3 Materials and Methods
4.4 Differences in the histopathology of inoculated and prion free mice
4.4.1 Characterization of brain pathology
Typical hallmarks of prion pathology are vacuolation and astrocytic gliosis in affected brains (Prusiner, 1998). The histological examination of brains from inoculated mice revealed a similar pattern of pathological alterations in terminally ill animals, harboring Prnp 114-121, as well as in non-transgenic mice, both on the Prnp-wt background. Both genetic mouse types suffered from severe vacuolation in the cerebellum, especially in the white matter; in the medulla oblongata; the pons and the midbrain; at the corpus callosum and, to a lesser extent, in the cerebral cortex. The vacuolation was accompanied with a severe astrocytic gliosis in the entire brain, especially in all layers of the cerebellum, in the hippocampus and in the cerebral cortex (Figure 40a, b). These histopathological changes were associated with behavioral deficits such as itching, reduced body weight, lethargy, hunchback, ataxia, paresis of the extremities and prolapse of the penis in male mice.
Interestingly, the examination of brains from aged Prnp knockout mice harboring the transgene also revealed pathological markers characteristic of encephalopathy in both transgenic lines. These mice developed similar behavioral abnormalities as compared to the inoculated animals on a wild-type background, especially ataxia, itching, prolapse of the penis and hind-limb paresis and paralysis (see also Chapter 4.2.9) accompanied by histopathological changes, such as vacuolation, mainly in the white matter of the cerebellum; in the medulla oblongata; the pons and the midbrain. Astrocytic gliosis was present in some brain areas, most prominent in the white matter of the cerebellum and at the corpus callosum. The examination of non-inoculated age-matched transgenic mice on the knockout background resulted in the same type of pathology, both in terms of histological pathology as well as in terms of behavioral disorders (Figure 40c, d; see also Chapter 4.2.9).
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Figure 40a: Prion brain pathology of a terminally ill Prnp-wt mouse; 208 days post infection (p.i.) Top: HE staining displayed strong vacuolation of various brain regions, most notably in the white matter (WM) of the cerebellum but also in the pons, the corpus callosum (CC) and in the cerebral cortex.
Bottom: GFAP staining revealed severe astrocytic gliosis in most brain regions, especially in all layers of the cerebellum and also in HC and the cerebral cortex; whereas CC was nearly free from activated astrocytes.
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Figure 40b: Pathology of a terminally ill transgenic mouse on Prnp-wt background (231 days p.i.) Despite prolonged survival, transgenic mice on the wild-type background displayed the same kind of severe pathology at the terminal stage of disease as their non-transgenic littermates, exhibiting severe vacuolation and astrocytic gliosis in the cerebellum, the cerebrum and other brain regions. Interestingly, in contrast to Prnp-wt mice, these animals were also affected by pronounced astrocytic gliosis in the corpus callosum.
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Figure 40c: Pathology of a terminally ill tg mouse (498 days p.i.) on the Prnp-ko background
Compared to inoculated mice harboring Prnp-wt, inoculated transgenic animals on the Prnp-ko background not only survived considerably longer, they also exhibited a different type of histopathology, demonstrated by an overall milder vacuolation and astrocytic gliosis; except for the corpus callosum, which exhibited severe astrocyte activation, thereby contrasting the pathological pattern of Prnp-wt mice.
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Figure 40d: Pathology of a non-inoculated tg mouse (473 days old) on the Prnp-ko background Aged non-inoculated transgenic mice on the knockout background exhibited the same kind of pathology as inoculated animals of the same genotype, displaying mild vacuolation in the cerebellum and the pons in addition to astrocytic gliosis in the cerebellum and especially in the corpus callosum.
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103 4.4.2 Distinctive differences in the pathology of Prnp-wt and -ko mice The histopathological pattern of mice devoid of Prnp-wt differed markedly from inoculated mice on a Prnp-wt background with regard to the degree of vacuolation and astrocytic gliosis.
In all mice examined, the white matter of the cerebellum exhibited notable signs of vacuolation. In mice on the Prnp-ko background, the vacuolation was significantly less severe as compared to mice on a Prnp-wt background, both in terms of vacuole number and vacuole size. An overall reduced severity of vacuolation could also be observed in other affected brain regions, exhibiting even no vacuolation in the cerebral cortex. In addition, differences in brain pathology between mice harboring Prnp-wt and mice on the knockout background became manifested by the level and the regional distribution of astrocyte expression in the brain. In transgenic mice on the knockout background, astrocytic gliosis in the cerebellum was restricted to the white matter and did not affect the molecular and granular layers.
