The Strain-Encoded Relationship between PrPSc Replication, Stability and Processing in Neurons is Predictive of the Incubation Period of Disease

Prion strains are characterized by differences in the outcome of disease, most notably incubation period and neuropathological features. While it is established that the disease specific isoform of the prion protein, PrPSc, is an essential component of the infectious agent, the strain-specific relationship between PrPSc properties and the biological features of the resulting disease is not clear. To investigate this relationship, we examined the amplification efficiency and conformational stability of PrPSc from eight hamster-adapted prion strains and compared it to the resulting incubation period of disease and processing of PrPSc in neurons and glia. We found that short incubation period strains were characterized by more efficient PrPSc amplification and higher PrPSc conformational stabilities compared to long incubation period strains. In the CNS, the short incubation period strains were characterized by the accumulation of N-terminally truncated PrPSc in the soma of neurons, astrocytes and microglia in contrast to long incubation period strains where PrPSc did not accumulate to detectable levels in the soma of neurons but was detected in glia similar to short incubation period strains. These results are inconsistent with the hypothesis that a decrease in conformational stability results in a corresponding increase in replication efficiency and suggest that glia mediated neurodegeneration results in longer survival times compared to direct replication of PrPSc in neurons.


Introduction
Prion diseases are a group of transmissible, fatal neurodegenerative diseases, which include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep. The prion agent is comprised mainly, if not entirely, of PrP Sc which is an abnormal isoform of the host encoded prion protein, PrP C [1,2,3,4,5,6]. Prion propagation is thought to occur in a two-step process where PrP Sc first binds to PrP C followed by a conformational conversion of PrP C to PrP Sc [7,8,9]. This conversion results in a change in physical properties of PrP C that include an increase in b-pleated sheet content, decreased solubility in non-denaturing detergents and increased resistance to proteolytic degradation [3,10,11].
Prion strains are operationally defined by characteristic incubation periods and neuropathological features that are maintained upon experimental passage [12,13]. The distribution of PrP Sc in organs and neuronal populations can differ between strains, suggesting that PrP Sc has a distinct strain-specific cellular tropism [14,15,16,17]. The initial uptake of PrP Sc by different celllines appears to be independent of the particular strain [18,19] and suggests that cellular factors are responsible for prion strain tropism [17,20], however, this has not been confirmed in vivo [21].
Prion strain diversity may be encoded by unique strain-specific conformations of PrP Sc [15,22,23,24,25,26]. Consistent with this, strain specific differences in the molecular weight of PrP Sc following limited PK digestion, the relative resistance of PrP Sc to degradation by PK, the relative alpha helical and beta sheet content of PrP Sc , the resistance of PrP Sc to PK digestion in increasing concentrations of a protein denaturant (i.e. conformational stability), and the aggregation state of PrP Sc have been observed [15,22,27,28]. The mechanisms underlying how strainspecific conformations of PrP Sc result in the distinct biological properties of disease are poorly understood.
The published reports on the relationship between the conformational stability of PrP Sc and the length of the incubation period of disease between prion strains are contradictory. In murine prion strains and during adaptation of synthetic prions, a decrease in the conformational stability of PrP Sc correlates with a corresponding decrease in the incubation period [5,24,29,30]. One explanation for this observation is that a decrease of PrP Sc stability increases PrP Sc fragmentation resulting in an increase in agent replication that produces a correspondingly shorter incubation period [31,32,33]. Consistent with this, a decrease in Sup35 fiber stability corresponds to an increased rate of fibril fragmentation in yeast prions [33,34]. These data contrast with what has been observed in hamster-adapted prion strains. Short incubation period prion strains have PrP Sc that is conformationally more stable compared to PrP Sc from strains with a relatively longer incubation periods in hamsters [27]. However, a direct comparison between PrP Sc replication rate and conformational stability has not been investigated.
Both the prion strain and the cell type infected can influence the processing of PrP Sc . Studies of sheep infected with different prion strains, either naturally or experimentally, have identified strainspecific patterns of PrP Sc truncation in both neurons and glia [35,36]. Within a given strain the PrP Sc truncation pattern can differ between glia and neurons suggesting that factors in addition to the conformation of PrP Sc contribute to PrP Sc truncation. While it is thought that replication in neurons is more important to disease development compared to glia, the effect of strain-specific processing of PrP Sc in these cell types is less clear [37,38,39].
To better understand the strain specific relationship between the agent and the host, we evaluated PrP Sc amplification efficiency, conformational stability of PrP Sc , and susceptibility of PrP Sc to endogenous proteolytic processing in vivo in several cell types, of eight hamster-adapted prion strains. Our data indicate that short incubation period strains have correspondingly more efficient replication, a higher conformational stability, and intrasomal accumulation of PrP Sc in neurons compared to long incubation period strains. These data suggest that the relationship between agent replication and clearance influence the progression of disease.

