein (PrPC) to disease-associated abnormal PrP aggregates. The abnormal aggregates consist of a diverse array of disease related isoforms; some have the propensity to form fibrils whereas others coalesce to multimeric assemblies or exist as oligomers. The existence of a toxic species of PrP, PrP lethal (PrPL), was first hypothesised from work in experimental animal models of subclinical prion infection, where mice inoculated with hamster prions develop high levels of infectious prions but do not succumb to disease. Further work within the Unit showed that prion propagation in a mouse model of scrapie occurs in two stages where infectivity is uncoupled from toxicity.
To characterise prion-induced toxicity, we have developed a multi-parametric assay comprising live cell imaging (Benilova, Reilly et al PNAS 2020) and high-content image analysis (Reilly et al Sci Rep 2022), https://www.ucl.ac.uk/prion/news/2021/nov/dont-catch-me-if-you-can-toxicity-highly-infectious-prions-under-microscope.
This has enabled us to demonstrate that exceptionally pure intact high-titre infectious prions are not directly neurotoxic and that toxicity present in infected brain tissue can be distinguished from infectious prions. Our long-term goals are to purify the toxic species from prion-infected brain tissue and unravel the neurotoxic signalling triggered by it.
Rotation Project (3 months) will provide training in preparation and culturing of primary mouse neuronal cells from E17 day embryos and in extraction of specific toxicity from prion-infected brains (these two techniques are widely used across different projects within the Institute). You will also get trained in IncuCyteS3 live cell imaging and in multi-parametric fluorescent toxicity assay using high content Opera Phenix microscopy. You will also be involved in setting up a synaptotoxicity assay using our recently acquired multi-electrode array (MEA) system to measure field potentials in cultured cells.
Using our established imaging-based assays of neuronal toxicity and a MEA platform, you will then screen a selection of pharmacologically active compounds that block or exacerbate neurotoxicity thereby identifying potential signalling pathways impacting prion-induced neurotoxicity.
PhD project: Our long-term goal is to purify and characterise the toxic species from prion infected brain homogenates and determine how it causes neurotoxicity. The cause of neurotoxicity in protein misfolding diseases remains unclear. To test the two-phase hypothesis of prion pathogenesis, we will assay toxicity in brains of RML prion-inoculated wild type mice; a prion disease model with a well-characterised incubation period and time-course of infectivity. Toxicity will be measured using IncuCyte live cell imaging to ascertain at what time after inoculation with RML toxicity begins to accumulate and how it relates to the time course of infectivity.
Purification, identification and characterisation of PrPL will utilise a combination of enzymatic and detergent treatments, centrifugation, ultra-filtration and asymmetrical flow field-flow fractionation. This is being undertaken by Programme 8.
In parallel, we will use semi-purified prion infected brain homogenates to investigate the toxic pathways triggered in embryonic neurons by the neurotoxic species, making use of the imaging-based assays of neuronal toxicity as well as the MEA platform. Small molecule and/or genetic inhibitors of candidate pathways will be tested determine if neurotoxicity can be blocked.
Defining the cellular interactome of the prion protein critical for prion propagation
Background: Prion diseases like Creutzfeld-Jakob disease (CJD) involve accumulation of aberrantly misfolded conformers of the cellular prion protein (PrPC) upon template-assisted recruitment and propagation of disease-associated PrP. They are unique in that the disease-associated protein shares 100% identity with PrPC, in primary but not secondary structure. The cellular pathways involved in prion propagation are unknown; however, PrPC expression is crucial for prion propagation and development of prion disease.
We have identified a group of seven amino acids within the unstructured amino terminus that are required for propagation of multiple prion strains in mouse cells. Moreover, PrP with alanine replacements of these amino acids can dominantly inhibit prion propagation in cells expressing wild type PrPC but cannot block prion propagation when expressed in chronically prion-infected cells. These results indicate efficient prion propagation is dependent upon these amino acids acting at the infection stage.
Aim: Identify the interactome of these amino acids; elucidate their role in prion propagation and relevance to prion disease
Rotation Project: Prepare retroviral expression constructs that transduce mouse PrPC tagged at aa30 with either myc (EQKLISEEDL) or STrEP (WSHPQFEK) and mutated to eliminate the N-terminal amino acids critical for prion propagation. Reconstitute PK1 neuroblastoma cells in which endogenous PrPC has been stably silenced with these expression constructs and challenge them with RML prions in scrapie cell assays (SCA) to determine if they propagate prions. The SCA measures infectivity by determining the number of spots of Proteinase K-resistant PrP obtained as a result of prion propagation. The rotation project will provide training in molecular biology, retroviral packaging and infection and stable transduction of cells as well as the SCA.
PhD project: If the reconstituted cells recapitulate our previous data, clonal cell lines expressing each alanine mutant that inhibits prion propagation with a myc or STrEP- tag as well as myc or STrEP- tagged wild type PrPC will be prepared and analysed to identify proteins that co-purify with wild type PrPC but not with the mutants. The STrEP tag is particularly useful since it can be used to purify associated proteins in a single step under physiological conditions, thereby preserving native complexes.
One of the problems associated with proteomic studies is their tendency to identify “sticky” proteins as interacting partners. However by only looking for proteins which differentially co-purify, the number of the potential targets should be minimised. Interacting proteins will be identified by MALDI-MS mass fingerprinting and LC-MS/MS. We will also investigate if the identified interacting proteins are altered upon prion infection and whether they bind to PrPC in CAD5 and LD9, cell lines that propagate different prion strains. The differential interactions will be validated by western blotting of co-immunoprecipitated proteins using antibodies, if available, or by development of specific antibodies.
The next step will be to undertake functional studies to determine if these interacting proteins are required for highly efficient prion infection/propagation in cells.
If we identify proteins that affect prion infection/propagation in cells, we will generate mice lacking the identified interacting proteins to determine if they are required for prion disease.