Programme 2: Uncoupling infectivity and toxicity in in vitro models of prion-induced toxicity

Background: Prion diseases are fatal progressive neurodegenerative diseases initiated upon conversion of the cellular prion protein (PrPC) to disease-associated misfolded isoforms. This conversion is an absolute necessity for pathology as PrP-null mice are resistant to developing prion disease. PrPC, itself, is posited as an essential mediator of neurotoxicity in prion disease. Conversion products consist of a diverse array of disease related isoforms; some have the propensity to form fibrils whereas as others coalesce to multimeric assemblies or exist as oligomers. A subset of conversion products is likely to be the cause of pathology by toxic signalling.

The first evidence to hypothesise a toxic species of PrP, PrP lethal (PrPL), was from work suggesting that toxicity and infectivity can be uncoupled. In experimental animal models of subclinical prion infection, mice inoculated with hamster prions develop high levels of Proteinase K (PK)-resistant PrP scrapie (PrPSc) but remarkably do not succumb to disease. Recent work within the Unit has shown that in a mouse model prion disease progresses through two distinct mechanistic phases of PrP conversion. The first phase: an autocatalytic production of infectivity, dependent on PrPC expression, but independent of expression level. The second phase: a conversion process tightly regulated by PrPC expression level. Pathology commences only during the second phase and is coupled to a linear increase in PK-sensitive PrP isoforms to a toxic threshold. These isoforms may exert a general or specific toxicity elicited by structurally distinct subpopulations.

Aim: To test the hypothesis that infectivity can be uncoupled from toxicity in vitro.

Rotation Project Dissect and culture neurons from E17 or P0 mouse hippocampi and cortices from WT, PrP null and PrP overexpressing mice. Treat cells with brain homogenates from mice infected with Rocky Mountain Laboratory (RML) prions or control uninfected mice. Examine formation of proteolysis-resistant PrP conformers in cell lysates by Western blotting. Determine what time of incubation with RML-infected brain homogenate is required for prion propagation and production of PrPSc in vitro. Use toxicity assays, to determine if there is an overlap between PrPSc appearance and prion-induced toxicity. Next, use hamster prions that are not known to propagate in vitro in mouse PK1 neuroblastoma cells to determine if they are toxic in the absence of prion propagation. The hamster prion infected mouse neuronal cultures will be tested to identify if there is any prion propagation albeit at a low level. Finally, inhibit prion propagation pharmacologically and assess the effect of this inhibition on the toxic response.

The project will provide training in the following techniques: preparation and culturing of primary mouse neuronal cells; handling mouse and hamster prions; western blotting; multi-parametric fluorescent toxicity assay; high content Opera Phenix microscopy.

PhD project The origin of neurotoxicity in protein misfolding diseases remains unclear.  In the case of Alzheimer’s disease (AD), for example, various amyloid-beta oligomers and aggregates have been isolated from transgenic mouse and patient brains, but their true pathological relevance is uncertain.  There is a great advantage in studying the nature of toxicity in animal models of prion disease; wild-type animals, within their life span, develop full characteristics of a human disease, unlike AD and other neurodegenerative diseases, in which animal models usually overexpress proteins of interest.

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 a time-course of infectivity.  Toxicity will be measured using our novel multi-parametric in vitro assay to ascertain at what time after inoculation with RML toxicity begins to accumulate.  We hypothesise this will occur after infectious titre has saturated.  Infectivity will be quantified by means of the mouse scrapie cell assay and prion pathological markers analysed by histology.

The next goal will be identification, purification and characterisation of PrPL utilising a combination of enzymatic and detergent treatments, centrifugation, ultra-filtration and asymmetrical flow field-flow fractionation.  Ultimately we will perform functional studies to determine the mechanism by which PrPL causes toxicity to identify potential therapeutic targets.

Programme 2: 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 conversion 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 two groups of amino acids within the unstructured amino terminus of PrPC, required for propagation of multiple prion strains in mouse cells.  Moreover, mutation of these amino acids inhibits prion propagation in the presence of wild type PrPC but cannot block prion propagation when expressed in chronically prion-infected cells.  Together, these results indicate efficient prion propagation is dependent upon these amino acid groups acting at the infection stage.

Aim: Identify the interactome of the amino acid groups, elucidate their role in prion propagation and determine their relevance to other protein misfolding disorders.

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 two groups of N-terminal amino acids that are 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 of the alanine mutants as well as myc or STrEP- tagged wild type PrPC will be prepared and analysed to identify proteins that co-purify with wild type PrP 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 proteomics 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 of the identified proteins to determine whether these interactions are required for highly efficient prion infection/propagation in cells. 

If we identify proteins that effect in vitro prion infection/propagation upon silencing, we will generate mice lacking the identified interacting proteins to determine if they are required for prion infection/propagation in mice.