Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPreslevel disparity
© Lewis et al; licensee BioMed Central Ltd. 2012
Received: 11 November 2011
Accepted: 26 April 2012
Published: 26 April 2012
Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution.
Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication.
This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels.
KeywordsPrion protein Prion infectivity Prion disease Protease resistance Subcellular localisation Fractionation
Prion diseases constitute a group of unique neurodegenerative disorders, which naturally afflict a number of mammalian species including humans. Although our understanding remains incomplete, considerable evidence supports the "protein-only" hypothesis, which purports that the agent ("prion") responsible for both transmission and consequent pathogenesis is predominantly composed of misfolded conformers of the normal cellular prion protein PrPC . Additional discriminating features of the aberrant prion protein include increased β-sheet content [2, 3], reduced solubility and increased tendency to aggregate, and typically heightened protease resistance [4–6]. Due to the characteristic protease resistant core, limited proteolysis with proteinase K (PK) truncates the N-terminus of the misfolded protein, producing PrPres, whilst PrPC is completely degraded, allowing a convenient biochemical differentiation of these two prion protein isoforms.
An intriguing but somewhat perplexing aspect of prion biology is the several instances in transmission studies, encompassing many prion strains, where infectivity titres and PrPres levels (as detected by biochemical assessment of inocula) do not faithfully correlate. Illustrating this are pre-clinical prion infections after low dose transmissions , BSE infectivity in tongue and nasal mucosa  and slowly sedimenting high titres of infectivity separated from PrPres in 'fast' prion strains . Further examples have occurred during cross species transmissions including intracerebral inoculation of hamster prions to mice , primary passage of bovine prions to rodents [11, 12], scrapie prions peripherally introduced into mice  and transmission of three distinct prion strains (human, hamster scrapie, murine scrapie) into transgenic mice expressing the murine equivalent of a human prion protein gene mutation . In addition, PrPres generated through protein misfolding cyclic amplification (PMCA) evinces a longer incubation period (indicative of a lower titre) despite western blot detection levels equivalent to those observed in the original seeding inoculum . This PMCA study suggests that a component within the original inoculum, which perhaps does not propagate or amplify as well as PrPres, may contribute to the more efficient transmission. Although PrPres is inextricably linked to prion infectivity, these numerous examples clearly illustrate the poorly understood complexities of this relationship.
The precise cellular location of PrPC misfolding and conversion also remains speculative (reviewed in ), as does the contribution of cellular co-factors to conversion efficiency, although the participation of a species-specific protein [17–19], or negatively charged macromolecules such as nucleic acids [20–24] and glycosaminoglycans [25–29] has been posited. In contrast, evidence exists correlating the efficiency of prion propagation and transmission with the size of prion multimers serving as templates for conversion [30, 31]. Acknowledging the aforementioned uncertainties, the current study investigated whether such observed disparities between infectivity titres and PrPres levels could be resolved to a subcellular level and thereby provide a useful model for insights into the molecular basis of this observation. To address this aim, we utilized fractionation of MoRK13 cells infected with M1000 prions to explore the contributions of subcellular co-factors and cognate prion protein species to the efficiency of transmission.
Prion protein conformers reside predominantly in lipid rafts in MoRK13 and MoRK13-inf cells
High levels of prion infectivity are present in lipid raft and ER/MT marker enriched fractions of MoRK13-inf cells
ER and MT marker enriched fractions show disparity between PrPres levels and infectivity titres in vitro and in vivo
To confirm genuine prion infectivity, selected fractions were bioassayed in Tga20 PrPC over-expressing mice. The fractions were chosen to provide a range of PrPres and infectivity level combinations; ie high PrPres and infectivity levels (fraction #4), low PrPres and infectivity levels (fraction #10), and low PrPres but high infectivity levels (fraction #8). Controls included inoculating mice with whole cell lysate from naive MoRK13 and MoRK13-inf cells, as well as M1000 brain homogenate, in order to make comparisons with the original prion strain. Figure 4B depicts the incubation periods for the selected fractions and control mice. Mice exposed to uninfected MoRK13 cell lysate were symptom free at 245 days post-inoculation. Mice inoculated with 0.01% M1000 brain homogenate had a significantly shorter incubation period than mice inoculated with MoRK13-inf whole cell lysate. Importantly, concordant with the in vitro cell culture transmissions, mice inoculated with fractions #4 and #8 had indistinguishable incubation periods, despite the significantly different PrPres levels contained within these fractions. Mice inoculated with fraction #10 had significantly longer incubation periods than mice infected with the other two fractions, again concordant with the relative infectivity levels determined by the cell culture transmissions. For illustrative purposes, this 35 day extension in incubation period approximates a 3 log reduction of infectious titre when modelled on a time interval assay developed in Tga20 mice inoculated with M1000 brain homogenate derived prions (Additional file 4: Figure S4). In summary, the in vivo findings faithfully recapitulate and validate the MoRK13 in vitro model.
