Fig 1.
PrPres electrophoretic pattern in brains and spleens of high expresser tg338 mice inoculated IC with LAN, CH1641-like isolates, and tg338-passaged LAN prions.
(A) PrPres profiles after challenge with representative LAN and CH1641-like isolates. The 21K-pattern observed after inoculation with PG127 sheep scrapie isolate is shown as control. The PrPres profile in the sheep brain is shown for comparison on the left side of the western blot. Note here and in the subsequent figures that 21K-spleen PrPres migrates slightly faster than 21K-brain PrPres, as observed previously. Typically, spleen PrPres migration is 0.5–1 kDa faster than brain [10, 21, 28, 49, 51, 75]. (B) PrPres profiles after challenge with tg338-passaged LAN (6th passage; LAN → tg338), cloned LA19K and LA21K fast prions. MM: molecular mass markers. (C) Ratio of diglycosylated versus monoglycosylated PrPres in the spleen of mice after challenge with tg338-passaged LAN (gray symbol), LA21K fast (green symbol) and PG127 (orange symbol) prions (n = 5 spleens analyzed at the 6th passage, data plotted as mean ± SEM).
Table 1.
PrPres electrophoretic pattern in the brain and spleen after intracerebral inoculation of sheep scrapie isolates to tg338 mice.
Fig 2.
Strain phenotype of prions replicating in tg338 mouse spleens on LAN serial passage.
(A) Summary of the transmissions (IC route) of brain or spleen extracts from tg338-passaged LAN (3rd passage, LAN → tg338) to high expresser (tg338) or low expresser (tg143+/-) mice. Transmission with brain or spleen extracts are indicated with black and red lines, respectively. The number of affected/inoculated mice and the mean survival times in days ± SEM are indicated for each inoculated group. Segmented, doubled circles are used to indicate the proportion of mice with dominant 19K PrPres (blue) or 21K PrPres signature (grey), either in the brain (inside of the circle, black lines) or in the spleen (outside of the circle, red lines). Data in italic are from [29]. Tg143+/- mice do not express PrPC in the spleen, hence the absence of representation. (B) Representative immunoblot of PrPres profiles in brain and spleen from high and low expresser mice, depending on whether tg338-passaged brain (Br) or spleen (Sp) material was used for inoculation. The immunoblots were revealed with Sha31 and 12B2 anti-PrP antibodies, as indicated. MM: molecular mass markers. (C) Representative histoblots at the level of the striatum (left panel) and the brain stem (right panel) showing the distribution of PrPres deposits in the brains of high expresser tg338 mice inoculated with brain or spleen extracts. Scale bar, 1 mm.
Fig 3.
Time course analysis of PrPres accumulation in the brain and spleen of tg338 mice on LAN serial passage.
PrPres profiles obtained after challenge with tg338-passaged LAN (5th passage; LAN → tg338). Three mice were analyzed at each time point. The immunoblots were revealed with Sha31 and 12B2 anti-PrP antibodies, as indicated. Cloned LA19K and LA21K fast PrPres were used as 19K and 21K controls. The amount of mg tissue equivalent loaded in each lane is indicated. At each time point, PrPres quantification was performed on mouse triplicates. Results are expressed as a percentage (± SEM) of the amount found in the ad hoc tissue at the terminal stage of disease.
Fig 4.
PrPres electrophoretic pattern in brains and spleens of high expresser tg338 mice inoculated IP with LAN, CH1641-like isolates, and tg338-passaged LAN prions.
