5 research outputs found
Western blot analysis showing amplification of protease-resistant CerPrP by serial PMCA.
<p>A: Serial PMCA of 04-22412 CWD using Tg(CerPrP)1536<sup>+/−</sup> brain homogenate. CWD prions in a 10% brain homogenate of diseased mule deer 04-22412 were diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536<sup>+/−</sup> mice. Following a round of PMCA, the sample, containing amplified protease-resistant CerPrP, was diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536<sup>+/−</sup> mice for a further round of PMCA. This process of serial PMCA was repeated for 22 rounds. PK-treated samples from each of the first 10 rounds were analyzed by Western blotting. In the final lane, a sample from Tg brain homogenate without PK treatment was loaded. B: Serial PMCA of RML using Tg(CerPrP)1536<sup>+/−</sup> brain homogenate. Mouse RML prions in a 10% brain homogenate from a diseased wild type FVB mouse were diluted 10-fold into 10% brain homogenate from perfused Tg(CerPrP)1536<sup>+/−</sup> mice. Serial PMCA was repeated for 22 rounds. PK-treated samples from each of the first 7 rounds were analyzed by Western blotting. The unamplified RML seed that produced protease-resistant PrP following PMCA in round 1 was loaded in the first lane, while a sample from Tg brain homogenate without PK treatment was loaded in the final lane. C: Western blot quantification of protease-resistant PrP in inocula used to challenge Tg(CerPrP)1536<sup>+/−</sup> mice. Samples were PK-treated as indicated. Ratios indicate the fold dilution of the original preparation. In the final lane, a sample from CWD brain homogenate without PK treatment was loaded.</p
Regional distribution of CerPrP in the CNS of diseased Tg(CerPrP)1536<sup>+/−</sup> mice infected with mouse RML prions, in vivo-adapted Cer/RML prions, and PMCA-adapted Cer/RML prions.
<p>PK-treated histoblotted coronal sections though, from top to bottom, the region of the septum, hippocampus, anterior midbrain, posterior midbrain, pons, and oblongata from diseased Tg(CerPrP)1536<sup>+/−</sup> mice inoculated with mouse RML prions, Cer/RML-4827, or PMCA Cer/RML prions. Sections were prepared from two different Tg(CerPrP)1536<sup>+/−</sup> mice inoculated with mouse RML prions (#4825 and #5302) and two different Tg(CerPrP)1536<sup>+/−</sup> mice inoculated with PMCA Cer/RML prions (#5294 and #5295). Lateral areas of the posterior midbrain section of mouse #4825 were lost during tissue processing. Histoblots of sections through the anterior and posterior midbrain from an asymptomatic age-matched Tg(CerPrP)1536<sup>+/−</sup> mouse inoculated with PBS, shown to the left, demonstrate the specificity of immunostaining with Hum-P anti-PrP recombinant Fab.</p
Immunohistochemical detection of CerPrP<sup>Sc</sup> and spongiform degeneration in the brains of diseased Tg(CerPrP)1536<sup>+/−</sup> mice.
<p>A, B, and D are sections through the hippocampus of non-diseased or diseased Tg(CerPrP)1536<sup>+/−</sup> mice; section C is from the cerebral cortex. A, absence of spongiform pathology and immunohistochemically-reactive PrP in the hippocampus of an asymptomatic PBS-inoculated Tg(CerPrP)1536<sup>+/−</sup> mouse; B, accumulation of plaques in the hippocampus of diseased Tg(CerPrP)1536<sup>+/−</sup> mouse inoculated with naturally occurring 04-22412 CWD prions; C, accumulation of plaques in the cerebral cortex of a diseased Tg(CerPrP)1536<sup>+/−</sup> mouse inoculated with naturally occurring 04-22412 CWD prions; D, accumulation of plaques in the hippocampus of diseased Tg(CerPrP)1536<sup>+/−</sup> mouse inoculated with PMCA CWD prions; E, high magnification of a large plaque aggregate rimmed by vacuoles; F, absence of spongiform pathology and immunohistochemically reactive PrP in the medulla of asymptomatic PBS-inoculated Tg(CerPrP) 1536<sup>+/−</sup> mouse; G, diffuse PrP accumulation in the medulla of diseased Tg(CerPrP)1536<sup>+/−</sup> mouse #5297 inoculated with mouse RML prions; H, high magnification of a section thought the hippocampus of diseased Tg(CerPrP)1536<sup>+/−</sup> mouse #5300 showing PrP accumulation in small plaques; I, diffuse PrP accumulation in the medulla of a diseased Tg(CerPrP)1536<sup>+/−</sup> mouse inoculated with PMCA Cer/RML prions. Hematoxylin was used as counterstain. Bar = 100 µm in A–D; Bar = 50 µm in E–I.</p
Assessment of the conformational stability of PrP<sup>Sc</sup> in the brains of diseased mice.
<p>In A and C, densitometric analysis of immunoblots shows the percentage of protease-resistant CerPrP<sup>Sc</sup> as a function of GdnHCl concentration. The sigmoidal dose-response was plotted using a four-parameter algorithm and non-linear least-square fit. Each point shown is the mean value derived from densitometric quantification of PK-resistant PrP in three diseased Tg(CerPrP)1536<sup>+/−</sup> mouse brain extracts in each study group. Error bars indicate the standard error of the mean which, in some cases, was smaller than the size of the symbols used to indicate the mean. A, Tg(CerPrP)1536<sup>+/−</sup> mice inoculated with 04-22412 CWD prions (green filled circles), or PMCA-derived CWD prions (green filled squares); C, Tg(CerPrP)1536<sup>+/−</sup> mice inoculated with Cer/RML-4827 prions (blue filled circles), or PMCA Cer/RML prions (blue filled squares). For comparison, the conformational stability of MoPrP<sup>Sc</sup> in the brains of wild type FVB mice infected with RML prions is shown (black diamonds). In B and D, representative immunoblots of protease-resistant PrP following PK treatment are shown. The mean GdnHCl<sub>1/2</sub> value, representing the concentration at which half the PrP<sup>Sc</sup> in each series was denatured, is also shown.</p
Generation and characterization of PMCA-derived prions using Tg(CerPrP)1536<sup>+/−</sup> mice.
<p>Each symbol represents an individual mouse. Closed symbols indicate diseased mice and open symbols indicate asymptomatic mice. Green symbols indicate prions originating from CWD; blue symbols indicate prions originating from RML; circles indicate in vivo-derived prions; squares indicate PMCA-derived prions. The blue circle surrounded by the red square signifies mouse #4827 that was the origin of Cer/RML-4827 prions, while the green and blue squares signify mice #5302 and #4825 respectively, the brains of which were analyzed by histoblotting. Incubation times are expressed as the mean±standard error of the mean; listed in parenthesis is number of diseased mice/ number of mice inoculated.</p