Article Figures & Data
Figures
- Figure 1.
Generation and identification of MBP-PrP transgenic mice. A, Schematic drawing of the MBP-PrP transgene used for generation of transgenic mice. Arrows and parenthetical numbers indicate the respective restriction sites. The second intron of rabbit β-globin was placed between the cap site and MBP cDNA to improve transgene expression. mMBP, Mouse MBP; mPrnp, mouse Prnp; SV40, simian virus 40. B, Genomic Southern blot analysis of MBP-PrP transgenic mice. EcoRI exactly excises the PrP ORF, confirming the presence of the transgene. (All tested mice showed the same banding pattern.) The BglII and the BamHI-ClaI digestions verify the integrity of the 5′ and 3′ ends, respectively. C, Northern blot analysis of brain RNA. The numbers above each lane indicate the various transgenic lines analyzed. RNA from wild-type (Prnp +/+) and Prnp o/o brains was used as a control. D, PrP C expression in brains and sciatic nerves of hemizygous (hem) or homozygous (hom) tg640 transgenic mice. Expression levels were estimated by comparison with serial twofold dilutions of wild-type brain homogenate. E, Brain PrP C quantification by chemiluminescence analysis. The abscissa displays the amount of protein in micrograms; the ordinate shows chemiluminescence intensity (arbitrary units). F, Anatomical dissection of the CNS in the cerebellum, brainstem, spinal cord, olfactory bulb, and gray or white matter (cortex, subcortex) revealed strongest expression of PrP C in white-matter or white-matter-rich regions of the CNS. MBP-PrP mice display a dominant unglycosylated PrP C band, whereas wild-type mice show a dominant diglycosylated band. To facilitate sample comparisons, only 25% of total protein was loaded in wild-type samples, resulting in PrP C bands of similar intensity. The uppermost bands represent actin. WT, Wild type.
- Figure 2.
Localization of PrP mRNA in MBP-PrP transgenic mice. In situ hybridization of forebrain sections (A-I) with corpus callosum and of sciatic nerve sections (J-L) of adult Prnp +/+, Prnp o/o, and MBP-PrP transgenic mice is shown. Expression of PrP mRNA occurs in neurons of wild-type (Prnp +/+) forebrains (A) and in Prnp +/+ Schwann cells (J), whereas Prnp o/o brains (B) and nerves (K) lack any signal. In contrast, strong PrP signals are visible in callosal oligodendrocytes (C) and sciatic Schwann cells (I-L) of transgenic mice. D-F, Sense probes did not reveal any signals in brains and sciatic nerves. G-I, The oligodendrocyte-specific PLP antisense probe revealed strong mRNA signals in the forebrains of all genotypes investigated. Scale bars, 50 μm.
- Figure 3.
Localization of PrP C in MBP-PrP mice. A-C, Cellular distribution of PrP C with in the white matter of adult mice revealed by immunoelectron microscopy of gold-labeled anti-PrP antibody. Arrows indicate the location of the black gold particles. MBP-PrP mice display abundant decoration of myelin with gold particles. Scale bar, 5 nm. D-F, Immunofluorescence of primary optic nerve oligodendrocytes cultured from 7- to 8-d-old mice. Top row, expression of PrP is detectable only in transgenic oligodendrocytes (F), whereas the early oligodendrocyte marker MAG is expressed on all oligodendrocytes (bottom row). Scale bar, 5 μm.
- Figure 4.
Absence of conversion of PrP C into PrP Sc after scrapie challenge of MBP-PrP mice. A, Western blots of homogenized brain material electrophoresed natively (-) or after digestion with proteinase K (+). Large amounts of PK-resistant prion protein (PrP Sc) are detectable in brains of wild-type mice (Prnp +/+) that developed terminal scrapie 167 d postinoculation. No PrP Sc is visible in Prnp o/o or MBP-PrP mice that had been challenged intracerebrally with a high dose of the RML strain. Molecular weight markers are indicated on the left. B, Histoblot analysis of PrP Sc expression. Protease-resistant PrP was detectable only in intracerebrally infected Prnp +/+ mice. MBP-PrP and Prnp o/o mice did not accumulate PrP Sc. As reported previously (Brandner et al., 1996b), myelinated structures show faint homogeneous background staining in inoculated Prnp o/o mice.