Results
The molecular weight and abundance of PrP Sc from multiple hamster-adapted prion strains is similar Brain tissue from hamsters at terminal disease infected with either the HY TME, 263K, HaCWD, 22AH, 22CH, 139H, DY TME or ME7H agents was digested with proteinase K and 250 mg equivalents were analyzed by Western blot (Figure 1). Western blot analysis of PrP Sc indicated that the unglycosylated PrP Sc glycoform of each strain migrated at 21 kDa with the exception of DY PrP Sc , which migrated at 19 kDa ( Figure 1A, Table 1). The abundance of PrP Sc was determined for each strain (n = 4) and there was less than a 25% difference in the abundance of PrP Sc per mg brain equivalent for each prion strain analyzed ( Figure 1B).
Short incubation period strains replicate PrP Sc more efficiently compared to PrP Sc from long incubation period strains To determine if differences exist in the rate of PrP Sc replication between strains, protein misfolding cyclic amplification (PMCA) was performed on eight hamster-adapted prion strains. Brain homogenates were prepared from animals at the clinical stage of disease or from an uninfected (mock) negative control and serial 10-fold serial dilutions of these homogenates were analyzed by Western blot prior to (Figure 2A and 2C) or after one round of PMCA ( Figure 2B and 2D). PMCA reactions that were initially seeded with 500 to 5610 22 mg eq of HY TME infected brain homogenate resulted in detectable amplification of PrP Sc , but amplification was not detected in PMCA reactions seeded with lower concentrations of HY brain homogenate ( Figure 2B). One round of PMCA using brain homogenate from DY TME infected animals amplified PrP Sc to detectable levels in reactions that were initially seeded with 500 or 50 mg eq, but was not detected in PMCA reactions seeded with lower concentrations ( Figure 2D). This was the general trend, as the short incubation period strains HY TME, 263K, and HaCWD resulted in detection of amplified PrP Sc in reactions seeded with lower ug eq of brain homogenate compared to the longer incubation period strains 22AH, 22CH, 139H, DY TME, and ME7H ( Figure S1, Table 1). These data demonstrate that the efficiency of PrP Sc amplification corresponds with the incubation period for the prion strains that were analyzed.
PrP Sc from short incubation period strains is conformationally more stable than PrP Sc from long incubation period strains The conformational stability of PrP Sc for each of the eight hamster-adapted prion strains was determined using either SDS or

Author Summary
Prion diseases are a group of infectious fatal neurodegenerative diseases that affect animals including humans. This unique infectious agent is the result of a post-translational conformational change of the normal form of the prion protein, PrP C , to an infectious form of the prion protein, PrP Sc . Different strains of the infectious agent result in characteristic incubation periods and neuropathological features within a single host species. These strain-specific differences in disease outcome are likely due to strainspecific conformations of PrP Sc , though the mechanisms by which different conformation can affect prion strain properties are not understood. The aim of this study was to investigate the relationship between the biochemical properties of PrP Sc to the corresponding neuropathological characteristics of eight hamster-adapted prion strains. Our findings indicate that PrP Sc from short incubation period strains were more efficiently replicated, had a more stable conformation, and were observed to be more resistant to clearance from the soma of neurons compared to prion strains with a relatively long incubation period. These results suggest the progression of prion disease is influenced by the balance between replication and clearance of PrP Sc in neurons.  Figure 3, Figure  S2). Similarly, there was a corresponding decrease in the [Gdn-HCl] 1/2 values with an increase in the incubation period. To demonstrate that the reduction in PrP Sc was not due to an inhibition of PrP Sc binding to the PVDF membrane due to the presence of SDS or Gdn-HCl, the PK digestion step of the conformational stability assay was omitted which resulted in the detection of PrP Sc (data not shown). Overall, this data demonstrates that PrP Sc from the short incubation period strains is more stable than PrP Sc from the long incubation period strains (Table 1, Figure 3, Figure S2).