Infectious prions from different subcellular fractions do not induce unique disease in vivo
The efficiency of M1000 prion transmission is not enhanced by exogenous fraction specific co-factors in vitro or in vivo
To further evaluate the possible contribution of cellular co-factors on the efficiency of prion infection, M1000 brain homogenate was diluted in PBS or selected subcellular fractions from uninfected MoRK13 cells (fractions #4, #8 and #10), and bioassayed in Tga20 indicator mice. M1000 was also diluted in 'empty' Nycodenz fractions (#4, #8 and #10), in order to control for any affect the Nycodenz gradient material itself may have on incubation period or the neuropathology. Neither the exogenous cellular co-factors in the selected fractions, nor Nycodenz alone, had any significant affect on the incubation time (Figure 6C) or overtly affected the neuropathology (Addtional file 8: Figure S8) of M1000 in Tga20 mice.
Discrepancies between PrPresand transmission efficiency are not due to protease-sensitive prions, and only in lipid raft enriched fractions is infectivity enhanced by sonication
Evidence suggests misfolded prion protein aggregate size correlates with efficiency of conversion or prion infection [30, 31]. As a consequence, in vitro conversion assays such as PMCA may be most efficient when incorporating sonication steps [52, 53]. To assess this possibility, selected fractions (#4, #8 and #10) were subjected to sonication (equivalent to one 'round' of PMCA) prior to using them as a source of infectivity in the MoRK13 cell culture model. As demonstrated by the representative cell blot in Figure 7C, quantified in Figure 7D, only subcellular fraction #4 had significantly increased levels of infectivity compared to its untreated fraction.
The protein-only hypothesis states that misfolded conformers of the normal cellular prion protein are the principal component of the agent responsible for transmitting prion disease . However, previously observed examples of high infectivity titres associated with very low or undetectable PrPres, the commonly utilized surrogate prion marker, suggest a poorly understood spectrum of infectious prions. Assessing the subcellular environment of the most infectious prions may provide information about optimal pH or metal content conditions, implicate membrane domains, or subcellular co-factors involved in localisation of highly efficient prions. Investigation of the subcellular distribution of prion infectivity and corresponding PrPres levels has been previously reported, albeit to a limited extent. However, absent in prior studies were attempts to explore what determines the intracellular topographical diversity of prions or the molecular basis of any observed discrepancies in PrPres and infectivity levels.
The present study clearly indicates that not all prion infectivity is associated with lipid rafts, although the significance of the lipid raft microenvironment in PrPC misfolding and prion conversion is yet to be resolved, with experimental evidence both for and against lipid raft localisation as an optimal site (reviewed in ). In complete agreement with the protein-only hypothesis, lipid raft and EE marker associated infectivity and PrPres were shown to correlate in the MoRK13-inf model. In contrast however, ER/MT marker enriched fractions contained much greater infectivity when reported to relative PrPres content. That lipid raft and ER/MT enriched fractions contain the same infectivity levels indicates some biological redundancy or relative inefficiency of the lipid raft localised prions, and/or higher efficiency of ER/MT localised prions, prompting further investigation.
