Impaired motor coordination on static rods in BSE-infected mice

Scrapie and bovine spongiform encephalopathy (BSE) are both progressive neurodegenerative diseases that are transmissible to mice. The onset of clinical symptoms is more subtle and variable in murine BSE than in murine scrapie. Assessment of behavioural changes that occur throughout disease would aid early diagnosis of disease so that more consistent end points could be made and potential therapies could be investigated. C57BL/6J mice inoculated via the intraperitoneal route with 301C BSE or control inoculum were monitored on a fortnightly basis. The end point was when a mouse showed clinical signs as opposed to behavioural signs of BSE for two consecutive observations. Significant loss of motor function, as assessed by mice balancing on a static rod, was observed consistently from approximately 40 days prior to death. No significant differences in home cage activity (locomotion, rearing) or cognitive function (T-maze alternation) were observed. However, there was an increase in digging by BSE-infected mice from an early stage. This data will aid the standardisation of behavioural tests to characterise and assess the onset of BSE.     

Differentiation of prion protein glycoforms from naturally occurring sheep scrapie,sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies

A panel of ruminant brain tissues were subjected to a Western immunoblotting technique using two monoclonal antibodies (mAbs). The resultant prion protein (PrP) glycoforms showed that three distinctions can be made between natural ovine scrapie cases and sheep experimentally inoculated with bovine spongiform encephalopathy (BSE). Differentiation between BSE-infected cattle and natural cases of sheep scrapie was also possible using these two antibodies. There were subtle differences in the molecular weight positions of the di-glycosylated, mono-glycosylated and unglycosylated forms of the abnormal PrP (PrP(Sc)) associated with these ruminant transmissible spongiform encephalopathies. In particular, a distinct difference for the unglycosylated protein band was observed. For ovine scrapie samples, this band was noticeably of a higher molecular weight than that found for brain samples from the Romney and Cheviot breed sheep infected with BSE and, to a lesser degree, higher than that observed for bovine BSE samples. Using the comparison of glycoform ratios, the technique provided a distinction between the sheep experimentally infected with BSE and natural cases of sheep scrapie but did not provide a distinction between natural cases of bovine BSE and ovine scrapie. The sheep-passaged CH1641 scrapie strain gave molecular weights similar to, but not identical to BSE, and a glycoform ratio similar to ovine scrapie cases. The SSBP1 experimental scrapie strain gave molecular weights that were akin to natural scrapie cases but the glycoform ratio was different to that found for all the other samples. When mAb P4 was substituted for mAb 6H4 in the technique, only the natural scrapie samples and SSBP1 gave strong signals. BSE in sheep and the CH1641 strain gave weak reactions and PrP(Sc) from BSE-infected cattle could not be detected at all. The results suggest that this combination of molecular weight and glycoform ratio analyses, and differentiation with two specific antibodies could be used to provide a possible screening test for BSE in the UK sheep flock, if confirmed as accurate by bioassay and lesion profile analysis in mice inoculated with brain tissue from suspect field cases.     

Scrapie-specific neuronal lesions are independent of neuronal PrP expression

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.     

Exploratory behavior of F2 crosses of mouse lines selected for different brain weight: a multivariate analysis

Principal component analysis of behavioural measures together with body and brain weight of hybrid F2 mice crosses between two lines selected for large (LB) and small (SB) brain weight yielded eight-factor solution explaining 75.1% of total variance. Two of eight factors had sufficient loading on brain weight and several behavioural measures. The factor analysis showed that, among F2 hybrids, mice with larger brain weight were characterised, in open-field test, by higher scores of locomotion in the periphery of arena and of rearing, as well as less frequent grooming and freezing than mice with smaller brain weight. F2 hybrids with larger brain weight moved faster and displayed stereotyped behaviour in the cross-maze test more frequently. In general, this diversity is in accord with the behaviour differences between parent LB and SB lines. The results show that, in mice fear-anxiety and stereotypic behaviours, which are known to interfere with normal exploration and learning of the environment, are causally connected with brain weight.     

Comparisons between C57BL/6J and A/J mice in motor activity and coordination,hole-poking,and spatial learning

The C57BL/6J (B6) inbred mouse strain was compared to the A/J inbred strain for motor activity in an open-field, exploration of a hole-board, motor coordination in the coat-hanger test, and spatial learning in the Morris water maze. B6 mice displayed a higher number of segment crossings in the open-field and of hole-poking responses than A/J mice. The performance of B6 mice was superior to that of A/J mice not only in the submerged but also in the visible platform version of the Morris water maze. By contrast to their hypoactivity, the A/J strain had shorter movement times in the coat-hanger test, indicating faster motor speed, although the groups did not differ in latencies before falling. These results indicate that recombinant inbred or congenic strains derived from B6 and A/J mice offer considerable potential for discerning the genetic basis of several behavioral phenotypes.     