Furthermore, no astrocyte activation was observed in the striatum and in the cerebral cortex.
Although slightly more pronounced in the transgenic mice, this mild form of pathology could also be observed in an age-matched, non-inoculated Prnp knockout control mouse devoid of Prnp 114-121, which also exhibited increased vacuolation and astrocytic gliosis as compared to a Prnp wt mouse of the same age (Figure 41).
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Figure 41: Comparison of brain pathology with regard to mouse genotypes and prion inoculation The most severe pathology, concerning vacuolation and astrocytic gliosis, was observed in inoculated mice on the wild-type background both, transgenic or non-transgenic. Aged inoculated transgenic mice on the knockout background displayed an overall less severe expression of the pathological features. This moderate pattern of pathology could thereby also observed in non-inoculated mice on the knockout background, both in transgenic and also to a lesser extent in non-transgenic controls.
Sagittal overview from: (Allen_Brain_Atlas, 2007)
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105 4.4.3 Characteristic astrocyte expression at the corpus callosum
Interestingly, the reduced expression of activated astrocytes in most brain regions of transgenic mice on the knockout background was contrasted by a severe astrocytic gliosis in the corpus callosum, especially towards its anterior end (genu). This type of pathology was observed independently of prion inoculation or Prnp 114-121 expression since a non-inoculated knockout control mouse also exhibited the same kind of astrocytic gliosis.
Inoculated Prnp-wt mice, however, were nearly free from astrocytes in this area (see also Figure 40a) but exhibited notable astrocytic gliosis in the adjacent cerebral cortex, whereas the additional expression of Prnp -121 on the wild-type background rendered also the corpus callosum susceptible for astrocytic gliosis (Figure 42).
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Figure 42: Distinct pattern of astrocytic expression at the corpus callosum
Upon inoculation with RML prions, non-transgenic Prnp-wt mice exhibited only moderate astrocytic gliosis at the corpus callosum, therefore showing strong astrocyte activation in the adjacent cerebral cortex. In contrast, transgenic and non-transgenic mice on the knockout background displayed a notable astrocytic gliosis at the corpus callosum, whereas the cerebral cortex remained almost free from astrocytes, also after prion infection.
Interestingly, inoculated transgenic mice on a Prnp-wt background exhibited astrocytic gliosis in both the corpus callosum and in the cerebral cortex.
Sagittal overview from: (Allen_Brain_Atlas, 2007); Arrows mark the corpus callosum.
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107 4.4.4 Quantitative parameters for the evaluation of brain pathology
The differences in the vacuolation of certain brain areas and the distinct pattern of astrocytic gliosis at the corpus callosum and the cerebral cortex were characteristic for the differences in the pathology of prion-inoculated and non-inoculated mice.
These pathologic properties were therefore used as parameters for the quantitative discrimination between the pathological pattern elicited either by prion inoculation or by PrPSc-independent mechanisms.
The cerebellum of inoculated Prnp wild-type mice, for example, displayed a significantly higher number of vacuoles than mice on the knockout background (Figure 41, Figure 43a).
With regard to astrocytic gliosis, the proportional comparison of astrocytic GFAP staining intensity in the cerebral cortex versus the corpus callosum (CC) resulted in a high cortex/CC ratio for these mice, exhibiting a prominent astrocytic gliosis in the cortex, whereas the corpus callosum was hardly affected. On the other hand, old prion protein knockout mice showed the opposite pattern: a strong astrocytic gliosis in the corpus callosum, but nearly no staining for GFAP in the cerebral cortex (Figure 42), leading to a ratio below one when assessing the cortex/CC GFAP ratio (Figure 43).
Interestingly, inoculated transgenic mice on a wild-type background exhibited a significantly lower cortex/CC GFAP ratio than non-transgenic Prnp-wt animals due to a pronounced astrocytic gliosis in both brain regions, thereby resembling a “mixed” pathological phenotype of the prion associated and the prion independent pathology.
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Figure 43: Quantification of brain histopathology
The different pathological pattern between inoculated mice on the Prnp-wt and the ko background led to quantitative differences of cerebellar vacuolation (a) and of astrocytic gliosis at the corpus callosum and the cerebral cortex (b).
