Transgenic mice over-expressing wild-type PrP develop a neurological illness
We previously established three transgenic mouse lines over-expressing epitopically tagged wild-type PrP derived from the mouse Prnpa
allele [Tg(WT) lines E1, E3 and E4] (Chiesa et al., 1998
). Mice from each of these lines that carried the transgene array in a hemizygous state developed normally and did not show any neurological dysfunction as they aged (Chiesa et al., 1998
). Tg(WT) mice of each line were subsequently interbred to render the transgene array homozygous and analyzed for the presence of neurological symptoms. We observed that homozygous mice of the Tg(WT-E3) line, which displayed the highest level of PrP expression in the brain (see below), developed a progressive neurological illness, the earliest symptom of which was a tremor that was apparent when the mice initiated walking. This symptom was followed by the appearance of kyphosis, ataxia and paresis of the hind limbs, which increased in severity to total paralysis as the mice aged (). Animals in the advanced stage of the disease showed dramatic thinning of the posterior part of the trunk, reduced body weight, ruffled coat due to poor grooming, and inability to right themselves from a supine position (). The animals were humanely sacrificed when they became unable to feed themselves.
Neurological symptoms in Tg(WT-E3) mice
The mean age of onset of the neurological signs in Tg(WT-E3+/+)/Prnp0/0 mice was 159 ± 23 days and the mean age at death was 409 ± 42 days (). The duration of the symptomatic phase of the illness was quite prolonged (250 ± 32 days; ). In three Tg(WT-E3+/+) mice on the Prnp+/+ background that we analyzed, the mean age of disease onset was 161 ± 1 days.
Clinical illness in Tg(WT-E3+/+)/Prnp0/0 mice
Significantly, neurological symptoms developed only in Tg(WT-E3+/+) mice that were homozygous for the transgene array. Tg(WT-E3+/-) mice have remained healthy for >750 days. In addition, no neurological abnormalities were observed in homozygous Tg(WT) mice of the E1 and E4 lines, which over-expressed wild-type PrP at lower levels (see below).
Analysis of PrP levels in brain
We previously determined the level of transgenically encoded PrP relative to endogenous PrP by Western blotting of post-nuclear supernatants from brain, using the antibody 3F4 (Chiesa et al., 1998
). We have now performed a more accurate analysis of transgenic PrP expression by comparing the levels of PrP in the brains of Tg(WT-E3) mice to those in non-transgenic C57BL/6J x CBA/J mice as well as Tga
20 mice that over-express non-epitopically tagged, wild-type PrP (Fischer et al., 1996
). For these quantitative Western blots, we used 3F4 antibody, as well as an antibody directed against mouse PrP residues 45-66 that recognizes both transgenic and endogenous PrP. Moreover, to reduce the variability associated with the preparation of post-nuclear supernatants, we analyzed whole brain SDS extracts, and normalized the PrP signal to the level of actin. This analysis demonstrated that Tg(WT-E3+/+
) mice were the highest expressers, with brain PrP levels that were 9- or 12-fold higher than those in non-transgenic mice, depending on whether the mice had the Prnp0/0
background, respectively (). Tg(WT-E3+/-
) mice had PrP expression levels of 4X and 7X on the Prnp0/0
backgrounds, respectively ().
With regard to the other Tg(WT) lines, we found that Tg(WT-E1+/+
) mice expressed 3.6-fold more PrP than wild-type mice (data not shown), consistent with our previous analysis showing a PrP expression level of ~1.8X in Tg(WT-E1+/-
) mice (Chiesa et al., 1998
). In the Tg(WT-E4) line, the transgene was present on the X-chromosome, so that the only mice that were homozygous for the transgene were female. Due to X-inactivation, these animals had a PrP expression level (3X) similar to that of both male and female Tg(WT-E4+/-
) mice (data not shown). Thus, mice with PrP expression levels of 1.8X-7X remain healthy, while those with expression levels of 9-12X develop neurological symptoms.
