Primary transmission of classical CJD, GSSA117V and vCJD prions to transgenic mice expressing huPrP^117V,129V+/+ (117VV Tg31).

<p>IPD = inherited prion disease; IHC = immunohistochemistry; IB = immunoblotting; ND = not determined; GH = growth hormone; DM = dura mater.</p>*<p>According to classification of Hill <i>et al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat.1003643-Hill4" target="_blank">[53]</a>.</p>†<p>Positive either by clinical signs, western blotting and/or immunohistochemistry; primary antibody was either 3F4 or ICSM 35.</p>‡<p>One PBS-inoculated mouse culled at 582 days post-inoculation due to intercurrent illness, had minimal PrP plaques (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat.1003643.s001" target="_blank">Figure S1A</a>) in the anterior commissure but was negative by IB.</p>§<p>One mouse was positive by the presence of PrP21–30 kDa on immunoblot (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat-1003643-g003" target="_blank">Figure 3B</a>, lane 4) but tissue was unavailable for immunohistochemical analysis.</p>¥<p>Post-inoculation survival period (days): 292, 387, 450, 455, 498, 515 and 547.</p

Immunoblot analysis of abnormal PrP propagated in the brains of 117VV transgenic mice challenged with IPD A117V and classical CJD.

<p>Mice were inoculated with classical CJD and GSS A117V brain. Immunoblots were analysed by enhanced chemiluminescence with monoclonal anti-PrP antibody ICSM 35. The numbers in parentheses above relevant lanes, represent the number of days each mouse survived post-inoculation. The provenance of each brain sample is designated above each lane. (A) Immunoblots of sporadic CJD-inoculated 117VV Tg31 mice (lanes 3–7) showing PrP^Sc resistant to harsh proteinase K (PK) digestion performed with 100 µg/ml PK at 37°C for 1 h (lanes 3, 4, and 7). Positive control was from a transgenic mouse expressing wild type HuPrP-129MV challenged with the same CJD inoculum (lanes 1and 2). An uninoculated 117VV Tg31 mouse brain is shown in lanes 8 and 9. (B) Brain homogenate of a 117VV Tg31 mouse inoculated with iatrogenic CJD prions that died without clinical disease at 804 days post-infection, showing weakly detectable PrP^Sc partially resistant to harsh PK digestion of 100 µg/ml for 1 hour at 37°C (lane 4) compared to the same control as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat-1003643-g003" target="_blank">Figure 3A</a> (lanes 1 and 2). Brain homogenate of a mouse killed relatively early at 294 days post-inoculation, shows no detectable PrP^Sc (lane 3). (C) Immunoblots of brains of five separate 117VV Tg31 mice all inoculated with the same GSS A117V patient brain homogenate showing the presence of PrP^C, PrP^Sc and 8 kDa PrP fragment following harsh PK digestion at 100 µg/ml PK at 37°C for 1 hour (lanes 3–7). Under these conditions PBS-inoculated age-matched control 117VV Tg31 mouse brain shows only residual PrP^C signal on long exposure (lane 8). Brain homogenate of a mouse culled relatively early at 188 days post-inoculation, compared to the group mean survival post-inoculation of >616 days, showed no detectable PrP^Sc (lane 3). One 117VV Tg31 mouse was clinically sick at 673 days post-infection, and its brain sample shows complete digestion of PrP^C and the presence of classical PrP^Sc (lane 7, denoted by *) confirming adequacy of the PK digestion conditions. (D) Immunoblotting was repeated for all samples shown in lanes 4–7 of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat-1003643-g003" target="_blank">Figure 3C</a>. These samples had undergone only one further freeze–thaw cycle before PK digestion. Compared with the readily detectable abnormal PrP signals seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat-1003643-g003" target="_blank">Figure 3C</a>, only one sample (denoted by *) now showed the presence of classical PrP^Sc but at reduced signal strength, and only after using PK at a reduced concentration of 10 µg/ml. In other samples, only an 8 kDa PrP fragment could be variably detected but after using reduced PK concentrations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat.1003643.s002" target="_blank">Figure S2B</a> lanes 3 and 4). (E) Immunoblot showing only the 8 kDa PrP fragment associated with A117V-challenged 117VV Tg30 mouse brains analysed with 50 µg/ml PK at 37°C for 1 hour (lanes 7 and 8), whereas PrP in brain homogenates of PBS-challenged Tg30 mice (lanes 3–6) and uninoculated age-matched Tg30 mouse brain (lane 9) is completely digested under the same conditions. Positive control in lanes 1 and 2 is brain homogenate of a transgenic mouse expressing wild type HuPrP (129MM Tg35) that was challenged with classical CJD.</p

Primary transmission of classical CJD, Inherited Prion Disease A117V and vCJD prions to transgenic mice expressing huPrP^117V,129V+/+ (117VV Tg30).