Cell-free conversion studies have shown there is a role for cellular co-factors, such as nucleic acid or other polyanionic molecules, in the efficiency of prion conversion and propagation [21–23, 29, 54]. Perhaps militating against a prominent role of specific co-factors contributing to the transmission efficiency of the MoRK13-inf subcellular fractions, infectivity of brain derived M1000 prions was not significantly differentially enhanced by dilution across various subcellular fractions. However, an explanation for this result is that the transmissible prions within M1000 brain homogenate were already largely in an optimal state, pre-formed and associated with the necessary co-factors required for infection. Therefore additional exogenous co-factors supplied via the MoRK13 or vecRK13 subcellular fractions were somewhat superfluous. Alternatively, the fractionation procedure itself may have inactivated any critical co-factors, such that they were not able to significantly enhance the infectivity of M1000 brain homogenate. In fact, there was a clear trend for increased PrPres propagation by recipient cells after infection with M1000 diluted in subcellular fractions compared to the lysis buffer only control, independent of whether the fractions contained PrPC. This may indicate that incompletely defined but relatively ubiquitous 'cellular co-factors' contribute to the efficiency of in vitro prion transmission, which would be consistent with previous studies.
Numerous prion strains exist, evident in both naturally occurring human [55, 56] and animal [38, 57–60] prion disease, as well as those adapted to laboratory based animal models. One hypothesis for what determines different strains is the tertiary structure of the prion conformer, possibly affected by metals, co-factors or binding partners . It is also believed that prions may adopt various stable tertiary conformations, and there is evidence of simultaneous propagation of more than one prion strain within the brain [55, 62, 63]. Furthermore, super-infection experiments indicate that the more infectious strain will predominate and determine disease expression [64–66]. Prion strains can be classified by their distinctive neuropathological lesion profiles, incubation periods, PrPres glycosylation patterns and electrophoretic mobilities [39, 42, 43, 46]. As RK13 cells are capable of supporting and maintaining propagation of many prion strains , and each fraction represented different subcellular localisations and potential binding partners, we explored the possibility that MoRK13-inf fractions contained structurally distinct prions of variable transmission efficiency. However histological and western blot analyses failed to detect any evidence of a subcellular divergence of prion strains, strongly militating against this as the explanation for the apparent increased relative infectivity in the ER/MT marker enriched fractions.
Previous experiments have shown that PrPres aggregate size affects the efficiency of conversion and prion infection, perhaps through effects on optimising the available templating surface [30, 31], with oligomers of five or fewer PrPres molecules and larger fibrillar aggregates of PrPres far less efficient than non-fibrillar particles of 14-28 molecules . There is also experimental evidence that a proportion of disease associated prions are protease sensitive [47–49], which may form low molecular weight aggregates [47, 49]. The results presented herein show that only a minor proportion of prion infectivity within MoRK13-inf fractions is protease sensitive, and is unlikely to account for the disconnect observed between PrPres levels and infectivity in the ER/MT and lipid raft enriched fractions. Rather, the sonication results are in keeping with a greater proportion of multimeric assemblies, fibrils or aggregated species of prions existing in the lipid raft compared to ER/MT marker enriched fractions, with sonication increasing the number of replication-competent prion oligomeric strand ends which are then more efficient at transmission and inducing prion propagation. Recent publications provide credence to this hypothesis [68, 69], with direct visualisation of the fragmentation of recombinant PrP after sonication. However, the current study does not exclude any positive effect that sonication may have had on other cellular components contained within the fraction mileu or interactions between the prion protein and other molecules. In fact another recent publication  found that sonication also fragments purified liver RNA, to a size that has previously been shown to stimulate prion conversion in PMCA assays. However the RNA sonication produced optimal (sized) RNA after approximately 8 cycles, whereas our sonication experiment was equivalent to one cycle, giving some support to the plausibility of our former hypothesis. Ongoing studies, including the utilisation of techniques such as the conformation dependent immunoassay (CDI) to measure prions in the fractions , and sophisticated size fractionation techniques, will help clarify the exact biophysical nature of the variably efficient prion species in the lipid raft and ER/MT enriched fractions.