Absence of PrP Sc from the soma of neurons corresponds with increased survival times
Immunohistochemistry was performed on CNS tissue of hamsters using a panel of six monoclonal anti-PrP antibodies whose epitopes span the length of the hamster PrP protein ( Table 2). Immunohistochemistry using this panel of six anti-PrP antibodies on mock-infected tissue sections containing red nucleus neurons failed to detect PrP Sc , indicating the specificity of the antibodies for PrP Sc ( Figure S3). PrP Sc deposits were detected perineuronally and within the neuropil of the red nucleus with every anti-PrP antibody tested in animals infected in the sciatic nerve with the DY TME agent at clinical disease suggesting the presence of full length PrP Sc ( Figure 4A to 4F). Within the soma of neurons, the three antibodies whose epitopes are at the N terminus of PrP (8B4, BE12, and POM3) failed to detect PrP Sc deposition ( Figure 4A to 4C). The three antibodies whose epitopes are located toward the C-terminal region of PrP (3F4, 6H4, and POM19) occasionally identified PrP Sc deposition within the soma of these neurons, however, these DY PrP Sc deposits appeared diffuse and faint compared to the PrP Sc immunoreactivity in the neuropil ( Figure 4D to 4F, Table S1). This same pattern of PrP Sc distribution was also observed in VMNs of the lumbar spinal cord, and the neurons in the interposed nucleus, red nucleus and hind limb motor cortex throughout the course of disease and in  animals inoculated by the i.c. route at clinical disease (data not shown). The distribution of PrP Sc in the red nucleus of hamsters i.c. inoculated with the long incubation period strains 22AH, 22CH, 139H or ME7H was indistinguishable from DY TME agent infected animals ( Figure S4, Table S1).
To extend these studies on a short incubation period strains, we performed PrP Sc immunohistochemistry on the red nucleus from animals infected in the sciatic nerve with the HY TME agent at clinical disease to ensure a direct comparison could be made with DY TME agent infected animals. The deposition of PrP Sc in the neuropil and soma of neurons was similar to what was observed following infection with the DY TME agent and the other the long incubation period strains using the anti-PrP antibodies whose epitopes are located N-terminal to the HY PrP Sc PK cleavage site ( Figure 4, Panels G-H). However, when using antibodies located C-terminal to the HY PrP Sc PK cleavage site, intrasomal and perinuclear PrP Sc deposition was detected that was similar in intensity to PrP Sc deposition in the neuropil ( Figure 4I to 4L, Table S1). This HY TME specific pattern of PrP Sc deposition was observed in animals inoculated by either the sciatic nerve or i.c.
routes of inoculation at early and late time points post-infection and in the same brain regions that were examined in the DY TME infected animals. This pattern of HY PrP Sc truncation was also observed in animals inoculated with the short incubation period strains 263K and HaCWD by the i.c. route at clinical disease ( Figure S4, Table S1). These data reveal similarities in the PrP Sc deposition patterns in the neuropil and somata of neurons of animals infected with either long or short incubation period strains.

Truncation of HY PrP Sc within the soma of neurons
The absence of PrP Sc immunoreactivity using antibodies directed against the N-terminal regions of PrP Sc suggests that truncated PrP Sc is present in these cells. To investigate this possibility, serial sections of red nucleus from clinically-ill HY TME infected hamsters were processed using either the BE12 or POM3 antibodies whose epitopes are N-terminal and C-terminal to the HY PrP Sc PK cleavage site respectively. Fiduciary marks, such as blood vessels and white matter tracts, were used to increase the likelihood that the same neurons were analyzed in both sections. The BE12 antibody detected punctate HY PrP Sc deposits in the neuropil and perineuronally in the red nucleus but failed to detect intrasomal PrP Sc ( Figure 5A). However, the POM3 antibody detected coarse, intrasomal PrP Sc deposits in these same three neurons ( Figure 5B, arrowheads). Additionally, the intrasomal PrP Sc formed large perinuclear aggregates ( Figure 5B). This demonstrates that the loss of N-terminal epitopes of PrP Sc and the aggregation of the Cterminal PrP Sc fragments occurs within the same neuron.