- Figure 5.
Oligodendrocytic PrP C does not support intraneural prion transport. Embryonal PrP-overexpressing tga20 neural tissue was transplanted into Prnp o/o (left) or MBP-PrP (right) mice. Hosts were inoculated with scrapie prions intraocularly (i.o.), intraperitoneally (i.p.), or intracerebrally (i.c.). Neuropathological signs of scrapie (gliosis visualized by overexpression of glial fibrillary acid protein, synaptic loss shown by loss of synaptophysin, and deposition of PrP) were only visible in grafts with hosts that had been inoculated intracerebrally. In contrast, neural grafts remained free of disease after intraocular or intraperitoneal inoculation. The trauma deriving from transplantation procedure induced a slight gliosis in all grafts. HE, Hematoxylin and eosin. Scale bars, 100 μm.
Tables
Mouse genotypes MBP-PrP+/o MBP-PrP+/+ Prnp+/+ Prnpo/o Type of prion challenge (dose) Attack rate Disease latencya Attack rate Disease latencya Attack rate Disease latencya Attack rate Disease latencya Intraperitoneal (6 logLD50) 0/11 >641 0/12 >467 6/6 195 ± 3 0/4 >641 Intracerebral (3 × 105 LD50) 0/10 >641, 2 × >210b 0/10 >467, 1 × >197b 6/6 163 ± 4 0/5 >467 1 × >243b Intraocular (1 × 105 LD50) 0/6 >531c 0/7 >410, 1 × >376d 7/7 194 ± 11 ND -
Only control wild-type mice (Prnp+/+) developed scrapie after intraperitoneal, intracerebral, and intraocular prion challenge, whereas mice carrying the MBP-PrP transgene hemizygously or homozygously and Prnpo/o mice never developed clinical signs of scrapie. Average incubation times and SDs were calculated for the groups of mice that developed scrapie. For mice that remained free of disease, the total observation time is reported. All mice were bred to a similar mixed genetic background (C57BL/6 × 129Sv). ND, Not determined.
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↵ a Average ± SD.
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↵ b Clinically healthy mice were killed at the time points indicated, and organs were used for infectivity analysis.
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↵ c One mouse died 24 hr after inoculation.
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↵ d Death of one mouse 376 d after inoculation resulting from abdominal tumor growth.
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Transmission of brain to indicator mice Transmission of spleen to indicator mice Mouse genotypes Days after inoculation; route of infection Brain infectivity (logLD50 per gram) Attack rate (mean ± SD days) Spleen infectivity (logLD50 per gram) Attack rate (mean ± SD days) MBP-PrP 35 d; intraperitoneal <1.5 0/4 <1.5 0/4 <1.5 0/4 <1.5 3/4 (99,148,150)a 140 d; intraperitoneal <1.5 0/4 <1.5 0/4 <1.5 0/4 <1.5 0/4 210 d; intracerebral <1.5 0/4 <1.5 0/4 <1.5 0/3 <1.5 0/4 Prnp+/+ 35 d; intraperitoneal <1.5 0/4 5.0 4/4 (73 ± 5) <1.5 0/4 5.1 4/4 (72 ± 4) 140 d; intraperitoneal 5.3 4/4 (68 ± 6) 4.0 3/3 (84 ± 4) 4.9 4/4 (74 ± 7) 5.1 4/4 (72 ± 8) 167 d; intracerebral 6.3 4/4 (58 ± 6) 5.1 4/4 (72 ± 5) 6.1 4/4 (60 ± 7) 5.3 4/4 (70 ± 2) Prnpo/o 42 d; intraperitoneal <1.5 0/4 <1.5 0/4 <1.5 0/4 <1.5 1/4 (99) -
Wild-type (Prnp+/+) and tg640 mice homozygous for the MBP-PrP transgenic cluster were inoculated intraperitoneally and intracerebrally and killed for analysis at the time points indicated.
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↵ a Prion disease in three of four tga20 indicator mice that had received spleen extracts from a 35 dpi-challenged tg640 mouse is most likely a result of prions persisting from the inoculum.
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