(a) The quantification of vacuoles in the cerebellum revealed a significant difference between inoculated mice on the wild-type background and of transgenic mice on the knockout background, inoculated or free from prions (***: p<0,001).
(b) The ratio of the GFAP staining in the cerebral cortex versus the corpus callosum (CC) further reflected the distinct pathological pattern observed.
Inoculated Prnp-wt mice exhibited a stronger staining in the cerebral cortex, induced by the pronounced astrocytic GFAP expression in this region in contrast to a rather weak GFAP staining in CC. However, inoculated or prion free mice on the knockout background exhibited intense GFAP expression in CC and a low staining in the cerebral cortex, leading to an opposite ratio of cortex/CC staining intensity below one (***: p<0,001).
Interestingly, inoculated transgenic mice on the wild-type background exhibited a strong GFAP expression in the cortex and a slightly weaker staining in CC, resulting in a significantly different cortex/CC GFAP ratio than for inoculated wild-type mice (##: p<0.01). Therefore, these animals resemble a “mixed” pattern between the pathology caused by prion inoculation in mice harboring Prnp-wt and the prion independent pathologic pattern, observed in old transgenic and non-transgenic animals.
The quantitative differences of vacuolation and GFAP intensity were analyzed using one-way ANOVA in combination with Tukey’s multiple comparison test. For the histopathological analysis of the non-inoculated Prnp-wt and ko controls, only one age-matched animal, respectively, was available (grey columns). Therefore, these controls were not included in the statistical assessment of brain pathology.
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109 4.4.5 PrP 114-121 does not elicit PrPSc pathology upon prion inoculation The comparable type of pathology in inoculated and in prion free transgenic mice on the knockout background argues against the activation of an additional toxic effect of PrP 114-121 upon prion inoculation.
In addition, the different pathological pattern of prion-inoculated mice on the wild-type background on the one hand and of inoculated but also of prion free animals on the knockout background on the other hand (see Table 11 for summary) indicate that two distinct kinds of pathology emerged in the different genotypes, elicited by PrPSc in the former case and by prion independent mechanisms in the latter.
Vacuoles Astrocytic Gliosis
Pons Cerebellum CC Cerebr. C Pons Cerebellum CC Cerebr. C
wt, no inoc. + - - - + + + -
ko, no inoc. ++ + + - + ++ +++ -
wt, RML +++ +++ +++ + +++ +++ + +++
wt + tg, RML +++ +++ +++ + +++ +++ ++ +++
ko + tg, RML ++ + + - + ++ +++ -
ko + tg, no inoc. ++ + + - + ++ +++ -
Table 11: Quantitative overview of the brain pathology in different genotypes
The histopathological parameters vacuolation and astrocytic gliosis were evaluated applying the quantitative assessment of Chapter 4.4.4. (-) represents less than 2 vacuoles per field analyzed, or hardly any GFAP staining intensity; (+) represents 2-7 vacuoles per field, or weak GFAP intensity. (++) denotes 8-13 vacuoles per field, or strong GFAP intensity. (+++) denotes over 13 vacuoles per field, or very strong GFAP intensity.
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5 Discussion
More than 25 years after Stanley Prusiner’s posting of the protein-only hypothesis (Prusiner, 1982), considerable progress has been made with regard to the characterization of prion conversion and the associated structural changes of PrPC, the pathway of prion infection, the common pathological features of various neurodegenerative diseases, such as prion disease, Alzheimer’s or Huntington’s disease, the cellular trafficking of PrPC and PrPSc, the management of the risk to the public health by BSE infected beef, etc. (Aguzzi & Polymenidou, 2004; Ryou, 2007; Winklhofer et al., 2008; Zou & Gambetti, 2007).
Despite all these impressive scientific advances every new discovered piece of the prion puzzle led to new, more complex questions: How can the phenomenon of different prion strains be explained? What is the smallest infectious unit? Why is the generation of infectious prions in vitro that complicated? What are the co-factors of prion infection? What is the risk of CWD to public health in North America? What is the actual cause of prion pathology and how can it be counteracted? And finally what is the actual physiological function of PrPC? In order to gain insight into some of these complex questions, a set of micro-deletions within the most highly conserved region of PrPC, the hydrophobic core (HC), was applied in the context of the present PhD project. This experimental approach is based on the hypothesis that an evolutionary necessity must have prevented any changes of this region, which points to an important function of the prion protein, embodied in this region.