PrPSc of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification

Virus Research 141 (2009) 26–33

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PrPSc of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification Song Shi, Chen-Fang Dong, Gui-Rong Wang, Xin Wang, Run An, Jian-Ming Chen, Bing Shan, Bao-Yun Zhang, Kun Xu, Qi Shi, Chan Tian, Chen Gao, Jun Han, Xiao-Ping Dong ∗ State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Ying-Xin Rd 100, Beijing 100052, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 1 September 2008 Received in revised form 21 December 2008 Accepted 22 December 2008 Available online 20 January 2009 Keywords: Transmissible spongiform encephalopathy Prion Spleen Muscle Protein misfolding cyclic amplification

a b s t r a c t Transmissible spongiform encephalopathies (TSEs), or prion diseases, are transmissible neurodegenerative disorders of protein conformation. This group of diseases is caused by infectious agents, termed prions, which can convert normal conformation (PrPC ) into misfolded protein (PrPSc ). The infectivity of non-neuronal tissues has been wildly addressed, but the propagating features and the biochemical properties of prion generated from these tissues are only partially settled. In this study, utilizing protein misfolding cyclic amplification (PMCA), the in vitro conversion of PrPC into PrPSc in spleen and muscle tissues can be induced by PrPSc produced in vivo. The further propagation of newly formed PrPSc in normal brain and some of the biochemical properties of new PrPSc are similar as the brain-derived prions, implying the naturally infectious pathway of prion from peripheral generation to neuro-invasion. However, compared with the brain-derived PrPSc , the weaker resistance of new PrPSc to some inactivated agents, i.e. sodium hydroxide and thermal inactivation, are observed. Our data provide the reliable evidence that the brain-derived PrPSc can utilize the PrPC from non-neuronal tissues for its propagation. Similarity of the replicative ability in PMCA in vitro and the infectivity in vivo highlights the possibility to use PMCA instead of bioassay to investigate the propagation of prion. © 2009 Published by Elsevier B.V.

1. Introduction Transmissible spongiform encephalopathies (TSEs), or prion diseases, are transmissible neurodegenerative disorders of protein conformation. This group of diseases such as bovine spongiform encephalopathy, sheep and goat scrapie and human Creutzfeldt–Jakob disease are caused by unique infectious agents, termed prion (Prusiner, 1982). Prion is considered to consist mainly of a misfolded scrapie-associated, aggregated proteinase K (PK)resistant isoform of the cellular prion protein (PrPC ) and the pathological isoform presented in the tissues of infected individual is called PrPSc (Prusiner, 1998). The “protein-only” model of the prion hypothesis implies that the infectious process is an autocatalytic mechanism in which PrPC is converted from normal conformation into misfolding state by PrPSc (Deleault et al., 2003; Lucassen et al., 2003; Saborio et al., 2001). Thereafter, not only initial prions, but also new generated PrPSc have the ability of converting on PrPC . For decades, the investigation of propagation of prion has relied on the assay in experimental animals. Although bioassay mimics

∗ Corresponding author. E-mail address: [email protected] (X.-P. Dong). 0168-1702/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.virusres.2008.12.010

maximally the natural process of TSEs pathogenesis, the extremely long incubation period of TSEs limits its usage. Since 1994, a methodology called cell-free conversion has been applied in TSEs studies, mostly using radioactivity-labeled recombinant PrPC protein as the substrate (Kocisko et al., 1994, 1995). However, the low and unstable yields of newly formed PrPSc , as well as unsatisfied sensitivity, make this technique not be widely applied. Recently, a new technique, named protein misfolding cyclic amplification (PMCA) has been described (Saborio et al., 2001) which provides an efficacious, unique and convenient experimental approach to evaluate properties of replication (Bieschke et al., 2004; Weber et al., 2006) and even the infectivity of newly formed PrPSc in vitro (Castilla et al., 2005a,b). By incubating PrPSc taken from infected animals with PrPC from normal homologous brains, large amount of normal PrPC can be converted into PrPSc in short period after cyclic processes of alternative sonication and incubation (Castilla et al., 2005a,b; Saborio et al., 2001). This technology has been considered to be most possible approach to reveal many fundamental mechanisms involving in PrPSc propagation, aggregation and neuro-invasion in vivo (Eiden et al., 2006). TSEs agents can infect susceptible individuals through many pathways. Although the direct infection to central nerve system (CNS) is the most efficient way, the naturally occurred prion diseases are usually acquired through peripheral systems (Bellworthy

