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Animal Model |
From the Department of Neurological Science,*Tohoku University Graduate School of Medicine, Sendai, Japan; the Laboratory of Biomedicine,Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; CREST,Japan Science and Technology, Kawaguchi, Japan; and Creutzfeldt-Jakob Disease Surveillance Unit,Western General Hospital, Edinburgh, Scotland
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Abstract |
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150 days, familial CJD with M232R mutation, and dura graft-associated CJD (dCJD) without amyloid plaque; less susceptible to MM2-type sporadic CJD and variant CJD, with some mice lacking any sign of transmission; and totally resistant to VV2 type sporadic CJD and dCJD with amyloid plaque. The rather short incubation time achieved by Ki-ChM mice suggests new approaches to produce mice that develop prion disease with very short incubation periods. We compared the characteristic susceptibility profile of Ki-ChM with those of other precedent transgenic mice and discussed, including the prospects in developing genetically engineered mice susceptible to human prions.
Experimental transmission of human prions to animals still plays the major role in the investigations of human prion diseases, because the most important feature of prion, infectivity, cannot be fully reproduced or even evaluated precisely in vitro. However, the long intervals between the inoculation of prions into the animal and the development of illness have made transmission studies so time consuming as to hamper efficient investigation of prion diseases and make screening for prions by bioassay impractical. So, attempts to produce mice with short incubation periods have been incessantly made, and several lines of transgenic (Tg) mice have been developed.8-13 By transmitting different kinds of human prion diseases to such susceptible mice, comparative study of human prion strains could be efficiently performed and much about human prions has been revealed: for example, the importance of PrPSc conformation for the prion properties,2,5 and the origin of variant CJD being bovine spongiform encephalopathy.8,9 On the other hand, as with the case of chimeric-PrP-expressing Tg mice transmission studies, which indicated the existence of the third component involved in the prion replication,11 developing such mice itself may give insight into the molecular mechanism of prion propagation.
We recently produced a line of knock-in mice expressing a unique human-mouse chimeric PrP (ChM) that has a rather long central human insert and the same length of C-terminal mouse sequence with the chimeric MHu2M PrP; in other words, it has mouse sequence practically only toward the end of the C-terminal part, from codon 215 to codon 231, after posttranslational modification,14,15
and herein we transmitted different human prion diseases to the mice to test its versatility as a research and diagnostic tool. They proved to be highly susceptible to some types of human prion diseases and developed clinical disease within rather shorter incubation periods, 150 days, compared with the precedent Tg mice, while less susceptible or totally resistant to other types of prion diseases. The results of the present study with the novel genetically modified mouse confirm the importance of the PrPSc conformation in propagation of prions, and at the same time provide practical implications for developing mice with shorter incubation times and constructing systematic bioassay systems.
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Materials and Methods |
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The frontal cerebral cortical tissues were isolated at autopsy, on informed consent for research use, from patients with sporadic, inherited, and iatrogenic (dura-graft-associated CJD; dCJD) prion diseases and variant CJD (vCJD). Parts of the tissues were immediately deep-frozen for biochemical study and the other parts were formalin-fixed for pathological assay. The formalin-fixed tissue blocks were then immersed in 90% formic acid for 1 hour at room temperature to attenuate the infectivity.16
The diagnosis of prion disease was confirmed by identifying characteristic neuropathological findings such as spongiform change, gliosis, and PrP deposition of formalin-fixed brain sections,4,17
and by identifying PrPres extracted from the brain tissues on Western immunoblots as described.16
Typing of PrPres was performed based on the classification proposed by Parchi and colleagues:3,7
for sporadic CJD cases, PrPres of 21 kd was designated as PrPres type 1, and PrPres of 19 kd as PrPres type 2 (Figure 1A)
. PrP genotyping of patients was performed by polymerase chain reaction-direct sequencing using genomic DNA purified from brain tissues or peripheral blood leukocytes.18
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, Table 1
). All of the cases of vCJD, dCJD, and inherited prion diseases were homozygous for methionine at codon 129; as for the another polymorphic codon, codon 219, all of the cases collected were homozygous for glutamate, except for GSS102, which had lysine at codon 219 on the allele without P102L mutation.
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The production of the knock-in mouse expressing human-mouse chimeric PrP (Ki-ChM) used in the current transmission study has been described in detail before.14,15
Briefly, the chimeric molecule is based on mouse PrP sequence, in which the sequence between SmaI site and BstEII site were replaced by the corresponding human PrP sequence with methionine at codon 129. Consequently, after processing of the N-terminal signal peptide (residues 1 to 22) in the posttranslational modification, the molecule practically has mouse-specific PrP residues only at the C-terminus (Figure 2)
. The relative expression level of chimeric PrP in the brain ofKi-ChM was assessed by Western blot and proved similar to that of endogenous PrP in wild-type mice. A 10% (w/v) homogenate of the brain tissue from frontal cortex of each human prion disease was prepared in sterile phosphate-buffered saline using a disposable glass homogenizer and 20 µl was inoculated. The mice were examined daily after appearance of central nervous system dysfunction and were sacrificed if any sign of distress appeared or the diagnosis of prion disease was made. The criteria for clinical diagnosis of mouse prion disease were as described.19
At autopsy, the whole brain was cut mid-sagitally into two halves: the right half was immersion-fixed in 10% buffered formalin for histopathological analysis and the left half was immediately frozen for Western immunoblots.