The association of prion infectivity with MT and ER has been previously investigated with conflicting results. One study showed that purified mitochondria and mitoplast fractions from scrapie infected hamster brain contained infectivity titres equivalent to those determined for crude brain homogenate, yet mitoplast fractions were not associated with detectable levels of PrP , in keeping with the findings of the present study. These results, which suggested an association of high levels of scrapie infectivity with the inner mitochondrial membrane or mitochondrial matrix, are broadly consistent with the characteristics of MoRK13-inf ER/MT enriched fractions, which co-localised with the mitochondrial membrane marker Bcl-2 . An integral and unique (to mitochondria) lipid component of the inner mitochondrial membrane is cardiolipin, a form of dimeric phosphatidylglycerol . Interestingly, utilizing serial PMCA, researchers have recently been able to produce protease resistant, infectious prions from recombinant PrP mixed with RNA and the synthetic phosphatidylglycerol, POPG (1-palmitoyl-2-oleoylphosphatidylglycerol) . The authors state that the POPG and RNA additives to their PMCA may be mimicking factors which facilitate the conversion process in vivo, which is entirely consistent with the results presented here implicating mitochondrial component enriched fractions as containing highly efficient infectious prions.
Somewhat incongruent with these observations, a much earlier study examined the infectivity of scrapie within membrane fractions and found that brain derived purified mitochondrial fractions were associated with very little infectivity . Nevertheless, similar to our results, Millson and colleagues  did find both brain and spleen derived subcellular fractions containing elevated enzyme activities usually associated with the ER and plasma membrane were associated with high scrapie infectivity. Conversely, Alais and colleagues  found fractions enriched in the ER marker Bip harboured no infectivity, despite containing moderate levels of PrPres. This result, whilst presenting another example of discrepancy between PrPres levels and infectivity, clearly contrasts with what was observed in the MoRK13-inf fractions, perhaps reflecting the different methods and prion strain-cell model employed.
Through the use of both in vitro and in vivo transmission studies, we have corroborated previously reported discrepancies between absolute PrPres levels and infectivity and provided insight into the basis of this phenomenon. Through subcellular separation of infectivity, our data indicates that a substantial amount of infectivity is contained outside of buoyant lipid raft fractions and importantly showed that the most transmission efficient prions per detectable PrPres unit were associated with either ER and/or MT membranes or proteins. We established that the high transmission efficiency shown by the ER/MT containing fractions was not due to the simultaneous separation of a more potent prion strain. As critical co-factor enrichment could not be completely excluded, it remains to be determined whether cellular microenvironments directly but variably contribute to the transmission efficiency of resident prions, or only passively serve as sequestration sites for the different prion species. Overall the current study broadly aligns with the notion that rather than absolute levels of PrPres, intrinsic prion properties may dictate or be dictated by the ultimate subcellular localisation of infectious prions, with transmission efficiency likely correlating best with optimal prion oligomeric state for template directed conversion. Importantly, through the development and validation of a tractable model, further detailed exploration of these fundamental aspects of prion biology, including assessment of other cell line-prion strain combinations to determine the breadth of applicability of our observations, can be undertaken.
Rabbit kidney epithelial cells which have no detectable endogenous PrPC protein, were stably transfected to over-express murine PrPC (MoRK13) or the empty vector (vecRK13)  and mouse hypothalamic GT1-7H cells were maintained as described previously , in a humidified incubator at 37°C with 5% CO2.
A method of non-toxic/non-detergent cell lysis and subcellular separation was necessary to allow subsequent use of fractions for infecting recipient cells or mice. Also, due to the possible involvement of lipid rafts in prion conversion, a lysis method was chosen in order to maintain lipid raft integrity and buoyancy , with minor modifications. Briefly two confluent T175 cm2 flasks (approximately 4 x107 cells) were washed twice with 20 mls ice cold lysis buffer (20 mM Tris-Cl pH 7.8, 250 mM sucrose, 1 μM CaCl2, 1 μM MgCl2) and harvested by scraping into a further 20 ml of lysis buffer and pelleting at 700 × g for 3 minutes. The cell pellet was re-suspended in 500 μl cold lysis buffer. Cells were lysed on ice, by passing the cell suspension through a 22 g needle exactly twenty times, and the crude lysate was centrifuged at 1000 × g, 10 minutes at 4°C. The post-nuclear supernatant was retained on ice and the extraction repeated on the pellet. The lysate was then assayed for total protein content by performing a bicinchoninic acid (BCA) assay (Pierce, Thermo Scientific, Scoresby, VIC, AUS) as per the manufacturer's instructions, and adjusted with lysis buffer to 1.8 mg/ml. The Nycodenz (HistoDenz™, Sigma-Aldrich, Castle Hill, NSW, AUS) density gradient fractionation method was adapted from a published protocol , to suit a Beckman Optima Max-E Benchtop Ultracentrifuge and MLS-50 rotor. Nycodenz solutions were prepared in TNE (25 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM EDTA) and an 8-35% linear step Nycodenz gradient was poured (400 μl of each of 8%, 12%, 15%, 18%, 20%, 22.5% and 25%) with 1.1 ml of a 35% Nycodenz cushion consisting of equal parts ice cold 70% Nycodenz and cell lysate (1 mg total protein) pipetted to the bottom of the gradient. In some cases, an 'empty Nycodenz' gradient was poured, whereby the 35% Nycodenz/lysate cushion mixture was substituted for 35% Nycodenz alone. Nycodenz gradients were centrifuged at 200,000 × g (average) for 342 minutes at 4°C. Following centrifugation, 10 equal volume fractions of 390 μl were collected and stored at -80°C or kept on ice for immediate use.
