Transgenic mice

The relevance of the hydrophobic core with regard to PrP^C function and to prion conversion was further investigated in vivo. Two lines of transgenic mice (F902 and M630) had been created, each one harboring one copy of the deletion mutant Prnp 114-121 (see Chapter 4.2.3) under the control of the prion protein promoter (Borchelt et al., 1996). The generation of these mice was carried out by Dr. Christine Brabeck, a former group member, in collaboration with the German Cancer Research Center (DKFZ, Heidelberg, Germany), using pronuclear microinjection. These mice were then backcrossed with Prnp knockout mice in order to obtain transgenic mice solely expressing Prnp 114-121 without the co-expression of the wild-type alleles. Besides Prnp-wt and –ko mice as controls, transgenic mice on the wild-type and on the knockout background, respectively, were used to determine the expression pattern of the mutant protein and its effect on the physiological PrP^C function.

In addition, the impact of the hydrophobic core on prion conversion was assessed by the inoculation of these mice with RML prions (see Chapters 4.3 and 4.4) (Figure 24).

Figure 24: Transgenic mice harboring Prnp 114-121

Genotypes of mice analyzed in this study. Besides Prnp-wt mice and Prnp-ko mice, two lines of transgenic animals were created, each one harboring one copy the deletion mutant Prnp 114-121, either on the wild-type or on the knockout background.

: Mouse genotypes used in addition for inoculation experiments with prions (Chapters 4.3 and 4.4)

78 4.2.2 Genotyping of transgenic mice

For the identification of transgenic offspring, DNA was extracted from hair roots, and the genotype was identified using two specific PCRs. Due to different sizes of the produced PCR fragments, both forward primers for Prnp-wt and the neomycin cassette, which was applied as selection marker in the Prnp-knockout mice, could be applied in one PCR, whereas the transgene Prnp 114-121 was detected in a separate PCR reaction. An example for a gel representing different genotypes is shown in Figure 25.

Figure 25: Assessment of tg mouse genotype

After DNA extraction from mouse hair roots and subsequent PCR, the resulting fragments were separated according to their size on an agarose gel. A DNA band (337 bp) in the first lane of each sample demonstrated the existence of the transgene Prnp 114-121 in the mouse genome. The second lane assessed wild-type (351 bp) and knockout (192 bp) alleles. Last sample: Negative control.

Results

79 4.2.3 Assessment of Prnp 114-121 copy number

The integration of the transgene and the number of Prnp 114-121 copies in the genome had been assessed by Southern blotting (Baumann et al., 2007) and by quantitative PCR. For both transgenic lines a single copy of Prnp 114-121 was integrated into the mouse genome (Figure 26).

Figure 26: Quantification of Prnp 114-121 expression

The copy number of Prnp 114-121 (tg) was assessed by quantitative PCR from mice with different genetic genotypes, harboring the transgene (dark grey columns) in comparison to non-transgenic animals (light grey).

In both transgenic mouse lines (F902 and M630), the transgene was integrated with one copy into the genome.

80 4.2.4 Spatial distribution of PrP 114-121

Due to the incorporation of the Prnp promoter in the applied vector, genes constructed behind this promoter should be expressed in the same tissues and cell types as Prnp-wt, in particular in most neurons of the brain with the exception of cerebellar Purkinje cells (Borchelt et al., 1996; Fischer et al., 1996). The brain is also the area, where prion diseases exert the strongest pathology (Prusiner et al., 1998). The analysis of the spatial PrP 114-121 distribution was therefore focused on the brains of adult transgenic mice. The probing of sagittal sections of brains from different genotypes with an anti-PrP antibody on histoblots revealed a general decrease in the level of mutant protein expression compared to PrP-wt, especially in the cerebellum, whereas the overall regional distribution in the brain seemed not to be changed (Figure 27). The pronounced decrease in the cerebellum can be explained by the absence of transgene expression in the Purkinje cell layer due to the vector construct used (Fischer et al., 1996), expressing normally high levels of PrP^C in wild-type brain (Allen_Brain_Atlas, 2007).

Results

81 5 mm

A B

Figure 27: Spatial PrP 114-121 expression in transgenic mouse brains

Histoblot analysis of PrP 114-121 in the brains of transgenic mice on a Prnp-ko background.

(a) For both transgenic lines, the level of mutant protein expression was reduced as compared to PrP-wt, especially in the cerebellum.

(b) The exposure time of the histoblots from (a) was increased for both transgenic lines and the knockout control (+). The equal PrP level intensities displayed a similar PrP 114-121 expression pattern in the brains of both transgenic lines as compared to PrP-wt.

82 4.2.5 Expression level of PrP 114-121

The quantification of prion protein expression in transgenic mice by densitometric slot blot analysis (Figure 28) confirmed the reduced mutant PrP expression observed in histoblots.

