Figure 1.
The PrPC expression pattern in different brain regions of Tga20 mice differs from that in C57BL/6 mice.
PrPC expression in six brain regions was monitored using Alexa 555-labeled D18 anti-PrP antibody in six brain regions: A, cortex; B, cerebellum; C, brain stem; D, striatum; E, thalamus. All photos except the ones labeled “C57+” were taken at the same settings. “C57+” are photos of the same area as “C57”, but taken at longer exposures. F,G: PrPC expression in Purkinje cells. PrPC and calbindin in the cerebellum were immuno-labeled using Alexa 555-tagged D18 anti-PrP antibody (red) and Alexa 488-tagged anti-calbindin rabbit polyclonal antibody (green), respectively. F: C57BL/6 mouse cerebellum, G: Tga20 mouse cerebellum. PrPC is stained red, calbindin green. The overlay on the right shows PrPC-expressing Purkinje cells in yellow. M, molecular layer; G, granular layer of the cerebellum; P, Purkinje cell layer. Bars represent 20 µm.
Figure 2.
Biochemical analyses of PrPC and PrPSc from Tga20 and C57BL/6 brains.
All samples were analyzed by western blotting and the quantification of the blots is summarized in Table 2. A. PK digestion of PrPC. A1: Uninfected Tga20 brain homogenates were diluted 1∶8 into PrPo/o brain homogenate to adjust PrPC to the same levels as in C57BL/6 homogenates. Serial 1∶2 dilutions of the two preparations, compared by western blot analysis, showed indistinguishable PrPC levels. A2: C57BL/6 and 1∶8 diluted Tga20 brain homogenates were digested for the times indicated with 40 µg PK/ml at 56°C. A3: The same samples as in (A2) were digested with PK at the concentrations indicated at 37°C for 60 min. B: Thermolysin digestion of brain homogenates from C57BL/6 or Tga20 mice infected with 22L, RML or ME7. Homogenates were treated with 25 µg TL/ml for one hour at 70°C and serial dilutions analyzed. C: Proteinase K digestion of brain homogenates from C57BL/6 or Tga20 mice infected with 22L, RML or ME7. Homogenates were treated with 25 µg PK/ml for one hour at 37°C and serial dilutions were analyzed. D: Proteinase K digestion of 22L-, RML- or ME7-derived PrP27-30. PrP27-30 was prepared as described in the Methods section and digested with the PK concentrations indicated for 60 min at 37°C.
Table 1.
Expression levels of PrPC in different brain regions of Tga20 relative to C57BL/6 mice.
Table 2.
PrP levels in prion-infected brains of C57BL/6 and Tga20, and specific infectivities of PK-resistant PrP.
Figure 3.
The Cell Panel Assay of four prion strains propagated in Tga20 or wild-type mice results in similar relative Response Indices on four cell lines.
CAD5 (red), PK1 (blue), R33 (green) and LD9 (violet) cells were exposed to 1∶3 serial dilutions of 0.1% brain homogenates infected with 22L (a,b), RML (c,d), ME7 (e,f) or 301C (g,h) propagated in Tga20 mice (a,c,e,g) or wild-type mice (b, f, h, C57BL/6; d, CD1). The number of PrPSc-positive cells (“spots”) is plotted against log[dilution] of the brain homogenate. The Response Index (RI1.5%/3) of a cell line for a prion strain (vertical dotted line) is the concentration (reciprocal of the homogenate dilution) that yields 300 spots per 20'000 cells (1.5% PrPSc-positive cells) after the 3rd split (horizontal dotted line). The “RI ratio” of a strain is the RI on a cell line relative to that on LD9; it was very similar for each strain regardless of whether it was propagated in wild-type or Tga20 mice (see Table S1 for RI values).
Table 3.
Characteristic parameters of various prion strains in C57BL/6 and Tga20 mice.
Figure 4.
PrPSc deposition pattern in C57BL/6 and Tga20 mice infected with three scrapie prion strains.
Although PrPC levels are higher in all brain regions of Tga20 as compared to C57BL/6 mice, PrPSc levels were lower in Tga20 mice except for the brainstem and thalamus (see also Fig. S1). PrPSc was detected by IHC using anti-PrP mouse monoclonal antibody Bar 233 (SpiBio) and secondary anti-mouse IgG antibody, as described in the methods section. Bar 233 stains PrPSc deposits with various morphologies in the brains of C57BL/6 and Tga20 mice infected with ME7, 22L and RML scrapie strains. Only faint diffuse cytoplasmic and neuropil staining was seen in uninfected mouse brains (Fig. S1). The secondary antibody alone stains only plasma in the blood vessels, as can be seen in large vessels in the last pictures on the right of A, D and E. A, striatum; B, brain stem; C, cortex; D, cerebellum; E, hippocampus. Table 4 summarizes the findings. Bars represent 50 µm. F: PET-BLOTS of horizontal brain sections from Tga20 and C57BL/6 mice infected with the 22L strain and the corresponding uninfected control mice. Arrows show the cortexes, hippocampi and cerebella.
Table 4.
Strain-specific diagnostic features in Tga20 mice.
Figure 5.
PrPSc deposition does not correlate with PrPC overexpression in Tga20 mice and is strain specific.
Schematic drawing of selected brain regions showing the intensity of PrPC overexpression (top row), PrPSc deposition for three different scrapie strains (middle row) and the merged view (bottom row). PrPC is shown in blue, PrPSc in red. This scheme summarizes the data described in Fig. 1, Table 1 (PrPC), Fig. 4, Fig. S1, Tables 4 and 5 (PrPSc).
Table 5.
Comparative neuropathology in Tga20 and C57BL/6 mice infected with three prion strains.