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et al., 2005; Race et al., 2000). In some prion diseases, such as scrapie and variant CJD, PrPSc can be detected in some non-neural tissues with a low degree (Archibald, 2004; Bosque et al., 2002; Wadsworth et al., 2001). However, the rarity of PrPSc in peripheral tissues of TSEs individuals lets it difficult to assess whether the tiny PrPSc is the replicating consequence of the original PrPSc using local PrPC , or just a transient trace of brain-derived PrPSc . Although many non-neuronal tissues, such as spleen, tonsil, skeleton muscle and even leukocytes in blood are able to express detectable PrPC (Vostal et al., 2001), and the skeleton muscle is intrinsically capable of propagating prions (Bosque et al., 2002), whether the PrPSc generated in peripheral tissues of wild-type animal shares the similarly biological characteristics with brain-derived PrPSc , such as propagation, biochemical features and resistance to inactivation, are only partially understood. In present study, the replication activity of native PrPSc from the hamsters infected with scrapie strain 263K in non-neural tissues from normal hamsters, i.e. spleen, kidney, small intestine and hindlimb muscle, were evaluated with the established PMCA system. We found that the native PrPSc converted efficiently the spleen- and muscle-derived PrPC into PrPSc in PMCA. Compared with the PrPSc generated from brain in vitro and in vivo, the PMCAgenerated PrPSc from spleen and muscle showed the same abilities to change the conformation of PrPC in brain. We also proposed the data that the PMCA-generated PrPSc possessed the biochemical features like the native PrPSc from scrapie 263K, including glycosylation pattern, detergent insolubility and PK-resistance. The propagating activity of PrPSc in PMCA was totally abolished when exposed to thermal treatment and highly concentrating sodium hydroxide. These results suggest that the PrPSc generated from peripheral tissues can further propagate in brain and thus provide some of the molecular evidences for initial stage of TSEs before neuro-invasion. 2. Materials and methods 2.1. Infection of hamsters with scrapie and preparations of scrapie brain homogenate In previous study (Gao et al., 2004), Syrian hamsters were injected intracerebrally with 20 ␮l of 1% brain homogenate prepared from 263K-infected hamsters in the terminal stage of disease. The animals exposed to the infectious agent developed clinical signs of disease 66.7 ± 1.1 days (mean ± standard error) after inoculation. The brains were removed and the homogenates (10%, w/v) were prepared in PMCA conversion buffer, containing 1× PBS (pH 7.2), 1% Triton X-100, 5 mM EDTA, 150 mM sodium chloride, protease inhibitor cocktail tablets (Roche Applied Science) as described elsewhere (Castilla et al., 2004). Crude homogenates were centrifuged at 5000 × g for 5 min, the supernatant was frozen into −80 ◦ C immediately as the stock seeds for subsequent experiments. 2.2. Preparations of normal tissues homogenates Hamsters were anaesthetized and sacrificed quickly by using cervical dislocation to avoid excessive pain. Whole brain, spleen, hindlimb muscle, kidney and small intestine and were immediately removed surgically. Particularly, the non-neuronal tissues were removed carefully to avoid of contamination of peripheral nerves. After washed thoroughly with 10 volumes of cold 1× PBS containing 50 mM EDTA, tissues were dried by filter paper to remove liquid. 10% (w/v) homogenates of normal tissues were prepared in the PMCA conversion buffer and were further centrifuged at 2000 × g for 10 s. Supernatants were collected for PMCA and Western blotting assays.