Processing of PrP Sc in astrocytes and microglia is nearly uniform between strains
The deposition of PrP Sc in astrocytes and microglia was investigated using the same panel of anti-PrP antibodies in combination with anti-GFAP and anti-Iba-1, which label astrocytes and microglia, respectively. As negative controls, reactive astrogliosis, microgliosis or PrP Sc immunoreactivity was not detected in mock-infected animals ( Figure S5A to S5F). . HY PrP Sc is more conformationally stable than DY PrP Sc in either Gdn-HCL or SDS. A) HY or DY TME infected brain homogenate was treated with increasing concentrations of either SDS or Gdn-HCL, digested with PK and the remaining PrP Sc was detected using a 96 well immunoassay. The concentration of either SDS or Gdn-HCL required for a 50% reduction in PrP Sc is greater for HY TME (panels B and D) compared to DY PrP Sc (Panels C and E). doi:10.1371/journal.ppat.1001317.g003  . Strain specific differences in the clearance of PrP Sc in neurons of hamsters infected with either the DY or HY TME agents. PrP Sc immunohistochemistry was performed on CNS from hamsters infected with either the DY (panels A-F) or the HY TME (panels G-L) agents. PrP Sc deposits are detected in the neuropil of hamsters infected with the long incubation period strain DY TME, however, PrP Sc was rarely detected in the soma of neurons (panels A-F). In hamsters infected with the short incubation period strain HY TME, PrP Sc is detected in the neuropil with all antibodies used (panels G-L). In contrast to the DY TME infected brain, HY PrP Sc was detected in the somata of neurons with anti-PrP antibodies whose epitopes are C-terminal to the in vitro PK cleavage site (panels I-L). The yellow region in the schematic insets in panel A depict the location in the brain area that was imaged for every panel. Additionally, non-specific binding of the monoclonal antibodies or the fluorescently conjugated secondary antibodies was not detected ( Figure S5G and S5H). The anti-PrP antibodies 8B4, BE12, and POM3 failed to detect PrP Sc within astrocytes ( Figure 6A to 6C), while the antibodies 3F4, 6H4, and POM19 detected coarse punctate PrP Sc deposits in astrocytes of hamsters infected with the DY TME agent at clinical disease ( Figure 6D to 6F, Table S1). The anti-PrP antibodies POM3, 3F4, 6H4, and POM19 detected PrP Sc within astrocytes, while the antibodies 8B4 and BE12 failed to detect PrP Sc within astrocytes of hamsters infected with the HY TME agent at clinical disease ( Figure 6G to 6L, Table S1). The same PrP Sc truncation pattern detected in astrocytes of HY TME infected animals was also observed in animals infected with the 263K, HaCWD, 22AH, 22CH, 139H or ME7 agents ( Figure S6, Table S1). The anti-PrP antibodies 8B4 and BE12 failed to detect PrP Sc in microglia, while the anti-PrP antibodies POM3, 3F4, 6H4, and POM19 detected coarse punctate PrP Sc deposits within these cells (Figure 7, Figure  S7, Table S1).