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Neuropathological Examinations of Ki-ChM Mice
Formalin-fixed mouse brains were treated with formic acid to inactivate infectivity, embedded in paraffin, and then cut into 5-µm sections. Deparaffinized sections were pretreated with hydrolytic autoclaving before immunohistochemistry to enhance the immunoreactivity.20 PrP-N antiserum,21 rabbit-derived polyclonal antibodies raised against synthetic polypeptides corresponding to the N-terminal residues 25 to 49 of human PrP, was used as the primary antiserum and anti-rabbit immunoglobulins conjugated with peroxidase-conjugated dextran polymer, Envision (DAKO, Glostrup, Denmark), was used as the secondary antibody. Hematoxylin and eosin and Congo red staining was performed with standard techniques. The mice were judged neuropathologically positive when fine-granular PrP-immunopositive deposits, so-called "synaptic-type deposits," in the gray matter, or plaque-like PrP-positive deposits are recognized.
Western Immunoblotting of Ki-ChM Mouse-Derived PrPres
PrPres was extracted from the mouse brain tissues as described22 with a little modification. Briefly, brain tissues were homogenized in 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L NaCl, and 10 mmol/L MgCl2 with glass homogenizers. DNase1 (50 U, Takara, Bio Inc, Otsu, Japan) was added to the homogenate and incubated at 37°C for 60 minutes. Then 30% N-lauroylsarcosine (Sarcosyl) was added to final concentration of 10% and rehomogenized. The homogenate was centrifuged at 22,000 x g for 30 minutes. The supernatant was digested with proteinase-K (20 µg/ml; Wako Chemicals, Osaka, Japan), at 37°C for 60 minutes, and then centrifuged at 453,000 x g for 60 minutes (CP100H model, RP100AT6; Hitachi Koki Co, Tokyo, Japan). The final pellet was resuspended in 1x Laemmli’s sample buffer and boiled. For deglycosylation, PrPres denatured in 1x Laemmli’s sample buffer was treated with N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) in 100 mmol/L sodium phosphate (pH 7.5)/2% Nonidet P-40. The 3F4 monoclonal antibody23 was used as the primary antibody. Horseradish peroxidase-conjugated anti-mouse IgG was used as the secondary antibody. Enhanced chemiluminescence (Amersham Biosciences, UK) was used to visualize the blots on Hyperfilm ECL (Amersham Biosciences UK); for analysis of PrPres glycoform ratios images obtained through an imaging device, Versa Doc 5000, were quantified with Quantity One software (Bio-Rad Laboratories, California, USA).
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Results |
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The Ki-ChM mouse was highly susceptible to prions from patients with MM1-sCJD and MV1-sCJD with all of the mice inoculated succumbing to disease within quite short incubation periods, 150 days (Table 1)
. Ki-ChM mice proved to be also highly susceptible to prion diseases of other etiologies; after inoculation of np-dCJD and CJD232, an iatrogenic CJD and a inherited disease respectively, all of the mice developed clinical disease in 160 to 180 days. Although the incubation period for GSS102 was relatively long, 360 days, the occurrence of the clinical disease suggests fair susceptibility of the mice to this type of inherited prion disease. On the other hand, the Ki-ChM mouse was much less susceptible to the prions from MM2-T-sCJD and vCJD, with a few mice developing clinical prion disease only after more than 500 days after inoculation. Although the occurrence of clinical disease was rather low, many of the mice inoculated with vCJD prions showed pathological evidence of transmission as described below. However, among the mice inoculated with either MM2-T-sCJD or vCJD, there were individuals that completely lack any sign of transmission. As for VV2-sCJD and p-dCJD, Ki-ChM mice seemed totally resistant to them with none of the mice showing clinical or pathological sign of transmission. We graded the susceptibility of Ki-ChM mouse to a given human prion as either susceptible (when all of the mice inoculated developed clinical illness), less susceptible (when some mice survived without any clinical or pathological signs of prion disease), or resistant (when none of the mice had even pathological sign of prion disease).
End-Point Titration of MM1-sCJD Prion
After the inoculation with serially-diluted H-3, all mice developed clinical disease at dilution rates lower than 105: at the dilution of 10-1, the incubation time was 141 ± 9.2 days; at 10-2, 153 ± 7.3 days; at 10-3, 198 ± 12.3 days; at 10-4, 243 ± 58.5 days (Table 2)
. At dilution rates more than 105, only a fraction of mice inoculated developed illness, but the incubation time exceeded 300 days only toward the end-point: at 10-5, two of five mice inoculated developed illness with incubation time, 269 days; at 10-6, only one succumbed at 348 days. The infectivity titer of the brain tissue of the MM1-sCJD, H-3, was calculated as 7.8 logLD50 U/g.