Scrg1 is induced in TSE and brain injuries, and associated with autophagy

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.     

A rapid accurate culture assay for infectivity in Transmissible Encephalopathies

The molecular and structural features of infectious agents that cause CJD, scrapie and BSE remain controversial. A major impediment for agent resolution is the very long and expensive animal assays of infectivity. It is crucial to develop a rapid and broadly applicable cell culture assay to titer and compare different TSE agent strains. Because we found GT1 hypothalamic cells, unlike neuroblastoma N2a clones, were highly susceptible to a variety of TSE agents, and could stably produce high agent titers for >1 year, we studied the progressive display of abnormal prion protein (PrP-res) in GT1 cells following exposure to serially diluted 22L scrapie brain homogenates; PrP-res was used as a surrogate, but non-quantitative marker of GT1 infection. Even as early as the first cell split after 22L exposure, GT1 cells produced their own PrP-res bands that were clearly different than brain bands. Plots from passages 3–7 showed a good discrimination of 3 fold differences in titer over a range of > 2 logs, with the same endpoint sensitivity (2 × 10⁸ LD₅₀/gm) as animal assays. Interestingly, the rapid production of de novo PrP-res suggested that GT1 PrP-res might be induced by interaction with an early-intermediate form of a particle that was not fully infectious. The GT1 assay here was also invaluable for rapidly identifying cell cultures with variant titers, even after detergent lysis. Additionally, in-situ PrP amyloid staining provided an independent measure of the minimum infectious dose per cell. Standardized GT1 assays can be used for direct comparison of different agent strains, and will facilitate the rapid isolation of essential agent components.     

Early behavioural changes in scrapie-affected mice and the influence of dapsone

Behavioural testing can reveal effects in scrapie-infected mice long before overt clinical signs appear (Betmouni et al., 1999, Psychobiology, 27, 63-71). These effects may be partly attributable to an early, atypical inflammatory response in the brain (Betmouni et al., 1996, Neuroscience, 74, 1-5). The present study replicated and extended these findings, and examined the effect of chronic treatment with dapsone. This anti-inflammatory compound has been reported to delay disease onset in a rat model of Creutzfeldt-Jakob disease (Manuelidis et al., 1998, Lancet, 352, 456). Although the doses used in the present study were higher than those of Manuelidis et al. (1998), no attenuation of the disease was seen in either behavioural or subsequent histological tests. Burrowing, i.e. displacing food pellets from a tube in the home cage, decreased from around week 12 in scrapie-infected mice, as did consumption of palatable glucose solution. Concurrently, ambulation in an open field increased, as did rearing at around week 17. Spontaneous alternation was impaired around this time. Around 18 weeks, motor performance on an inverted screen, horizontal bar, rotating rod and static rods decreased. Nest construction was impaired at 20 weeks. Overt clinical signs (reduction in mobility, hunched posture, poor coat condition, bladder enlargement) only occurred after week 20, when the mice were prepared for histology. The ME7 scrapie-infected mice thus showed a characteristic complex of neurological and behavioural changes during the course of the disease that were not ameliorated by dapsone. These changes appeared well before clinical signs were prominent.     

Comparison of the neuropathological characteristics of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) in mice

Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) belong to a group of diseases called the transmissible spongiform encephalopathies (TSEs). Transmission studies in inbred mice (strain typing) provided overwhelming evidence that vCJD arose from BSE. In this study, we compare the patterns of neuropathology in a panel of three inbred mouse strains (RIII, C57BL and VM) and one cross (C57BL x VM) infected with either vCJD or BSE. For each mouse strain, patterns of abnormal prion protein (PrPres) deposition, astrocytosis and vacuolation were similar in the vCJD- and BSE-challenged mice. Prion protein (PrP)-positive plaques were prominent in the VM and C57BL x VM mice in addition to diffuse PrPres accumulation, whereas only diffuse PrPres labelling was observed in the RIII and C57BL mice. The hippocampus was targeted in all mouse strains, as was the cochlear nucleus in the medulla, both showing consistent severe vacuolation and heavy PrPres deposition. Although the targeting of PrPres was similar in the BSE- and vCJD-infected brains, the amount and intensity of PrPres observed in the brains treated with formic acid during fixation was reduced considerably. The distribution of astrocytosis was similar to the targeting of PrPres deposition in the brain, although some differences were observed in the hippocampi of mice challenged with vCJD. We conclude that there are no significant differences in the targeting of neuropathological changes observed in the BSE- and vCJD-infected mice, consistent with the previous evidence of a link between BSE and vCJD.     

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.     