Discussion
Here we show that short incubation period strains have a more stable PrP Sc conformation when compared to long incubation period strains. PrP Sc conformational stability assays using either Gdn-HCl or SDS as the denaturant found the same relationship between the conformational stability of PrP Sc and incubation period of disease indicating that this relationship is independent of the denaturant used (Table 1). This relationship between PrP Sc conformational stability and incubation period is consistent with previous work examining the conformational stability of purified PrP Sc from hamster-adapted prion strains [27]. In contrast to what is observed in hamsters, a decrease in the PrP Sc conformational stability correlates with a reduction in the incubation period in mice [24,29,40]. The results in murine systems suggest that decreasing PrP Sc stability increases the fragmentation of PrP Sc therefore allowing in the generation of more PrP Sc surfaces for PrP C to bind resulting in an increased rate of PrP Sc formation and subsequently shortening of the incubation period. Consistent with this hypothesis, studies examining Sup35, PrP, Tau, a-synuclein, and ß-amyloid demonstrate that less stable fibrils have a higher propensity to undergo breakage, thereby creating new seeds for conversion [33,34,41,42,43,44,45].
The PrP Sc conformational stability data presented here suggest that conformationally stable PrP Sc may also be more susceptible to fragmentation. SDS, like Gdn-HCl, can increase the susceptibility of PrP Sc to PK digestion and inactivate the agent [27,46,47]. Since treatment of PrP Sc that is enriched using detergent extraction and ultracentrifugation with SDS results in the disaggregation of PrP Sc and the production of smaller PrP Sc particles, SDS can affect the aggregation state of PrP Sc [32,48]. Therefore, the higher concentration of SDS required to increase the susceptibility of PrP Sc to PK digestion of short incubation period strains may be due to increased PrP Sc particle size compared to long incubation period strains.
Short incubation period strains have more efficient PrP Sc amplification compared to long incubation period strains. We used PMCA to determine the relative efficiency of PrP Sc conversion between hamster strains. We have previously shown that PMCA of HY and DY TME recapitulates the strain-specific properties of PrP Sc and faithfully replicates the HY and DY TME agents [49]. In examining the eight hamster strains we found that the efficiency of PrP Sc amplification correlated with the strains respective incubation periods, as the strains with more efficiently replicating PrP Sc had a shorter incubation period compared to long incubation period strains (Table 1). This is consistent with cellfree conversion experiments that demonstrated a faster rate of HY PrP Sc synthesis compared to the rate of DY PrP Sc synthesis [50]. The data presented here also indicate that conformationally more stable PrP Sc amplifies more efficiently compared to less stable PrP Sc . Interestingly, the short incubation period strain HaCWD has conformationally less stable PrP Sc in SDS compared to 263K and HY PrP Sc which corresponded with a lower amplification efficiency compared to the two other short incubation period strains. A possible explanation for the increased amplification efficiency of PrP Sc from the short incubation period strains is that this PrP Sc is more likely to fragment due to its large PrP Sc particle size compared to the longer incubation period strains used in this study. Alternatively, a minor subpopulation of PrP Sc that is conformationally less stable may be responsible for the highly efficient PrP Sc replication that was observed. This conformationally less stable subpopulation may be masked by an excess of conformationally more stable PrP Sc that replicates with lower efficiency [32,51,52]. PrP Sc immunohistochemistry was performed on serial sections with either an anti-PrP antibody whose epitope is either A) N-terminal (BE12) or B) C-terminal (POM3) to the HY PrP Sc PK cleavage site. Arrows indicate the same neurons in panels A and B. The yellow region in the schematic insets depict the location in the brain area that was imaged in each panel. Abbreviations: b.v., blood vessels; w.m., white matter. Scale bar, 50 mm. doi:10.1371/journal.ppat.1001317.g005 Figure 6. Strain-specific truncation of PrP Sc in astrocytes of hamsters infected with either the DY or HY TME agents. Dual immunofluorescence was performed on brains of DY TME (panels A-F) or HY TME (panels G-L) infected animals using antibodies directed against PrP (red fluorescence) and GFAP (green fluorescence). Dual PrP/GFAP immunofluorescence was performed on the reticular formation from DY TME (A-F) Strain and cell-specific variations in the proteolytic processing of PrP Sc have been observed in both brain tissue and cultured cells [36,53,54,55,56]. The results presented here are consistent with these findings and additionally suggest a relationship between the extent of truncation of PrP Sc within the soma of neurons and the strains respective incubation periods. The short incubation period strains, HY TME, 263K, and HaCWD, contained a longer portion of C-terminal protein intact and a large punctate deposition of PrP Sc within the soma of neurons, compared to the longer incubation period strains suggesting a strain-specific clearance of PrP Sc (Figures 8, Figure 9). Furthermore, the low immunoreactivity of PrP Sc in ME7H infected animals observed with all six anti-PrP antibodies and within all three cell types examined (Figure 8, Table S1) may represent the more efficient clearance of PrP Sc in both neurons and glia for this particular strain and account for its significantly longer incubation period. However, we cannot exclude the possibility that the inability to detect intense PrP Sc immunoreactivity in the soma of neurons from animals inoculated with the long incubation period strains is due to a failure of PrP Sc transport to the soma. This strain-specific truncation pattern was only observed in neurons, as the same Nterminally truncated PrP Sc species was detected in astrocytes and microglia for all strains examined, with the lone exception of the loss of the POM3 epitope from DY PrP Sc within astrocytes (Figure 8, Figure 9). These data support the hypothesis that direct infection of neurons leads to more rapid death of neurons resulting in shorter incubation periods, compared to indirect neuronal death via infection of astrocytes and microglia [37,38,57].
The results presented here suggest the strain-encoded relationship between PrP Sc replication, stability and processing in neurons is predictive of the incubation period of disease ( Figure 9). Here we show that strains with a short incubation period have conformationally stable PrP Sc that replicates efficiently. The fast replication and stable PrP Sc may be responsible for the accumulation of PrP Sc in the soma of neurons resulting in a shorter incubation period. The long incubation period strains displayed relatively less efficient PrP Sc replication and less stable PrP Sc . In these strains, the combination of a slower replicating agent and PrP Sc that is less stable may result in neurons to be able to more effectively cleared of PrP Sc resulting in longer incubation periods.