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2.3. Generation of scrapie in vitro PMCA was utilized to generate scrapie in this study. This procedure was performed on a water-bath sonicator (Misonix sonicator 3000). Aliquots of scrapie and normal homogenates were mixed in the volume of 100 ␮l and loaded onto 0.2-ml PCR tubes. The tubes were positioned on the plate holder of sonicator and programmed to automated amplification cycles. The detailed protocol of PMCA has been described elsewhere (Castilla et al., 2004; Saá et al., 2006b). Briefly, one cycle consisted of the step of sonication at 60% potency for 40 s and followed by the step of incubation at 37 ◦ C for 30 min. For serial PMCA in this study, 48 cycles was defined as one round. After each round, 10 ␮l of amplified product was added into 90 ␮l fresh normal homogenates and subjected to next round. This procedure was repeated several times to reach an ideal amplification. 2.4. Proteinase K-resistance assay The assay of proteinase K-resistance for brain-derived and PMCA-generated PrPSc was determined by incubation at 37 ◦ C for 60 min with different concentrations of PK (Merck) ranging from 0 to 2000 ␮g/ml. The digestion was stopped by mixture of 2× electrophoresis loading buffer for Western blot. For other assays involving PK digestion, the samples were incubated consistently with 50 ␮g/ml of PK at 37 ◦ C for 60 min. 2.5. Gel electrophoresis and Western blotting assay Samples were separated in SDS-PAGE under reducing conditions and electronically transferred to PVDF membranes (Immobilon-P, Millipore). The primary antibody (dilution 1:3000) was monoclonal antibody 3F4 (Dako). The secondary antibody (dilution 1:10,000) was goat anti-mouse IgG-HRP (Boehringer). Detection of signals was performed with an ECL detection kit (Amersham-Pharmacia Biotech). Densitometric analysis was done by using a Gel-Pro analyzer (Binta 2020D). The relative gray values of PrPSc signals were standardized by division with the gray value of known concentration of recombinant PrP in the same gel. 2.6. Purification of PrPSc The purification of PrPSc followed the short purification process described elsewhere (Polymenidou et al., 2002). Briefly, 500 ␮l of each PMCA products was digested with PK at 37 ◦ C for 1 h. After stopping the reaction by adding 5 mM PMSF (final concentration), 500 ␮l of 1× PBS containing 20% sodium chloride and 1% sarkosyl was added into each preparation, making the final sodium chloride concentration to 10%. The tube was kept on ice for 10 min with gently rocking. Subsequently, the preparations were centrifugated 20,000 × g at 20 ◦ C for 30 min, the pellets were resuspended with Tris–HCl buffer (pH 6.8, containing 0.5% sarkosyl) followed by 20,000 × g of centrifugation at 20 ◦ C for 15 min. The resuspension process was repeated three times. Pellets were analyzed by Western blot as described above. Native PrPSc and new PrPSc from PMCA products of all origins were purified, concentrated and adjusted to similar concentration by densitometric analysis of blots. These purified PrPSc were utilized to investigate properties of deglycosylation pattern, PK-resistance and detergent insolubility in further assays. 2.7. Deglycosylation assay PrPSc generated from in vivo and in vitro of all origins were purified and adjusted to similar concentration as described above. After mixed with equal volume of glycoprotein denaturing buffer (New England Biolabs), preparations of PrPSc were heated at 100 ◦ C for