Genetic control of apomorphine-induced climbing behavior in two inbred mouse strains

Over a wide dose range (1–32 mg/kg), apomorphine-induced climbing behavior was significantly greater in the AKR/J than in the DBA/2J inbred mouse strain. A similar difference was found when apomorphine-induced stereotypy was examined. A cross-fostering study demonstrated that the strain difference in apomorphine-induced climbing behavior did not result from differences in post-natal rearing environment. After apomorphine administration, brain levels of the drug were similar in the two strains indicating that the difference in behavioral response to apomorphine in the two strains was not due to differences in metabolism or distribution of the drug. The climbing response to apomorphine was examined in the F1 cross of the two strains (AKD2F1/J) and in 10 AKXD recombinant inbred strains. Results suggested that the trait was partially dominant and not X-linked; furthermore, a few and possibly one locus was responsible for the differences in apomorphine-induced climbing behavior observed in the AKR/J and DBA/2J mice.     

Focal and asymmetrical vacuolar lesions in the brains of mice infected with certain strains of scrapie

Summary In mice experimentally infected with most strains of scrapie, vacuolar degeneration almost always shows a bilaterally symmetrical distribution in the brain. However, asymmetrical foci of vacuolation are frequently seen with a group of six mouse-passaged isolates from diverse natural sheep sources (designated 31A, 51C, 87A, 125A, 138A and 153A). As these isolates are similar in other respects they may be independent isolations of the same strain of scrapie. The distribution of focal vacuolar lesions in C57BL mice affected with 87A scrapie was found to depend on route of infection. In mice injected intracerebrally into the left or right hemisphere, all focal asymmetrical lesions occurred on the side of injection, in some cases intense vacuolation being associated with the needle scar. following midline intracerebral injection, focal lesions were evenly distributed between the two sides and were most frequent in the medial areas of the thalamus. In one mouse injected intraocularly, intense unilateral lesions were seen contralaterally in the major retinal projection regions. Asymmetrical lesions also occurred following infection by intraperitoneal, intravenous and subcutaneous routes, but were less frequent than after intracerebral infection. The most likely explanation is that focal asymmetrical lesions result from focal replication of scrapie infectivity in the brain. As all the scrapie strains which frequently produce asymmetrical vacuolation are also those that generate mutants, it is possible that focal lesions represent foci of the new mutant strain, replicating preferentially in areas with a low background level of the parent strain.     

Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model

Prion strains yield specific neuropathological features including spongiform degeneration and deposition patterns of pathological prion protein. Their invariant regional distribution, following variations in the infection route, has led to the proposal that prions replicate preferentially in defined neuro-anatomical areas. The molecular mechanisms underlying this apparent strain-specific neuronal tropism are currently unknown. However, a possible explanation may be that prion replication is relatively innocuous, resulting in long-term propagation, thus masking initial regional distribution variations linked to different infection routes. This “low neurotoxicity” may be imputable either to the rodent model used or the prion strain(s) inoculated. To investigate this possibility, we studied prion pathogenesis in a prototypal short-incubation disease model consisting of 127S scrapie strain propagated in tg338 transgenic mice expressing the VRQ allele of ovine PrP. This prion strain derives from a natural sheep scrapie isolate that was serially transmitted to tg338 mice without any obvious transmission barrier and biologically cloned by limiting dilution. We compared the pathology induced by the peripheral or intracerebral inoculation of 127S strain. Surprisingly, we found that the disease greatly differed in clinical signs, abnormal prion protein levels, and neuropathology among the routes of infection. Secondary transmission performed with brain material from mice inoculated either intracranially or intraperitoneally produced similar neuropathological features. These results therefore indicate that the route of infection can strongly influence the apparent phenotype of a scrapie strain.     

Characteristics of scrapie isolates derived from hay mites

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.     

Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice

Prion diseases are fatal neurodegenerative disorders of humans and animals, which include bovine spongiform encephalopathy (BSE) and its human form, variant Creutzfeldt-Jakob disease (vCJD). They are characterized by a prolonged incubation period, which is known to be influenced by polymorphisms in the prion protein gene. Previous studies of inbred mice have demonstrated that additional genetic loci also contribute to the observed variation in incubation period. However, a substantial transmission barrier between cow and mouse complicates studies using BSE. As a result, primary transmissions display large variations in incubation period and not all animals develop clinical signs of disease. To identify quantitative trait loci for BSE without the presence of a transmission barrier, we analysed 124 animals from an F2 intercross between CAST/Ei and NZW/OlaHsd mice and challenged them intracerebrally with a strain of BSE that was passaged twice through C57BL/6OlaHsd mice. Interval mapping identified two highly significant linked regions on chromosomes 2 and 11 with peak lod scores of 6.34 and 4.77, respectively. Composite interval mapping suggests that chromosome 2 includes three linked quantitative trait loci. Loci in the same position on chromosomes 2 and 11 were also identified in a previous study using the same mouse cross but infected with Chandler/RML scrapie prions. If these are the same loci, it suggests that these loci may be influencing incubation time independently of prion strain. This provides hope that it may be possible to identify human quantitative trait loci for prion incubation time using mouse models that may allow identification of at-risk individuals and the discovery of novel therapeutic targets. Electronic Publication     