Prion strain and cell infections
The M1000 prion strain used in this study was derived from a well characterised stock of pooled mouse brain homogenate . Recipient MoRK13 or GT1-7H cells were infected using an overlay technique as described previously . For comparisons of cell lysate and brain homogenate M1000 infectivity, cell lysates were prepared by harvesting and lysing in sterile phosphate buffered saline (PBS; Invitrogen, Mulgrave, VIC, AUS) by three cycles of freezing (10 minutes at -80°C) and thawing (3 minutes at 37°C), and centrifugation at 1000 × g for 3 minutes at 4°C to obtain a post-nuclear supernatant. The total protein content of the PBS supernatant and M1000 brain homogenate were determined by BCA assay, and the lysates and homogenate were balanced to the same protein concentration with PBS. Following this 100 μl of lysate or homogenate was mixed with 400 μl of complete medium and this was used to infect recipient cells. For fraction infections 100 μl fraction ('neat') or, where indicated, fraction which had been serially diluted in medium, was mixed with 400 μl complete medium, and used to infect recipient MoRK13 cells. For 'spiking' experiments, M1000 brain homogenate was diluted in 400 μl media and mixed with 100 μl MoRK13 or vecRK13 fraction (or as a control the lysis buffer used to prepare cells prior to fractionation) to give a final concentration of 0.05%, 0.025% and 0.0125% M1000, prior to being used to infect recipient MoRK13 cells.
Subcellular fraction pre-treatments
For PK digestion, 100 μl of a fraction was treated with a final concentration of 1 μg/ml PK for 8 hours at 37°C, conditions found to reduce PrPC by approximately 80% when tested on control fractions (data not shown). For sonication pre-treatment, 100 μl of a fraction in a 1.5 ml microfuge tube was subjected to 60 seconds at amplitude 70 in a S4000 sonicator (Misonix, Farmingdale, NY, USA) with microplate horn adapter in 300 mL water maintained at 37°C. The PK digested or sonicated fractions (and untreated controls) were mixed with 400 μl of complete medium and used for in vitro infections as described above.