Compared to the PrP expression in Prnp-wt mice, the level of PrP 114-121 was only around 20% in the brain excluding the cerebellum (medulla oblongata, pons, met- and prosencephalon) of both transgenic lines, with an additional decrease in the cerebellum to only 7% compared to PrP-wt.

Figure 28: Quantification of PrP 114-121 expression

(a) The level of prion protein expression was assessed by slot blot analysis with brain homogenates from the brain without the cerebellum and from the cerebellum only. For each genotype, samples from three different mouse brains were analyzed. Both lines expressing Prnp 114-121 on the knockout background exhibited a markedly reduced protein expression compared to PrP-wt levels.

(b) The ratio of mutant PrP expression versus the level of PrP-wt was determined by densitometric analysis of the slot blots from (a). Signal intensities were adjusted to the respective -tubulin loading controls.

Compared to wild-type expression, the level of PrP 114-121 was around 20% in both lines in the brain without the cerebellum, with a further significant decrease to only 7% in the cerebellum.

Statistical analysis of protein expression levels was performed by student’s t-test (*** represents p<0.001).

Results

83 4.2.6 Tissue and cell type specific expression of PrP 114-121

Detailed examination of the cerebellum and the hippocampal formation, i.e., two brain areas of particular interest with regard to PrP expression and prion pathology (Prusiner et al., 1998;

Wadsworth et al., 2003), further verified an equivalent tissue and cell type specific expression of PrP 114-121 in comparison to PrP-wt, albeit at reduced levels. For both transgenic mouse lines on the knockout background, sagittal cryosections of the brain revealed a considerable reduction of the PrP signals in both areas, especially in the cerebellum (Figure 29) by immunohistochemical examination, thereby corroborating the observations of low PrP 114-121 expression from the histo- and slot blot analyses in Chapters 4.2.4 and 4.2.5.

Figure 29: PrP 114-121 levels assessed by immunohistochemistry

The expression of PrP 114-121 was determined by anti-PrP antibody 6H4. In comparison to PrP-wt, the mutant protein was expressed at lower levels in the cerebellum and in the hippocampal formation.

Abbreviations: DG: dentate gyrus; GL: granular layer; HC: hippocampus; WM: white matter.

84 Co-staining experiments of sagittal brain sections with an anti PrP antibody, the neuronal marker NeuN and glial fibrillary acidic protein (GFAP), a marker for astrocytes, revealed a similar distribution of PrP 114-121 in the brain tissues compared to PrP-wt.

PrP-wt and PrP 114-121 were primarily expressed in the area of neuronal processes and in the surroundings of perikarya, such as in the neuropil of the hippocampal formation (Figure 30), mainly consisting of neuronal and also to a minor extend of astrocytic processes (Ventura

& Harris, 1999; Zhou et al., 2000). Although the resolution of the complex cellular network in cryo-sections of brain tissue is insufficient to assign PrP 114-121 to individual cells and cell types directly, the comparable expression of the mutant protein in the same areas as PrP-wt, predominantly consisting of neuronal processes, demonstrated that PrP 114-121 was correctly expressed in neurons and likely – albeit less strongly - also in astrocytes which is in accordance with previous studies applying the same vector for transgene expression (Borchelt et al., 1996; Fischer et al., 1996).

Results

85

Figure 30: Spatial distribution of PrP 114-121 at the hippocampal formation

Sagittal brain cryo-sections from Prnp-wt and transgenic animals were probed with antibodies directed against astrocytes (GFAP), neuronal nuclei (NeuN), prion protein (PrP) and with nuclear staining (Hoechst).

The sections displayed a similar distribution of PrP-wt and PrP 114-121 around neuronal perikarya and particularly in the neuropil, consisting of cellular processes. For a clearer assessment of PrP 114-121 distribution, the signal intensity of the PrP-Ab was increased in both transgenic samples to the intensity of the wt brain sample (PrP +).

86 4.2.7 PrP 114-121 lacks -cleavage in vivo

The effect of the hydrophobic core region on PrP^C -cleavage was also present in vivo. While an 18-kDa band was clearly detectable on western blots in non-transgenic Prnp-wt mice using the C-terminal specific antibody 6H4, the intensity of this fragment was dramatically reduced in both transgenic mouse lines expressing PrP 114-121 (Figure 31a).

For the characterization of the 18-kDa fragment, a similar experimental approach as for the immunoprecipitation in Chapter 4.1.3 was applied: When the blot was re-probed with an antibody directed against the N-terminus of PrP^C, the lower band was no longer detectable in the PrP-wt sample, thus validating the identity of the 18-kDa band as the N-terminally truncated C1 fragment (Figure 31c).