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Fig. 1. The hamsters’ brain-derived PrPSc of scrapie 263K could propagate in the homogenates of spleen and muscle tissues in PMCA system. (A) 10 ␮l of 10% (w/v) homogenates prepared from various tissues of normal hamsters were treated with (lanes 2, 4, 6, 8 and 10) or without (lanes 1, 3, 5, 7 and 9) PK, respectively. (B) 1 ␮l of 10% (w/v) scrapieinfected brain homogenates were serially diluted into various 10% (w/v) normal tissue homogenates (illustrated as 102 –108 ) in a final volume of 100 ␮l and subjected to 96 cycles of PMCA. Same aliquot of 10% scrapie-infected brain homogenate was directly diluted in PMCA conversion buffer and undergone this procedure simultaneously as the control. (C) Scrapie brain homogenate was mixed with 100-fold various10% homogenate and subjected to 48 cycles of PMCA as the first round. The amplified products were further mixed with 10-fold individual normal homogenate and subjected next round of PMCA. After several rounds, the PMCA products from 109 -fold dilution were mixed with 100-fold individual normal homogenate and subjected to serial PMCA to reach a 1027 -fold dilution of initial scrapie-infected brain homogenate, respectively. The dilutions of the input scrapie-infected brain homogenate were shown on the top. Brain, spleen and muscle on the left side represented the PMCA preparations containing individual normal tissue homogenate as substrates. The lane 10 marked PrPC represented individual tissue homogenate used as a reference for comparison of electrophoretic mobility. (D) Aliquots of each PMCA product of kidney and intestine from the preparation of 102 -fold dilution was mixed with 10-fold individual 10% homogenate and subjected to same procedure of serial PMCA described above to reach a 105 -fold dilution of initial scrapie brain homogenate, respectively. Kidney and intestine on the top represented the each normal tissue homogenate used in PMCA. PrPC on the top was the normal brain homogenate used as a reference for comparison of electrophoretic mobility. The molecular markers were shown on the right.

10 min. Subsequently, 50 mM sodium phosphate, pH 7.5, containing 1% NP-40 and 2 ␮l of N-glycosidase F (1,800,000 units/mg, New England Biolabs) were added into samples and the mixtures were incubated at 37 ◦ C for 2 h. PrP signals in each preparation were detected by Western blot. 2.8. Detergent insolubility assay Equivalent of purified PrPSc from all origins were added into the solutions containing different concentration of urea, including 0, 0.5, 1.0, 1.5 and 2.0 M. After incubated at room temperature for 2 h, each preparation was added 1% sarkosyl and centrifuged at 20,000 × g for 1 h. The distributions of PrPSc in the fractions of supernatants and pellets were monitored by Western blot. 2.9. PrPSc sodium hydroxide resistance assay Equivalent samples of PMCA products were incubated with sodium hydroxide at the final concentrations of 0, 0.5, 1 and 2 M at room temperature for 1 h. In parallel, scrapie-infected brain homogenates were treated with same sodium hydroxide solutions as control. After neutralized with equal molar of hydrochloride acid, each preparation was mixed with 100-fold brain homogenates of normal hamsters and subjected to 48 cycles of PMCA. After treated by PK, the signals of PK-resistant PrP were detected by Western blots.

2.10. PrPSc thermal stability assay PMCA products as well as scrapie-infected hamsters’ brain homogenates were exposed to different temperature conditions, including room temperature, 37 ◦ C, 100 ◦ C, 121 ◦ C (0.11 MPa, prevacuum autoclave) and 134 ◦ C (0.23 MPa, prevacuum autoclave), for 1 h, respectively. Each preparation was spiked into 100-fold brain homogenates of normal hamsters and subjected to 48 cycles of PMCA. The PK-treated samples were monitored by Western blots.

3. Results 3.1. PrPSc in scrapie 236K-infected hamsters’ brains can convert the PrPC in spleen and muscle tissues into PrPSc in PMCA system To evaluate the presences of PrPC in various organs and tissues of normal hamsters, equivalent volumes of 10% (w/v) tissues’ homogenates were employed into Western blots. Clear PrP signals migrating at the position from Mr 25–35 kDa were seen in the homogenates of brain, spleen and hindlimb muscle, but not in that of kidney and small intestine (Fig. 1A). In line with the observations of other studies (Bosque et al., 2002; Bellworthy et al., 2005), assays of limiting dilutions of tissues showed that the relative amounts of PrPC in spleen and muscle were notably lower than that in brain (data not shown). Because the concentration of PrPC in kidney and intestine was too low to be detected by our Western blot system, we