PrP in Tg(WT-E3) mice is mildly protease resistant and forms insoluble aggregates that are recognized by an aggregation-specific antibody
We previously reported that the development of neurological illness in Tg(PG14) mice is associated with accumulation in the brain of a non-infectious form of mutant PrP that is insoluble in non-denaturing detergents, and that yields a 27-30 kDa protease-resistant fragment upon digestion with low concentrations of PK (Chiesa et al., 1998
; Chiesa et al., 2000
; Chiesa et al., 2003
). We therefore tested whether Tg(WT-E3) mice accumulated an analogous form of WT PrP that was aggregated and weakly protease-resistant.
To test PrP solubility, detergent extracts of brain were subjected to ultracentrifugation and the amount of PrP in the supernatant and pellet fractions was evaluated by Western blot. As shown in , a small percentage of PrP was recovered in the pellet fraction of brain extracts from both Tg(WT-E3+/+) and Tg(WT-E3+/-) mice. In contrast, virtually no detergent-insoluble PrP was found in brain samples from non-transgenic mice expressing physiological levels of PrP (). The proportion of insoluble PrP was consistently higher in Tg(WT-E3+/+) mice than in Tg(WT-E3+/-) mice (5% compared to 10%). As expected, about 40% of the PrP from the brains of Tg(PG14) mice was found in the pellet fraction ().
A fraction of PrP in the brains of Tg(WT-E3) mice is insoluble and can be immunoprecipitated by 15B3 antibody
We have recently shown that the mAb 15B3, originally thought to specifically recognize PrPSc
, also detects a variety of misfolded and aggregated forms of PrP, including mutant PrP in the brains of Tg(PG14) mice (Biasini et al., 2008a
; Biasini et al., 2008b
). We therefore tested whether 15B3 reacted with WT PrP aggregates in Tg(WT-E3) mice. We found that 15B3 selectively immunoprecipitated the small amount of PrP found in the insoluble fractions of Tg(WT-E3) brain homogenates (). The much larger amount of soluble PrP found in the supernatant fractions from these animals was not recognized (). The amount of 15B3-reactive PrP was greater in Tg(WT-E3+/+
) mice than in Tg(WT-E3+/-
) mice, paralleling the larger amount of insoluble PrP in the former animals. As a negative control, no 15B3-reactive PrP was detected in either the soluble or insoluble fractions from non-transgenic mice (). Moreover, as found previously (Biasini et al., 2008a
; Biasini et al., 2008b
), insoluble PrP in the brains of Tg(PG14) mice was selectively immunoprecipitated by 15B3 ().
Resistance to digestion by PK is another property of aggregated PrP (Chiesa et al., 2003
; Biasini et al., 2008a
). To assay this property, detergent extracts of brain were incubated with 1-3 μg/ml of PK at 37°C for 30 min. We found that PrP from Tg(WT-E3+/+
) mice was mildly PK resistant (), yielding small amounts of a PrP27-30 fragment similar in size to that found in Tg(PG14) brains ().
PrP in the brains of Tg(WT-E3) mice is mildly protease resistant
In order to better detect weakly PK-resistant PrP, we carried out the protease digestion assay at 4°C, following a protocol that has been used to detect protease-resistant PrP in the brains of Tg(MoPrP-P101L) mice (Tremblay et al., 2004
). Using this assay, PK-resistant PrP could be readily visualized in brain samples from both Tg(WT-E3+/-
) and Tg(WT-E3+/+
) mice, with the amount being higher in the latter animals (). A small amount of PrP was also detected after cold PK digestion of brain extracts from non-transgenic mice; however, this material corresponded to residual full-length PrP since it did not show a shift in molecular weight typical of PrP27-30 (). As a positive control, PrP27-30 was also detected after PK digestion at 4°C of samples from Tg(PG14) brain ().