Confirming the results above (Chapters 4.2.4 - 4.2.6), the level of PrP 114-121 expression was markedly reduced in both transgenic mouse lines compared to PrP^C in wild-type mice (Figure 31a). In order to rule out that this low level of mutant protein expression caused the C1 band to be below the detection threshold, the blot was overexposed, resulting in a faint C1 band (Figure 31b). Thus, the reduction of the C1-fragment in the transgenic mice is an authentic result and not an artifact due to the limited quantity of PrP loaded on the gel.

The data from the densitometric analysis of the western blot were consistent with the in vitro topology results. In both experimental setups, PrP 114-121 displayed a near-complete loss of -cleavage compared to PrP-wt (Figure 31d).

Results

87

Figure 31: PrP^C -cleavage in vivo

(a) Western blot analysis of brain homogenates from PrP-wt and transgenic mice expressing PrP 114-121 on a Prnp-ko background (M630 and F902). For each genotype, equal amounts of brain homogenates from three different mice were either deglycosylated by PNGaseF or left untreated as a control. Actin was used as a loading control. Non-cleaved full-length PrP^C (“Full”) and the C1 fragment were visualized by probing the blot with the C-terminal antibody 6H4. A strong C1 band was only present in the PrP-wt samples.

(b) Section of the blot from panel (a). In order to rule out that the C1 bands of the transgenic mice were below the detection limit due to the reduced expression of PrP 114-121, the blot was overexposed. Very low levels of C1 became indeed visible in the brain extracts form mice expressing PrP 114-121.

(c) Section of the blot from panel (a). After removal of the C-terminal antibody 6H4, the blot was re-probed with an antibody directed against the N-terminus of PrP^C. Only full length PrP^C was visible. Due to its truncation after cleavage, the C1 fragment has lost the epitope for the N-terminally binding antibody (see also Figure 19).

(d) Quantification of the PrP^C fragments in vivo. The signal intensities of panel (a) were quantified by densitometry. The two bars on the right-hand side indicate the amount of C1 versus the non-cleaved fragments (^secPrP plus the transmembrane forms ^NtmPrP and ^CtmPrP) from the in vitro topology assay (see also Figure 21). The in vitro and in vivo results showed a comparable decrease in the -cleavage for PrP 114-121 (in vivo: transgenic line M630: 2.0%, line F902: 3.4% versus 1.5 % in vitro) compared to 33.7% C1 for PrP-wt in vivo and 33.6% in vitro.

88 4.2.8 Alterations of apoptosis-related proteins in transgenic mice

Prnp-ko mice have been reported to exhibit changes in the expression and the activity of various proteins involved in the regulation of cell stress, such as superoxide dismutase, p53, proteins of the MAP-kinase pathway, cyclin-D, Bcl-2 or Bax (Brown et al., 2002).

These findings suggest that PrP^C plays a role in cellular stress response and apoptosis. In order to assess the influence of Prnp 114-121 on these mechanisms, adult transgenic mice on the Prnp knockout background were examined for the expression of apoptosis-related proteins Bax and Bcl-2. Slot blot analysis of protein expression levels in the brain revealed that both, the pro-apoptotic protein Bax as wells as the anti-apoptotic protein Bcl-2, was elevated (Figure 32). This increase was not as pronounced as in non-transgenic knockout mice, which exhibited slightly higher levels of these molecules. Despite the elevated level of these two proteins, the Bax to Bcl-2 ratio, being a marker for the sensitivity to apoptotic insults (Mackey et al., 1998; Salakou et al., 2007), was not changed in the transgenic mice.

Results

89

Figure 32: Expression analyses of Bax and Bcl-2

(a) Slot blot assessment of Bax and Bcl-2 protein levels in the brain homogenates of transgenic and non-transgenic mice. For each genotype or non-transgenic mouse line, three animals were analyzed.

(b) Densitometric quantification of (a), adjusted to the -tubulin loading control. Bcl-2 and Bax were elevated in the two mouse lines F902 and M630, harboring Prnp 114-121 on a Prnp-ko background and increased even more in non-transgenic Prnp-ko control mice.

(c) Whereas non-transgenic Prnpknockout control mice exhibited a slight increase of Bax/Bcl-2, the ratio of Bax versus Bcl-2 remained unchanged in the transgenic mice.

Statistical analyses for (b) and (c): One-way ANOVA in combination with Tukey’s multiple comparison test, assessing the difference of protein levels in comparison to Prnp-wt mice (*: p<0.05; **: p<0.01;***: p<0.001) and in comparison to non-transgenic Prnp knockout control mice (#: p<0.05; ##: p<0.01; ###: p<0.001).

Im Dokument Impact of the Hydrophobic Core on PrP Function and Conversion (Seite 88-100)