Ethics statement
All procedures involving animals were approved by the Creighton University Institutional Animal Care and Use Committee and were in compliance with the Guide for the Care and Use of Laboratory Animals.

Animal inoculations
Sciatic nerve and intracerebral inoculations of the HY or DY TME agents were performed on male Syrian golden hamsters (Harlan-Sprague-Dawley, Indianapolis, IN) as previously described [21]. Groups of five hamsters were inoculated in the sciatic nerve or intracerebrally with 1 or 25 ml, respectively, of a 1% (wt/vol) brain homogenate from animals at the terminal stage of disease infected with either the HY TME, DY TME, 263K, HaCWD, 22AH, 22CH, 139H, or ME7H agents. Hamsters were observed three times per week for the onset of clinical signs as described previously [58]. Incubation period was calculated as the number of days between inoculation and onset of clinical signs.

Tissue collection
Tissue from infected and mock-infected hamsters was collected for either immunohistochemistry (IHC) or Western blot analysis. For IHC analysis animals were anesthetized with isoflurane and perfused transcardially with 50 ml of 0.01 M Dulbecco's phosphate-buffered saline followed by 75 ml of McLean's paraformaldehyde-lysine-periodate (PLP) fixative as previously described [21,59]. Brain was immediately removed and placed in PLP for 5 to 7 h at room temperature prior to paraffin processing. For Western blot analysis, animals were sacrificed by CO 2 asphyxiation, and the brain was rapidly removed and flash frozen and stored at 280uC.

Western blot analysis
Brain tissue and spinal cord tissue were homogenized to 10% w/v in Dulbecco's Phosphate Buffered Saline (DPBS) without Ca++ or Mg++ (Mediatech, Herndon, VA) containing protease inhibitors (Roche Diagnostics Corporation, Indianapolis, IN) by passing the tissue through a 26 g needle, followed by a 30 second incubation in a cup horn sonicator (Fisher Scientific, Atlanta, GA). The tissue was diluted to 5% w/v in DPBS containing proteinase K (PK) at a final concentration of 1 U/ml (Roche Diagnostics Corporation, Indianapolis, IN) and incubated at 37uC for 1 hour with constant agitation. The PK digestion was terminated by incubating the samples at 100uC for 10 minutes. SDS-PAGE and Western blot analysis were performed as described previously [49] using the anti-PrP antibody 3F4 (1:600; Chemicon; Billerica, MA). The blot was developed with Pierce Supersignal West Femto Maximum Sensitivity Substrate according to manufactures instructions (Pierce, Rockford, IL) and imaged in the linear range of detection on a Kodak 4000R Imaging Station (Kodak, Rochester, NY) and analysis was performed using Kodak Molecular Imaging Software v.5.0.1.27 (New Haven, CT) as described previously [49].