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used brain, spleen and muscle homogenates as substrates for our further studies. Normal homogenates of each origin were regulated by dilution of conversion buffer to reach approximately equivalent content of PrPC . To answer whether PrPSc could propagate in non-neuronal tissues, the normal homogenates from spleen and muscle were mixed with a serial diluted scrapie brain homogenates, the dilutions of scrapie brain homogenate were from 102 -fold to 108 -fold. The mixtures were subjected to PMCA process for 96 cycles. In parallel, same serial diluted scrapie brain homogenates mixed with normal brain homogenates or simply in conversion buffer were undergone the same procedure as the positive and negative controls, respectively. PK-digested Western blots identified clear PrPSc signals in the preparations of less than 106 -fold dilution of 10% brain homogenates, whereas in that of less than 104 -fold dilution of 10% spleen and muscle homogenates (Fig. 1B). In line with the observations of PrPC presences, PrPSc signals were only detected in the preparations of less than 102 -fold dilution of 10% kidney and intestine homogenates, which showed the similar patterns as the preparation of directly loaded PK-digested PrPSc (data not shown). These results strongly suggest that PrPSc in brain tissues can convert the PrPC of the peripheral tissues into PrPSc with PMCA. Due to the low concentration of PrPC , the PrPSc propagation could not be detected in the kidney and intestine. 3.2. The newly formed three kinds of PrPSc are able to propagate in vitro by PMCA In order to address the possible difference in replicating capacity of the newly generated PrPSc , scrapie brain homogenates were diluted 102 -fold into individual 10% (w/v) normal homogenates and subjected into one round PMCA (48 cycles). Consecutively, 10 ␮l of these products were employed and spiked into 90 ␮l of individually fresh homogenates for next round PMCA. This procedure was repeated several times and the amount of scrapie brain homogenates was evaluated to a 109 -fold dilution. The PrPSc of each sample could be detected by Western blots after PK digestion (data not shown). Then the PMCA products of each tissue were further amplified by 102 -fold dilution in individually normal homogenates in next round of PMCA. After several rounds, the final concentration of original PrPSc was calculated as 1027 -fold dilution. Clear PrPSc signals were identified in all preparations (Fig. 1C). It implies that the new PrPSc generated from hamsters’ normal brain, spleen and muscle by PMCA can utilize their own PrPC for replication. In parallel, 102 -fold diluted scrapie brain homogenates were mixed with 10% kidney or intestine homogenates and subjected to the serial PMCA by 10-fold dilution to reach 105 -fold dilution of scrapie brain homogenate. As expected, PrPSc signals were undetectable in the reactions containing less than 103 diluted scrapie brain homogenates (Fig. 1D). It also confirmed that the kidney and intestine could not be used as the substrates to reveal the propagation of prion in PMCA system. To see whether the PMCA-derived PrPSc from spleen and muscle homogenates could further replicate in brain tissues, the PrPSc from all origins (263K, in vitro-generated PrPSc from brain, spleen and muscle) were equilibrated (Fig. 2, lane 9 in all gels) with a recombinant hamster PrP in Western blot (Zhang et al., 2002). These preparations were mixed with 10% (w/v) normal brain homogenates in twofold serial dilutions from 1:100 to 1:6400, respectively. Meanwhile, normal brain homogenate (Fig. 2, lane 8 in first and second gels) and normal brain homogenate containing 1/100 (v/v) spleen or muscle homogenate respectively (Fig. 2, lane 8 in third and fourth gels) were undergone directly with the same procedure as negative controls. After 48 cycles of PMCA, PrPSc signals of all groups were detected until 3200-fold dilution with similarly decreasing tendency (Fig. 2, lane 6). No positive signal was detected

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Fig. 2. Spleen and muscle derived PMCA-PrPSc could replicate themselves with the normal brain homogenates in subsequent PMCA. Equivalent PrPSc (marked as PrPSc on the top) of all origins were mixed with 10% normal brain homogenates in twofold (v/v) serial dilutions from 1:100 to 1:6400. Normal brain homogenate and normal brain homogenate containing 1/100 (v/v) individual non-neural tissue homogenate were undergone directly with the same procedure as controls (marked as −PrPSc ). All amplified preparations were subjected to Western bolts after PK treatment. Brian, spleen and muscle on the left side represented the PMCA preparations containing individual input PMCA products as seeds. The molecular markers were shown on the right.