For determination of subcellular localisation of PrP and other proteins, fractions were mixed with 4X sample buffer and subject to PAGE (using either 4-20% or 10-20% Tris-glycine SDS or 4-12% Bis-tris NuPAGE pre-cast gels (Invitrogen), depending on the size of the proteins to be detected, and then transferred to PVDF membrane for western blotting of PrP as described previously  and organelle marker proteins using antibody dilutions as outlined in the manufacturer's instructions (BD Pharmingen™ Organelle Sampler Kit, BD Biosciences, North Ryde, NSW, AUS). For detection of the ganglioside GM1 (lipid raft marker), 3 μl of each fraction was spotted onto nitrocellulose membrane, and allowed to dry for 20 minutes at 37°C. The membrane was blocked for a minimum of 1 hour in 5% skim milk powder in PBS containing 0.05% (v/v) Tween-20 (PBST) and then incubated in 1:100,000 cholera toxin B subunit (CTB)-horseradish peroxidise (HRP) conjugate (Sigma, stock concentration 0.45 mg/ml CTB and 1 mg/ml HRP in H2O) solution in block for 1.5 hours at room temperature prior to chemiluminescent detection (ECL Plus, GE Healthcare, Rydalmere, NSW, AUS). For PrPres detection, fractions, cell lysates or brain homogenate were digested with a final concentration of 50 μg/ml PK, 1 hour at 37°C before SDS-PAGE and western blotting. Dot blots of fractions were also carried out for PrPres detection, whereby 5 μl of fraction was spotted onto nitrocellulose membrane, which was dried 30-60 minutes at 37°C and then treated in exactly the same manner as the cell blot assay nitrocellulose membrane. Cell blots for the detection of PrPres in recipient cells, at four passages (P4) post-infection were carried out as described previously . All immunoblotting (western blots and cell blots) for PrP species used the monoclonal antibody ICSM18, (D-Gen, London, UK). All chemiluminescent images were captured by a Fujifilm LAS-3000 (Berthold Australia, Bundoora, VIC, AUS).
In vivoprion transmissions
All animal experiments were carried out in strict accordance with the 'Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC)', with approval from the University of Melbourne Animal Ethics Committee (AEC #04154). Tga20 PrPC over-expressing mice  were anesthetised using methoxyfluorane and inoculated intracerebrally with 30 μl of 0.01% M1000 brain homogenate diluted in PBS or the appropriate fractions as indicated, or with 30 μl of 'neat' fraction or whole cell PBS lysate. Mice were provided food and water ad libitum and housed following routine animal husbandry practices. Mice were examined daily for symptoms of prion disease. Once mice developed persisting features of advanced prion disease, including impaired righting reflexes, hunched posture and hind limb paresis, they were culled by cervical dislocation under anaesthesia and the number of days post-inoculation was recorded. Brains were removed and sagittally hemi-sectioned, with half the brain fixed and stained to allow scoring of vacuolation, astrocytic gliosis and PrP deposition as described previously , and the other half made to 10% (w/v) homogenates in PBS, with homogenates stored at -80°C until required. Neuropathological scoring was performed on two separate occasions, blinded as to the inoculum group, to provide a semi-quantitative comparison of lesion profiles between the groups of animals. Stained sections were visualised using a Zeiss Axioskop 50 microscope with images captured using a Zeiss AxioCam HRC camera (Carl Zeiss, North Ryde, NSW, AUS).
Densitometry and statistical analysis
All densitometric analyses used the public domain ImageJ software (National Institutes of Health, USA). For determining relative levels of PrPres in fractions, each fraction PrPres dot blot signal intensity was measured, with the sum of the 10 individual PrPres levels providing the 'total PrPres'; each individual fraction was expressed as a percentage of the total PrPres. For determining relative levels of infectivity contained within MoRK13-infectious fractions, PrPres produced by MoRK13 cells exposed to each 'neat' fraction was measured at passage 4 (P4) post-exposure by cell blot signal intensity, with the sum of the 10 individual PrPres levels providing 'total PrPres'. Once again, each individual fraction was expressed as a percentage of the calculated total PrPres. All statistical analyses were performed in GraphPad Prism 4, with one way ANOVA and Tukey's multiple comparisons or two way ANOVA and Bonferonni post-tests used as indicated, unless stated otherwise.
Conformation dependent immunoassay
Cholera toxin B subunit
Phosphate buffered saline
Protein misfolding cyclic amplification
Cellular prion protein
Protease resistant prion protein.
The authors thank Professor Charles Weissmann for the gift of the Tga20 transgenic mice, the Animal Housing Facility staff in the Faculty of Medicine, Dentistry and Health Sciences, the University of Melbourne for their assistance with animal husbandry, and Ms Laura Leone for technical assistance with preparation of the mouse brains for neuropathological assessment. This work was funded by an Australian Government National Health and Medical Research Council (NHMRC) Program Grant (#400202). VL is supported by NHMRC Training Fellowship (#567123). SJC is supported by NHMRC Practitioner Fellowship (#400183). VAL is supported by The University of Melbourne CR Roper Fellowship. AFH is supported by an Australian Research Council Future Fellowship (FT10100560).
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