Protein misfolding cyclic amplification
Protein misfolding cyclic amplification (PMCA) was performed as previously described [1,49]. Briefly, uninfected brain was homogenized to 10% (w/v) in ice-cold conversion buffer [phosphate buffer saline (pH 7.4) containing 5 mM EDTA, 1% v/v Triton X-100, and complete protease inhibitor tablet (Roche Diagnostics, Mannheim, Germany)] using a Tenbroeck tissue grinder (Vineland, NJ). The brain homogenate was centrifuged at 5006 g for 30 seconds and the supernatant was stored at 280uC. PMCA was performed with a Misonix 3000 sonicator (Farmingdale, NY) with the sonicator output set to level 6 with an average output of 156 watts for each sonication cycle. All PMCA reactions were replicated in triplicate. One round of PMCA consisted of 144 cycles of a five-second sonication followed by a ten-minute incubation at 37uC. Before each PMCA round, an aliquot was placed at 280uC as an unsonicated control. Samples seeded with prion-infected brain homogenate were replicated using a minimum of three individual hamster brains to control for variation or HY TME (G-L) agent infected hamsters at the clinical stage of disease. The solid white circle located in the schematic inset is the location of the photographed images within the reticular formation. The schematic at the bottom of the figure represents the location of the anti-PrP antibodies and the HY and DY PrP Sc PK cleavage sites are depicted as solid and dashed lines, respectively. The HY and DY PrP Sc PK cleavage sites are also depicted as the solid and dashed lines respectively. Scale bar, 10 mm. doi:10.1371/journal.ppat.1001317.g006 Figure 7. Identical processing of PrP Sc in microglia of hamsters infected with either the DY or HY TME agents. Double immunofluorescence was performed using antibodies directed against PrP (red fluorescence) and Iba-1 (green fluorescence). (A-F) Immunofluorescence in the reticular formation of DY TME infected hamsters at the clinical stage of disease. (G-L) Immunofluorescence in the between animals. Samples containing uninfected brain homogenate in conversion buffer alone were included in every round of PMCA as a negative control. The amplification efficiency was calculated as the reciprocal of the mg equivalent of last dilution of prion-infected brain homogenate that resulted in detectable amplified PrP Sc following one round of PMCA.

Conformation stability assay
Brain homogenates [7.5% (w/v)] were diluted in either SDS (Fischer Scientific, Atlanta GA) to a final concentration of 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2% (w/v) or in Gdn-HCl (Sigma-Aldrich, St. Louis, MO) to a final concentration of 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 molar and were immediately heated at 70uC for 10 min. Proteinase K was added to 0.0625 U/ml (Roche Diagnostics, Indianapolis, IN) and the samples were incubated at 37uC for 15 min while shaking. All samples were brought to 200 ml in DPBS and the concentration of PrP Sc was determined using a 96-well immunoassay as described previously [60]. Gdn-HCL and SDS treated samples were performed in quintuplicate.
Serial two-fold dilutions of brain homogenate each strain were performed in triplicate to ensure that the PrP Sc levels of the SDS or Gdn-HCl treated samples were in the linear range of PrP Sc detection. Denaturation curves were generated by dividing the intensity of all samples by the average intensity of the 0% SDS or Gdn-HCl samples. A sigmoidal dose response curve with variable slope was fitted to the standardized values (Prism statistical software, GraphPad, La Jolla, CA). The [SDS] 1/2 and [Gdn-HCl] 1/2 values is the percentage of SDS or molarity of Gdn-HCl required for a 50% reduction in the PrP Sc signal intensity.