in the negative controls. This result highlights the propagation ability of PMCA-generated PrPSc from brain spleen and muscle is similar with native PrPSc . 3.3. The PMCA-generated PrPSc bears the biochemical features like the native PrPSc in scrapie-infected hamsters To test the similarities of PMCA-derived PrPSc with native PrPSc , the glycosylation patterns, detergent insolubility and PK-resistance of the 1027 -fold PMCA products were analyzed. Western blot showed that PMCA-PrPSc from brain, spleen and muscle shared the same electrophoresis patterns as the native PrPSc in scrapie brains. Treatments of PK-digested PMCA products with glycosidase produced one single PrP-specific band as in the preparation of native PrPSc , which were at the exactly same position in SDS-PAGE as the brain-derived PrPSc (Fig. 3A). It implies that the PMCA-PrPSc have the same glycosylated profiles as the native one. To compare the features of detergent insolubility of the PMCAPrPSc with brain-derived PrPSc , all preparations were purified and equilibrated (see Section 2). After resuspended in various concentration of urea containing 1% sarkosyl, each reaction was centrifuged at 20,000 × g and the distribution of PrPSc in the fraction of pellet was evaluated by Western blots (Fig. 3B). PrPSc signals were detected in all preparations at the condition of 0.5 M urea, and dropped down notably at the condition of 1.0 M. At the condition of 1.5 M urea, all PMCA-PrPSc were undetectable, whereas the native-PrPSc was observable though the amount was greatly reduced. PrPSc signal vanished in all preparations when the buffer containing urea higher than 2.0 M. These results indicate that the PMCA-formed PrPSc from brain and native PrPSc share similar solubility feature in urea, whereas PMCA-PrPSc from spleen and muscle are more soluble. To test the PK-resistant activity of PMCA-PrPSc , equivalently purified PrPSc of all origins were treated with PK from 50 to

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Fig. 3. The electrophoresis pattern, deglycosylation, solubility in urea and PK-resistance of all origins of PrPSc . (A) PMCA-generated PrPSc showed the similar electrophoresis patterns and deglycosylation properties as the native PrPSc . Equivalent of purified PrPSc of all origins (illustrated as 263K, brain, spleen and muscle) were employed in PK digestion and/or deglycosylation. PrP specific signals in each preparation were analyzed by Western blot. PNGase: peptide N-glycosidase F. The molecular markers were shown on the right. (B) Equivalent of purified PrPSc of all origins (shown on the left) were incubated with different concentration of urea from 0 to 2 M (indicated as 0, 0.5, 1, 1.5 and 2 on the top) for 2 h. Then preparations were centrifuged and PrPSc signals in pellets were detected by Western blots. (C) Equivalent of purified PrPSc were digested by different concentrations of proteinase K at 37 ◦ C for 1 h (indicated as 50–2000 ␮g/ml on the top). 2 ␮l of each sample without PK-treatment was directly loaded onto the lane 1 as the internal control (marked as zero on the top). The molecular markers were shown on the right.

Fig. 4. The influences of thermal treatments on the replicative capacities of PMCA-PrPSc . (A) Western blot analyses of the replicative capacities of the PMCA-PrPSc in PMCA after exposing to various thermal treatments. Three kinds of PMCA products (marked as brain, spleen and muscle) at the preparation of 1027 , as well as scrapie 263K-infected hamsters’ brain homogenates (marked as 263K) were exposed to different conditions, including 37, 100, 121 and 134 ◦ C for 1 h. Equivalent aliquot of each preparation was spiked into 100-fold (v/v) brain homogenates of normal hamsters and subjected to 48 cycles PMCA. The replicative capacities in PMCA were evaluated by Western blots after PK digestion. 263K, brain, spleen and muscle at the bottom represented the PMCA preparations containing individual input PrPSc as seeds. Different thermal conditions were illustrated on the top. The molecular markers were shown on the right. (B) The gray value of PrPSc signal was measured by a densitometry. The relative gray value of each preparation was standardized by division with the gray value of 250 ng of recombinant PrP. Each reaction was triplicate and the gray values were presented with as mean ± S.D.