<p>IPD = inherited prion disease; IHC = immunohistochemistry; IB = immunoblotting; ND = not determined; GH = growth hormone.</p>*<p>According to classification of Hill <i>et al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003643#ppat.1003643-Hill4" target="_blank">[53]</a>.</p>†<p>Positive either by clinical signs, western blotting and/or immunohistochemistry; primary antibody was either 3F4 or ICSM 35.</p>‡<p>Post-inoculation survival period (days): 388, 650, 627, 694,727, 799 and 811.</p

Immunoblot analysis of abnormal PrP propagated in the brains of 117VV Tg30 transgenic mice challenged with vCJD prions.

<p>All mice were inoculated with the same vCJD prion isolate. Immunoblots were analysed by enhanced chemiluminescence with monoclonal anti-PrP antibody ICSM 35. The provenance of the brain samples is designated above the lanes. (A) Lanes 3 and 4 show the predicted type 5 PrP^Sc bands seen when vCJD is propagated in transgenic mice expressing HuPrP with the codon 129VV genotype, compared with the detection of type 4 PrP^Sc in the brain of vCJD-inoculated 129MM Tg45 mouse (lanes 1 and 2). Type 5 shares the glycoform ratio of type 4 but differs in migrating more slowly on western blots because all 3 glycoform fragments of type 5 have higher apparent molecular masses than those of type 4. The lower signal intensity in lane 3 (100 µg/ml PK) compared to lane 4 (50 µg/ml PK) reflects PK-sensitivity of the vCJD-seeded 117V PrP^Sc. The 8 kDa PrP fragments can be seen associated with only PK-digested prion-infected 117V PrP-expressing mouse brain samples. These truncated 8 kDa fragments are absent from either vCJD infected Tg45 mice (lanes 1 and 2) or vCJD-infected 117VV Tg30 brain samples not digested with PK (lane 5).These data confirm that the 8 kDa human PrP fragment is a disease-associated PrP degradation product. (B) Variable PK resistance in brain of 117VV Tg30 mouse inoculated with vCJD. The 8 kDa PrP fragment is only seen in PK digested lanes 1–4, but is absent in the same sample undigested with PK (lane 5).</p

Overview of histological findings in 117VV HuPrP transgenic mice challenged with A117V prion isolates.

<p>The figures are schematic drawings reflecting the overall spatial distribution and intensity pattern of the gliosis or PrP deposition within the experimental groups. They are not meant to indicate precise representations of individual brains. * Definition of values for neuronal loss: NL0: No neuronal loss; NL+: Drop out of single neurones either focally or within the Ammon's horn (AH), leaving the AH continuity intact; NL++: Focal or regional drop out, interrupting the continuity of the AH and creating a small-medium gap (up to 1/3 of the length of the AH); NL+++: Neuronal drop out leaving gaps of more than 1/3 of the AH's length. Ratios represent the proportion of samples with the corresponding neuronal loss score.</p

Neuropathological analysis of transgenic mouse brain.

<p>Panels A, E, I and M, schematics showing regional distribution of abnormal PrP deposits. Note that these panels reflect the overall spatial distribution of neuropathology and are not meant to indicate precise representations of individual brains. Panels B, F, J and N, H&E staining demonstrating spongiform neurodegeneration in the hippocampal areas. Panels C, G, K and O, PrP immunohistochemistry using anti-PrP monoclonal antibody ICSM 35 demonstrates abnormal PrP immunoreactivity. Panels D, H, L and P, GFAP staining demonstrating gliosis in the hippocampal areas. A–D, IPD A117V prions inoculated to 117VV Tg31 mouse; E–H, sporadic CJD prions inoculated to 117VV Tg31 mouse showing distinctive diffuse synaptic PrP deposition characteristic of sCJD prions (panel G); I–L, IPD A117V prions inoculated to 117VV Tg30 mouse. M–P, vCJD prions inoculated to 117VV Tg30 mouse showing abundant non-florid PrP plaques (panel O). Scale bar = 500 µm for all panels except A, E, I and M.</p

Transmission of homogenous GSS-102L prions to wild type FVB mice or transgenic mice expressing PrP 102L or wild type human PrP.