(A) Mean ± SEM incubation durations (ID) following challenge of tg338 mice by IC route (green bar) and IP route (red bar) with PG127, LAN, CH1641-like sheep scrapie isolates, tg338-passaged LAN (LAN → tg338, 4th and 6th passage (4p, 6p)) and cloned LA19K prions (Cloned). The IDIP / IDIC ratio are indicated. The grey vertical bar indicates the time at which PrPres was first detected in the spleen (S2 Table). Segmented, doubled circles are used to indicate the proportion of mice with 19K PrPres signature (blue), 21K PrPres signature (grey) or absence of PrPres (white), either in the brain (inside of the circle, black lines) or in the spleen (outside of the circle, red lines). (B) Representative immunoblot of PrPres profiles in brains and spleens from tg338 mice inoculated IP with isolates from the LAN and CH1641-like groups, at the terminal stage of disease. The spleen and the brain from the same animal are presented, meaning that the brain can harbor a 21K or 19K PrPres signature whereas a unique 21K signature is detected in the spleen. The spleen PrPres profiles are also shown at early time points post-inoculation (from 50 to 200 days depending on the isolate, S2 Table). Patterns after PG127 infection are shown for comparison. (C) Representative immunoblot of PrPres profiles in brain and spleen from tg338 mice inoculated IP with tg338-passaged LAN (LAN → tg338, 4th and 6th passage (4p, 6p)) and cloned LA19K prions (cl), at the terminal stage of disease. The spleen and the brain from the same animal are presented, meaning that the brain can harbor a 21K or 19K PrPres signature (with the 6th pass, a double 21/19K signature was observed), whereas a unique 21K signature is detected in the spleen. The spleen PrPres profiles are also shown at early time points post-inoculation (from 20 to 50 days depending on the number of passages, S2 Table). Patterns after PG127 infection are shown for comparison.
Fig 5.
Strain phenotype of prions replicating in tg338 mouse spleen and brain on primary passage of LAN404 spleen.
(A) Summary of the transmissions by IC or IP route of brain or spleen extracts from LAN404 sheep scrapie isolate to tg338 mice. Legends are as in Fig 2 and Fig 4. Data in italic are from [29]. (B) Representative immunoblot of PrPres electrophoretic pattern in (Br) and spleen (Sp) from high expresser tg338 mice, upon inoculation with LAN404 spleen material by IP or IC route. PrPres electrophoretic patterns of PG127 and cloned LA19K in tg338 mouse brain are shown for comparison. The immunoblots were revealed with Sha31 and 12B2 anti-PrP antibodies, as indicated. MM: molecular mass markers.
Fig 6.
PrPC-level dependent selection of prions with differing splenotropism in ovine PrP transgenic mice.
(A) Summary of the transmissions (IC route) of brain extracts from tg338-passaged MM2-sCJD prions (4th passage), cloned T1Ov or T2Ov prions on low expresser tg335+/- mice, before back passage to tg338 mice. The mean survival time in days ± SEM and the number of affected/inoculated mice (mice with TSE and positive for brain PrPres by immunoblot) are indicated for each inoculated group. Segmented, doubled circles are used to indicate the proportion of mice with T2Ov (brown or clear brown depending on the accumulation levels) or T1Ov (yellow or clear yellow) PrPres signatures in their brains (inside of the circle, black lines) and spleens (outside of the circle, red lines). Data in italic are from [28]. (B) Representative immunoblot of PrPres profiles in brain and spleen from tg338 high expresser mice and tg335+/- low expresser mice infected with tg338-passaged MM2-sCJD (MM2 → tg338), cloned T1Ov or cloned T2Ov prions. The gels on the right side are portions of the gels (colored red and green lines) that are purposely overexposed to detect T2Ov PrPres in the spleen. (C) Representative immunoblot of PrPres profiles in brain and spleen from tg338 high expresser mice after one or two back-passage from tg335+/- low expresser mice infected with tg338-passaged MM2-sCJD (MM2 → tg338), cloned T1Ov or cloned T2Ov prions. PrPres signatures from T1Ov and T2Ov prions in tg338 mice are shown for comparison. (D) PrPres deposition pattern in the brain of tg335+/- low expresser mice infected with tg338-passaged MM2-sCJD, cloned T1Ov or cloned T2Ov prions. Representative histoblots of antero-posterior coronal brain sections at the level of the septum (i), hippocampus (ii), midbrain (iii) and brainstem (iv). Scale bar, 1 mm.
Fig 7.
Resistance of LA19K prions to proteolysis.
(A) Comparative PK digestion assay performed on brain material of tg338 mice inoculated with tg338-passaged PG127 prions (PG127 → tg338) or cloned LA19K prions. The homogenates were treated with increasing concentrations of PK, as indicated, and analyzed by western blotting. PrPSc quantification was performed on n = 5 independent experiments. Results are expressed as total PrPSc levels (arbitrary units (a.u.) ± SEM). (B) Representative immunoblot showing PrPres degradation in tg338 peritoneal macrophages exposed to tg338-passaged PG127 (PG127 → tg338) prions or cloned LA19K prions for the indicated period (duplicates analysis). PrPres quantification is expressed as total PrPres levels (arbitrary units (a.u.) ± SE).