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Fig. 5. The influences of sodium hydroxide on the replicative capacities of PMCA-generated PrPSc . (A) Equivalent samples of PMCA products (marked as brain, spleen and muscle) at the preparation of 1027 and native PrPSc (marked as 263K) were incubated with sodium hydroxide at the final concentrations of 0.5, 1, 1.5 and 2 M at room temperature for 1 h. After neutralized with equal molar of hydrochloride acid, each preparation was mixed with 100-fold (v/v) brain homogenates of normal hamsters and subjected to one round PMCA. The replicative capacity of each preparation in PMCA was detected by Western blots after PK digestion. Different sodium hydroxide concentrations were illustrated at the top. The molecular markers were shown on the right. (B) The gray value of PrPSc signal was measured by a densitometry. The relative gray value of each preparation was standardized by division with the gray value of 250 ng of recombinant PrP. Each reaction was triplicate and the gray values were presented with as mean ± S.D.

2000 ␮g/ml (Fig. 3C). Similar as the observation in the preparations of native PrPSc , PMCA-generated PrPSc signals were detectable in all preparations and the intensities of signals were fairly stable. These results indicate that the PMCA-generated PrPSc have similar PK-resistant feature as brain-derived PrPSc .

3.4. High temperature and high concentrating sodium hydroxide can inactivate the propagating activity of PrPSc in PMCA To estimate whether the PMCA-generated PrPSc had similar resistant property against thermal treatment as native PrPSc , equivalent PrPSc from scrapie brain homogenate and PMCA product (see Fig. 2, lane 9) was exposed to various temperatures for 1 h. Heattreated preparations were 100-fold diluted and employed in 48 cycles of PMCA using normal brain homogenate as the substrates (Fig. 4). All three PMCA-derived PrPSc showed the comparable replicating activities in PMCA as native PrPSc after maintained at 37 ◦ C. The replicating capacities of the PMCA-PrPSc from spleen and muscle declined after maintained at 100 ◦ C and lost their replicating capacities after exposed 121 ◦ C, while the native PrPSc and PMCAPrPSc from brain still partially maintained its propagation in PMCA. Both PMCA-derived PrPSc and native PrPSc failed to amplify when exposed to 134 ◦ C, which corresponded well with many observations for inactivation of prion propagation in study described elsewhere (Castilla et al., 2005a). To address the sodium hydroxide inactivation on all origins in PMCA, equivalent amount of three PMCA-PrPSc and native PrPSc (see Fig. 2, lane 9) were incubated with different amounts of sodium hydroxide at room temperature for 1 h. Thereafter, various preparations were 100-fold diluted and subjected to 48 cycles of PMCA using normal hamsters’ brain homogenates as substrates (Fig. 5).

Compared with the respective reactions of native PrPSc after treatment with 0.5 M, the amplification activities of the PMCA-PrPSc of all origins were similar. After treated with 1 M sodium hydroxide, PMCA-PrPSc of brain and native PrPSc maintained the activities to produce further PrPSc in PMCA, while other two were obviously lower. In the preparations after treatment with 1.5 M NaOH, native PrPSc from scrapie brain and PMCA-PrPSc of brain still produced, low amount PrPSc in PMCA, but PMCA-PrPSc of spleen and muscle lost their replicating capacities. As expected, all PrPSc lost their propagating capacity in PMCA after incubation 2 M sodium hydroxide.

4. Discussion The propagation and accumulation of PrPSc in CNS are main hallmark during TSEs pathogenesis. As a unique pathogen without detectable nucleic acid, the mechanism of prion propagation seems to be strictly dependent on the presence of PrPC as the substrates. In fact, the propagation can be considered as the conversion process from PrPC to PrPSc . Except for the traditional bioassays, some other techniques, e.g. TSEs cell-free conversion systems (Kocisko et al., 1994; Kirby et al., 2003; Safar et al., 1998; Zhang et al., 2002) and cell culture models (Bosque and Prusiner, 2000; Béranger et al., 2001; Lehmann and Harris, 1996; Nishida et al., 2000) have been developed for replication of prion. However, relatively low yields of newly generated prion and time-consume limit their usages for further studies. PMCA as a novel in vitro approach provides an efficient and stirring technology for exploring the mysterious prion propagation. Furthermore, The extremely high analytical sensitivity of the PMCA provides also allow to develop pre-clinical live animal tests for BSE, scrapie or CJD infections in cattle, sheep and humans,