<p>IHC = immunohistochemistry; IB = immunoblotting. Tg35c and Tg152c are congenic on FVB/N genetic background.</p><p>* Three IPD P102L patient brain isolates were passaged in 102LL Tg27 transgenic mice to generate GSS-102L prions. Details of these primary transmission are described in reference [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004953#ppat.1004953.ref033" target="_blank">33</a>].</p><p>^† All samples were analysed by both IB and IHC using monoclonal antibodies ICSM 18 and ICSM 35. In 102LL Tg27 tissues ICSM 35 serves as a control to define non-specific background.</p><p>^‡ Positive either by clinical signs, immunoblotting and/or immunohistochemistry.</p><p>^# All samples were positive for ICSM 18 but negative for ICSM 35.</p><p>^§ All samples were negative for both ICSM 18 and ICSM 35.</p><p>Transmission of homogenous GSS-102L prions to wild type FVB mice or transgenic mice expressing PrP 102L or wild type human PrP.</p

Serial transmission of CJD-102L prions in transgenic mice homozygous for 102L or wild type human PrP.

<p>IHC = immunohistochemistry; IB = immunoblotting; ND = not determined; Tg35c and Tg152c are congenic on FVB/N genetic background; sCJD = sporadic CJD; iCJD = iatrogenic CJD; DM, = dura mater; GH = growth hormone.</p><p>* All inocula were first passaged in 102LL Tg27 transgenic mice to generate CJD-102L prions.</p><p>^† All samples were analysed by both IB and IHC using monoclonal antibodies ICSM 18 and ICSM 35. In 102LL Tg27 tissues ICSM 35 serves as a control to define non-specific background.</p><p>^# Positive either by clinical signs, immunoblotting and/or immunohistochemistry.</p><p>^‡ All samples were positive for ICSM 18 but negative for ICSM 35.</p><p>^§ Only 1 sample was available for analysis by IB.</p><p>Serial transmission of CJD-102L prions in transgenic mice homozygous for 102L or wild type human PrP.</p

Immunohistochemical detection of abnormal PrP deposition in brains of transgenic mice challenged with CJD-102L prions.

<p>All images show abnormal PrP deposition in the thalamus stained with anti-PrP monoclonal antibody ICSM 18. The inoculum and line of transgenic mouse is designated to the left and above the panels respectively. All inocula were from primary transmissions of classical CJD prions from patient brain to 102LL Tg27 mice; sporadic CJD <i>PRNP</i> 129MM with type 1 PrP^<b>Sc</b> (sCJD T1 MM) (A-C), iatrogenic CJD <i>PRNP</i> 129MM with type 2 PrP^<b>Sc</b> (iCJD T2 (DM) MM) (D-F), iatrogenic CJD <i>PRNP</i> 129VV with type 3 PrP^<b>Sc</b> (iCJD (GH) T3 VV) (G-I), iatrogenic CJD <i>PRNP</i> 129MV with type 3 PrP^<b>Sc</b> (iCJD T3 (GH) MV) (J-L) DM, dura mater; GH, growth hormone. Scale bar, 160 μm in all images.</p

Summary of serial passages of IPD P102L prions (A) and classical CJD prions (B) to transgenic mice expressing human PrP 102L or wild type human PrP.

<p>The total number of prion-affected mice (both clinically and sub-clinically infected) is reported for each inoculated group: 102LL Tg27 mice (black), 129MM Tg35c mice (light grey) and 129VV Tg152c mice (dark grey). Animals were scored by clinical signs, immunoblotting for PrP^<b>Sc</b>, and/or PrP immunohistochemistry. The X in panel A denotes the marked transmission barrier that prevents GSS-102L prions from propagating in transgenic mice expressing wild-type human PrP. Primary transmissions of P102L and classical CJD prions have been published previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004953#ppat.1004953.ref033" target="_blank">33</a>].</p