Inhibition of prion conversion by PrP 114-121 in vivo

A previous study had demonstrated that the mutant PrP 114-121 exerts a dominant negative effect on the conversion of PrP^C into PrP^Sc in persistently infected cell cultures (Holscher et al., 1998). In order to verify this effect in vivo, the two lines of transgenic mice, both on a Prnp wild-type and a Prnp knockout background, and in addition PrP-wt mice were inoculated intracerebrally with the mouse adapted scrapie strain RML6 (Chandler, 1961); see also Figure 24. The inoculation of Prnp-ko control mice was not permitted by the authorizing agency due to earlier results showing that knockout mice are resistant to prion infection (Bueler et al., 1993). Inoculated mice were examined for the appearance of neurological symptoms and were sacrificed at the terminal stage of disease.

Transgenic mice on the wild-type background survived more than 10% longer than their non-transgenic littermates (Figure 35). This dominant negative effect of the transgene seemed therefore to reduce the kinetics of PrP^Sc conversion by slowing down the conversion process, which finally led to a prolonged survival time after prion infection. This effect on the onset of prion disease is even more remarkable, if one takes into account the considerably lower level of transgene expression in both lines compared to the endogenous PrP-wt.

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Figure 35: Survival of prion-inoculated mice on a Prnp-wt background

The survival of transgenic and non-transgenic littermates on a Prnp-wt background was compared for the genetic lines F902 and M630. Mice harboring the transgene Prnp 114-121 exhibited a significant longer median survival than their non-transgenic littermates (F902: p < 0.01; M630: p < 0.001).

Previous experiments showed that Prnp knockout mice are resistant to prion infection (Bueler et al., 1993). Based on the earlier findings that PrP 114-121 could not be converted into PrP^Sc in cell culture (Hölscher et al., 1998), mice harboring this transgene in a Prnp-ko background should survive prion inoculation. Indeed both transgenic lines on the knockout background survived substantially longer than mice harboring wild-type Prnp, reaching life times beyond 500 days (Figure 36).

Figure 36: Survival of inoculated mice on a Prnp-ko background

In comparison to mice on the Prnp-wt background, both lines of inoculated transgenic Prnp-ko mice lived more than twice as long, i.e., beyond 500 days post inoculation (p < 0.001 for F902 and M630).

94 4.3.2 PrP 114-121 is not convertible into a PK resistant form

Further evidence for the actual resistance of PrP 114-121 with regard to PrP^Sc conversion comes from the analysis of accumulated PrP^res in the brains of the various mouse lines by phosphotungstic acid precipitation (Wadsworth et al., 2001) and subsequent western blotting. At the terminal stage of disease, inoculated mice harboring Prnp-wt had accumulated high levels of PK resistant PrP conformations in their brains, termed PrP^res, representing the accumulation of PrP^Sc.

The level of PrP^res was comparable to the accumulation found in long-term survivors expressing additionally PrP 114-121 (Figure 37). The equally high levels of PrP^res in transgenic and non-transgenic mice on the wild-type background substantiated the hypothesis that the dominant negative effect of the transgene slowed down the conversion process of wild-type PrP^C to PrP^Sc. However, since this effect was only partial, presumably due to the low expression level of PrP 114-121, no complete inhibition of the conversion process but rather a delay in PrP^Sc accumulation was achieved, consequently resulting in late disease.

Figure 37: Analysis of PrP^res accumulation in inoculated mice

PrP^res from the brain homogenate of inoculated mice was enriched by phosphotungstic acid precipitation, proteinase K digested und subsequently analyzed by western blotting. In all mice on a Prnp-wt background, a strong signal was detectable in the PK-treated lanes, whereas there was no signal for PK digested material from both lines (F902; Ko-F and M: M630; Ko-M) of transgenic mice on a Prnp knockout background.

Sample 1: Positive control; tga-20 mouse, overexpressing Prnp-wt; Sample 2: Prnp-wt mouse harboring Prnp 114-121; Sample 3: Prnp-wt mouse without the transgene; Sample 4: Negative control; tga-20, not inoculated; Sample 5: Prnp-ko mouse harboring Prnp 114-121; line F902; Sample 6: Prnp-ko mouse harboring Prnp 114-121; line M630.