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which are based on blood or cerebrospinal fluid (Eiden et al., 2006; Saá et al., 2006a). It is widely accepted that the direct infection to CNS is the most efficient way, but the naturally occurred prion diseases, such as BSE in cattle and scrapie in sheep and goat, are mostly through peripheral ways (Race et al., 2000). Although, low amounts of PrPSc can be detected in other tissues except CNS, such as tonsil, spleen, rectum and muscle, etc. during the pathogenesis of various TSEs (Archibald, 2004; Bosque et al., 2002; Bellworthy et al., 2005; Horiuchi et al., 1995; Wadsworth et al., 2001), it is still uncertain that the prions observed in these non-neuronal tissues are the exotic one temporally accumulating in the local areas, or the endogenous one generated from local PrPC by PrPSc . In addition, it is believed that prion replication also requires the assistance of some other molecules besides PrPC , for example, polyanions (Deleault et al., 2003, 2007), which also widely exist in many other tissues. Our data supply the reliable evidences that the brain-derived PrPSc can successfully utilize the peripheral tissue-derived PrPC for its replication in PMCA. It indicates that the infectious TSEs agents may propagate and enrich themselves in peripheral tissues before reaching and orienting in CNS tissues. Moreover, it is confirmed that the newly formed PrPSc can further convert efficiently not only the PrPC from the individual tissues, but also that from CNS tissue. This proposes the molecular basis that the peripheral PrPSc generated during TSEs pathogenesis would sufficiently propagate in CNS tissues if they reach at brains. In line with other studies (Castilla et al., 2005a,b; Saborio et al., 2001), the PMCA-generated PrPSc in this study can propagate stably and infinitely in the following serial PMCA. The yields and the mainly biochemical features of PrPSc maintain almost unchanged after consecutive amplifications. In other words, PrPSc can replicate itself persistently in vitro if supplying with enough suitable substrates. This phenomenon corresponds well with the multiplicative characteristics of known microorganisms in vitro. Three new “prions” propagated from brain, spleen and muscle tissues share some alike replicative and biochemical features in PMCA, which are quite similar as the virus replication in various cell cultures. Although the infectivity of the PMCA-PrPSc still needs more precise investigation, fast and high yields of PrPSc through PMCA provide a reliable tool for prion propagation in vitro. Interestingly, corresponding well with our previous study (Yao et al., 2005), the physical and chemical factors (treated with 2 M sodium hydroxide or heated at 134 ◦ C for 1 h) that inactivate the infectivity of scrapie agents 263K in bioassays can also abolish the propagating activity of PMCA-PrPSc . Treatment of scrapie 263K with less strict conditions, i.e. incubated with 1 M sodium hydroxide and heated at 121 ◦ C for 1 h only partially reduces its replicative activity in PMCA. Our data highlights a good coincidence between the replicative activity in PMCA and infectivity in animal tests. Whether the conversion capacity of PrPC into PrPSc by native prion in PMCA can substitute the evaluation of infectivity of TSEs agent in bioassay needs further investigation. Nevertheless, PMCA technique may provide a hopeful time-save and economic methodology for estimating the inactivation on prion. Compared with the native PrPSc , all PMCA-generated PrPSc seem to be more sensitive to the inactive factors, whereas their PK resistances do not reveal any difference. Although the exact reason for this difference is not clear, one may argue that there will be slight difference in conformational conversion between short-time formed PMCA-PrPSc in vitro and long-time deposited PrPSc in vivo. Moreover, spleenand muscle-derived PrPSc from PMCA are slightly, but repeatedly, more vulnerable to the treatments of the physical and chemical agents than their brain-derived analog. The slight difference in several biochemical and biophysical properties among them might be related with the unknown intrinsic environments in various tissues. Whether these differences influence the poten-

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