IMMUNOSTAINING OF SCRAPIE CEREBRAL AMYLOID PLAQUES WITH ANTISERA RAISED TO SCRAPIE–ASSOCIATED FIBRILS (SAF)

Brain sections from 16 different mouse scrapie models were immunostained with antisera to scrapie-associated fibrils (SAF) from three experimental scrapie sources (hamster 263K, mouse ME7 and mouse 22L). These models involved seven strains of scrapie injected intracerebrally or intraperitoneally into a range of inbred mouse strains, producing a wide variety of neuropathological changes. The only brain structures which were positively immunostained were amyloid plaque cores in those models in which plaques could be readily identified using traditional amyloid stains. The intensity of immunostaining correlated with the density of amyloid in the cores, as detected by Congo red and thioflavine S staining. No differences in immunostaining specificity were found between antisera or between plaques in different combinations of scrapie strain and mouse genotype. There were also no differences in immunoreactivity between plaques in different parts of the brain. These results strongly suggest that SAF and histologically detectable amyloid in scrapie mice are derived from the same precursor protein. Scrapie-associated cerebrovascular amyloid and plaques in sheep and goats also gave positive immunostaining with SAF antisera, although the lesions in the natural disease could only be stained after formic acid pretreatment. Senile plaques in Alzheimer's disease and Down's syndrome, although structurally similar to scrapie amyloid plaques, were found to be completely negative for SAF, in agreement with previous biochemical and immunocytochemical findings.     

Appearance of tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie preceeds the onset of clinical disease

Summary We have consistently observed tubulovesicular structures in brain tissues during the terminal stages of naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species and virus strain. In NIH Swiss mice inoculated intracerebrally or intraocularly with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, tubulovesicular structures, measuring 20–50 nm in diameter, were particularly prominent in dilated, pre-and postsynaptic neuronal processes, occasionally being mixed with synaptic vesicles. These structures appeared 13 weeks following intracerebral inoculation, 5 weeks before the onset of clinical signs, when spongiform changes were also detected. The number and density of tubulovesicular structures increased steadily during the course of clinical disease, and were particularly abundant in mice 47 to 51 weeks after intraocular inoculation. In hamsters infected with the 263 K strain of scrapie virus, these structures were initially detected 3 weeks following intracerebral inoculation and increased dramatically at 10 weeks postinoculation. The appearance of tubulovesicular structures before the onset of overt disease in mice inoculated with CJD virus by either the intracerebral or intraocular route, and before the appearance of other neuropathological changes in hamsters infected with scrapie virus, indicate that they represent either a part or aggregate of the infectious virus or a pathological product of the infection.Presented in part at the 64th annual meeting of the American Association of Neuropathologists, held in Charleston, South Carolina, June 9–12, 1988 and at the 7th annual meeting of the American Society for Virology, Austin, Texas, June 12–16, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Ministry of Health and Social Welfare, Poland     

Brain glutamate decarboxylase and cholinergic enzyme activities in scrapie

C57BL/6J mice, age 6-8 weeks were inoculated intracerebrally with brain homogenate from mice previously infected with the 139A strain of scrapie; control mice were identically treated with brain homogenate from non-infected normal mice. The activities of choline acetyltransferase (CAT), acetyl cholinesterase (AChE), and glutamic acid decarboxylase (GAD) were determined in the forebrain and hindbrain of these animals after 67, 126 and 151 days post-inoculation. There were no significant differences in the activities of CAT and GAD between scrapie and control mice at early, middle or late stages of the disease in the scrapie-infected animals; there was an about 20% decline in AChE activity in the scrapie brain.     

Re-establishment of stimulatory estradiol effects on luteinizing hormone secretion in long term ovariectomized rats

The effects of the scrapie agent on the levels of monoamines and their metabolites, and on choline acetyltransferase (CAT) activity have been investigated in discrete brain areas in the rat. Two strains of scrapie (8745 from sheep brain and C506 M3 from mice brain) were inoculated. Scrapie-infected rats showed a reduction in the levels of serotonin (prefrontal cortex, hippocampus, striatum) and dopamine (striatum) and an elevation of 5-HIAA levels (cerebral cortex, striatum, thalamus). Noradrenaline levels were decreased only in the cerebral cortex and cerebellum of rats infected with the scrapie strain C506 M3. CAT activity remained unchanged. These data suggest that the scrapie agent causes a derangement of noradrenergic, serotonergic and dopaminergic systems in the rat brain.