Due to the lower mutant protein expression, samples 5 and 6 contained twice the protein amount in all lanes as compared to the controls and samples containing wild-type PrP.

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95 4.3.3 PrP^res detection was not impaired by low PrP 114-121 expression In order to detect also marginal amounts of PrP^res, five-fold amounts of PK digested brain homogenates were applied to the western blot analyses as compared to the amount of brain homogenate for undigested controls. In the case of Prnp-wt gene mice, this difference in the amount of protein loaded on the gel resulted in strong signals after PK digestion. In transgenic PrP 114-121 mice on the ko background however, no PrP^res was detectable.

The enrichment of PrP^res by phosphotungstic acid precipitation in addition to the only 20%

expression level of PrP 114-121 in the transgenic lines resulted in a considerably weaker band intensity of undigested PrP 114-121 as compared to the PrP-wt samples (Figure 37).

The five-fold protein overload in the PK digested samples however largely excluded the sensitivity of this assay to be below the level of detection due to the lower level of transgene expression. In case that PrP 114-121 was indeed convertible into a PrP^Sc like form, the band intensity of the PK digested homogenates would be expected to be at least as strong as those without PK.

In addition, a dilution series of PrP-wt brain homogenate was applied in comparison to a transgenic mouse sample on the knockout background. The same or even lower input of PrP-wt material as compared to PrP 114-121 still exhibited a strong and clearly detectable signal in the PK digested lanes, further demonstrating that the absence of PK resistant material in the brains of transgenic mice is indeed not the result of a PrP^res expression below the level of detection (Figure 38).

Figure 38: Dilution series of inoculated brain homogenate

In comparison to the 100% input of inoculated brain homogenate from a Prnp 114-121 mouse on the ko background, the diluted samples of a Prnp-wt mouse also displayed a clearly detectable PK resistant material even at a lower intensity than the non PK digested band (at 3% and 10% input), whereas the transgenic sample was lacking PK resistance.

96 4.3.4 Histological assessment of PrP^res in the mouse brain

In addition to western blotting, sagittal brain sections of prion-inoculated transgenic mice on either the knockout or the wild-type background were analyzed for PrP^res expression (Figure 39). Whereas the brain tissue of the Prnp wild-type background exhibited a pronounced staining for PrP^res in various brain regions, such as in the cerebellum, the hippocampus, or in the cerebral cortex, the sample of the transgenic mouse on the knockout background was nearly devoid of positive staining. The only staining observed was visible at border areas of the section and at blood vessels. A comparable staining pattern was also observed in a non-transgenic Prnp knockout mouse. In addition, the punctuate staining is known by other investigators (Dr. Hanns-Joachim Rziha, Friedrich-Löffler-Institut, Tübingen; personal communication) as an artifact and does not represent PrP^res, further validating that the brain tissue solely expressing the mutant PrP 114-121 was free from PrP^res.

Collectively, these analyses of the PrP^Sc expression in the brains of prion-inoculated mice clearly demonstrate that the transgene PrP 114-121 is not convertible into a protease K resistant PrP^Sc-like form.

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Figure 39: Histological assessment of PrP^Sc accumulation in the brain

Top, PrP^Sc staining of an aged mouse (498 days p.i.), harboring Prnp 114-121 on a ko background, inoculated with RML prions. No specific staining for PrP^Sc could be detected. The occasional staining of the tissue was a result of staining artifacts, mainly observed around blood vessels and also in a non-inoculated ko mouse.

Bottom, PrP^Sc staining of a tg mouse (231 days p.i.) on the wt background. PrP^Sc was present in many brain regions, such as cerebellum and cerebral cortex, forming dark stained aggregates. Black arrows point to a cluster of dying cells, leading to the formation of vacuoles within the brain tissue, a typical hallmark of TSEs.

Variations in the brightness of different regions of the brain overview are due to its assembly from several single pictures and are not the result of different staining intensities. Abbreviations: WM: white matter; GL:

granular layer; ML: molecular layer of the cerebellum. HC: hippocampus. CC: corpus callosum.

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