Tg(WT-E3) mice display punctate PrP deposits
The most striking neuropathological feature seen in Tg(WT-E3) mice was the presence of numerous, coarse deposits of PrP in the molecular layer of the cerebellum, as revealed by PrP immunohistochemistry using two different antibodies. PrP deposits were seen in both healthy hemizygous animals () as well as clinically ill homozygous animals (Fig. and ); however, in homozygous mice they were more numerous and larger in diameter. Very mild and diffuse PrP immunoreactivity could be also detected in the granule cell layer (Fig. and ). Punctate PrP deposits were also seen in the olfactory bulb, neocortex, hippocampus, basal ganglia and brain stem, especially in homozygous mice (not shown). PrP deposits were already evident in a Tg(WT-E3+/+) mouse that was 171 days old. None of the PrP deposits stained with thioflavin S (not shown), indicating that they are not amyloid.
Neuropathological findings in the cerebella of Tg(WT-E3) mice
PrP deposits are detected by two different antibodies in Tg(WT-E3) mice, and are absent in non-transgenic control mice
PrP deposits were not seen in non-Tg mice, even when the animals were very old (). PrP immunoreactivity was occasionally found in older Tg(WT-E1+/+) mice in the olfactory bulb, and in the cerebellar cortex at the level of the Purkinje cell layer (). However, the degree of PrP deposition in Tg(WT-E1+/+) mice was much lower than in Tg(WT-E3) mice, and was variable.
Tg(WT-E3+/+) mice also showed severe cerebellar atrophy, accompanied by marked reduction in the number of granule cells and thinning of the molecular layer (). Milder cerebellar degeneration was seen also in Tg(WT-E3+/-) mice, which displayed moderate reduction in thickness of the cerebellar cortex compared to Tg(WT-E1+/+) mice (compare ).
Immunohistochemical staining using anti-GFAP antibody demonstrated prominent astrocytosis, predominantly in the cerebellum, of Tg(WT-E3+/+) mice, while astrocytosis was mild or absent in other areas showing PrP deposits. Staining was most prominent in the white matter (not shown), and in the molecular layer, which displayed markedly hypertrophied Bergmann glial fibers (). Less prominent GFAP staining was observed in the cerebellum of older Tg(WT-E3+/-) and Tg(WT-E1+/+) mice, particularly in the Purkinje and granule cell layers (). However, similar GFAP immunoreactivity was also observed in older non-Tg mice (not shown).
Tg(WT-E3) mice show synaptic pathology
Electron microscopic examination of the cerebella of both Tg(WT-E3+/+) and Tg(WT-E3+/-) mice revealed two kinds of abnormalities. First, as expected from the light microscopic analysis, we observed degenerating granule neurons which displayed fragmented, osmiophilic nuclei characteristic of apoptosis (). Second, prominent synaptic abnormalities were observed. The neuropil of the molecular layer contained synaptic terminals that were markedly enlarged, and contained redundant, spiral, membranous structures (). These membranes surrounded mitochondria and large numbers of synaptic vesicles in the cytoplasm (). Pre- and postsynaptic membrane specializations were well preserved, and collections of vesicles could be observed adjacent to the presynaptic membrane ().
Ultrastructural abnormalities in Tg(WT-E3) mice
Tg(WT-E3) mice show inter-nucleosomal DNA cleavage in the cerebellum
To confirm that neuronal degeneration in the cerebella of Tg(WT-E3) mice was apoptotic, as suggested by electron microscopic analysis (), we checked for the presence of inter-nucleosomal cleavage of DNA. When DNA was extracted from cerebella of Tg(WT-E3) mice and analyzed by gel electrophoresis, a characteristic 200 base-pair ladder was observed in Tg(WT-E3) mice (). DNA laddering was less intense in Tg(WT-E3+/-
) compared to Tg(WT-E3+/+
) mice (compare lanes 4 and 6), consistent with the lower degree of cerebellar atrophy and granule cell loss observed in the hemizygous mice. In Tg(WT-E1+/+
) no inter-nucleosomal DNA cleavage was detected (), whereas intense DNA laddering was observed in Tg(PG14) cerebella (lane 8), as previously reported (Chiesa et al., 2000
; Chiesa et al., 2005
Cerebellar DNA from Tg(WT-E3) mice shows inter-nucleosomal cleavage