PrP Sc immunohistochemistry
PrP Sc IHC was performed as previously described [21,59]. Briefly, 7 mm tissue sections were deparaffinized and incubated in 95% formic acid (Sigma-Aldrich, St.Louis, MO) followed by blocking of endogenous peroxidases by immersion in 0.3% H 2 O 2 in methanol. Following blocking of non-specific staining with 10% horse serum, sections were incubated overnight with an anti-PrP antibody ( Table 2) at 4uC. The sections were then incubated with a reticular formation of HY TME infected hamsters at the clinical stage of disease. The solid white circle located in the schematic inset is the location of the brain area that was imaged in each panel. The schematic at the bottom of the figure represents the location of the anti-PrP antibodies and the HY and DY PrP Sc PK cleavage sites are depicted as solid and dashed lines, respectively. Scale bar, 10 mm. doi:10.1371/journal.ppat.1001317.g007 biotinylated horse anti-mouse immunoglobulin G conjugate and subsequent incubation with the ABC-horseradish peroxidase elite (Vector Laboratories, Burlingame, CA) staining kit. Sections were developed using 0.05% w/v 3,39-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) in tris-buffered saline containing 0.0015% H 2 O 2 and counterstained with hematoxylin (Richard Allen Scientific, Kalamazoo, MI). Microscopy was performed using a Nikon i80 microscope (Nikon, Melville, NY) and images were captured using DigiFire camera and ImageSys digital imaging software (Soft Imaging Systems, GmbH) and processed using Adobe Photoshop CS2 v9.0.1 (Adobe Systems Inc., San Jose, CA).
For double immunofluorescence, tissue sections were deparaffinized and treated with 95% formic acid as described above. The tissue sections were blocked with 10% goat serum in tris-buffered saline for 30 minutes at room temperature, followed by overnight incubation at 4uC with the same panel of anti-PrP monoclonal antibodies (Table 2) and anti-glial fibrillary acidic protein (GFAP; 1:16,000; Dako; Carpinteria, CA) or anti-ionized calcium binding adaptor molecule 1 (Iba-1; 1:500; Abcam; Cambridge, MA). Sections were then incubated with both Alexa Fluor goat antimouse 546 and Alexa Fluor goat anti-rabbit 488 (1:500; Invitrogen; Carlsbad, CA) secondary antibodies for one hour at room temperature. Slides were cover slipped using ProLong Gold antifade reagent with DAPI (Invitrogen; Carlsbad, CA).

Confocal laser scanning microscopy
Fluorescent images were captured on a Zeiss LSM 510 META NLO confocal scanning system (Carl Zeiss Jena; Jena, Germany) using a Plan Neo 406 1.3-NA DIC oil objective. Excitation of the Alexa Fluor antibodies and DAPI was achieved using an Argon laser at 488 nm, a Helium Neon laser at 543 nm, and a Coherent Chameleon near infrared tunable Ti:Sapphire laser. To increase the signal to noise ratio, each line was scanned 4 times and averaged. The pinhole aperture for each channel was adjusted so that an optical slice of 1.0 mm was imaged. In the profile view for each image, the line tool was used to draw an arbitrary line, and the relative fluorescent intensities along that line were compared to determine intracellular staining.

Semi-quantitative measurement of PrP Sc immunoreactivity
Semi-quantitative measurements of PrP Sc immunoreactivity was performed as previously described [61]. Briefly, captured images were randomized and the relative magnitude of neuropil, intraneuronal, intra-astrocytic, and intra-migroglial PrP Sc immunoreactivity was classified as absent (0), slight (1), moderate (2), or striking (3) from a minimum of 6 observations by three independent observers. The PrP Sc immunoreactivity scores were compared between strains and cell types and analyzed by two-way analysis of variance and Bonferroni post-tests for statistical significance (p,0.05). These tests were performed using the Prism 4.0 (for Macintosh) software program (GraphPad Software, Inc., San Diego, CA). Figure S1 PMCA replication efficiency of hamster adapted prion strains. Western blot analysis of PrP Sc following one round of PMCA that was performed on 10 fold serial dilutions of brain To determine co-localization of PrP Sc within astrocytes or microglia using confocal microscopy, the relative fluorescence intensities of GFAP (M) and PrP Sc (N) from the same 1 mm optical slice was merged (O) and a the relative intensities of the GFAP and PrP Sc signals were determined along a line through the length of the cell (P). The solid white circle located in the schematic inset is the location of the photographed images within the reticular formation. Scale bar, 10 mm.