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Pathologically, the brains of MM1- and VM1-sCJD-inoculated Ki-ChM exhibited diffuse spongiform changes typical of prion disease; iatrogenic CJD, np-dCJD, and inherited prion diseases, GSS102 and fCJD232, also exhibited similar changes (Figure 3B)
. These involved the cerebral cortex, caudate/putamen, hippocampus, thalamus, substantia nigra, superior colliculus, and to a lesser extent, the hypothalamus and pons. PrP deposition in the affected mouse brains was observed as fine granular synaptic-type deposits in gray matter (Figure 3, A and C)
. The intensity of the immunostaining grossly correlated with the severity of spongiform changes in multiple gray matter areas.
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; D to F). Some coarse granular PrP deposits were also observed in gray matter (Figure 3F)
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The pathology of vCJD-inoculated mice was unique. Spongiform changes and synaptic-type PrP deposition were observed only focally near the inoculation site (hippocampus, thalamus, or septal nuclei) and were much milder than those in MM1/VM1-inoculated mice (Figure 3; G, H, I, K, and L)
. Meanwhile, plaque-type PrP deposits were found in the periventricular white matter and in the gray matter near the inoculation site, mainly thalamus and hippocampus (Figure 3, G and J)
. When stained by Congo red dye, at least some of the plaques exhibited birefringence under polarized light, indicative of amyloid (data not shown).
Western Immunoblotting of Ki-ChM Mouse-Derived PrPres
To confirm the successful transmission of vCJD prions to Ki-ChM mice, PrPres in the brain tissue of vCJD-inoculated mice was examined by Western immunoblot (Figure 1B)
. Although PrPres was present also in the vCJD-inoculated Ki-ChM mouse as MM1- or MM2-T-sCJD-inoculated Ki-ChM mice, the unglycoform of the vCJD-inoculated mouse seemed to migrate slightly slower than that of the inoculated vCJD prion (Figure 1B
, lanes 5 and 6). The molecular mass of deglycosylated PrPres from the vCJD-inoculated Ki-ChM mouse was estimated as 20 kd because it took a mean position between those of MM1-inoculated mice (21 kd) and MM2-T-inoculated mice (19 kd) (Figure 1C)
; PrPres size was preserved through transmission for MM1- and MM2-T-sCJD but not for vCJD. The shift of PrPres size of vCJD prion through transmission to Ki-ChM mice may reflect the influence of the PrPSc conformation on transmission; a similar phenomenon was observed in transmission studies with Tg mice expressing PrP with 129V.9
Likewise, the relative ratio of PrPres glycoform was not always preserved through transmission. Although the dominance of di- and mono-glycoform relative to nonglycoform was preserved for vCJD, the glycoform ratios changed for MM1- and MM2-T-sCJD (Figure 1, A and B)
. The ratios of di-, mono-, and unglycoform of PrPres from MM1-sCJD patient (H-3) were 15%, 48%, and 37%, respectively, however, in the PrPres from the recipient Ki-ChM mouse the ratios of di-, mono-, and unglycoform were 51%, 42%, and 7%, and the diglycoform was the most abundant (Figure 1B
, lanes 1 and 2). Similarly, although the ratio of di-, mono-, and unglycoform of PrPres from MM2-T-sCJD (NK) was 8%, 44%, and 48%, respectively, diglycoform of the PrPres from the Ki-ChM accounted for 51% (Figure 1B
, lanes 3 and 4). As a possible explanation for the difference in glycoform ratios between PrPres from inocula and mice, the contribution of the effects of postmortem glycolytic changes cannot be ruled out because as for human samples the antemortem and postmortem conditions were not constant among cases, in contrast to those of mouse samples.
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Discussion |
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. Briefly, Tg mice expressing human PrP with 129V were highly susceptible to all of the human prions examined except for vCJD, although the incubation periods are not very short.8,9
Tg (HuPrP129M+/+ Prnp0/0) mice were as susceptible to prions with 129M as Tg mice expressing PrP with 129V but seem less susceptible to prions with 129V; as for vCJD, although they hardly developed clinical disease, all of the inoculated mice showed pathological signs of transmission of vCJD.12
Tg (MHu2M) Prnp0/0 mice were reported to be highly susceptible to some typical CJD with 129M, eg, MM1-sCJD, with short incubation periods, 200 days,10,11
and sporadic fatal insomnia as well,2
but seem less susceptible to prions with 129V5,11
and vCJD.13
Tg mice expressing another type of chimeric PrP, Tg(MHu2M, M165V,E167Q)Prnp0/0, which is distinct from MHu2M PrP in two residues at codons 165 and 167 showed dramatically short incubation periods, 110 days, for MM1-sCJD.13
Compared with those Tg mice, the susceptibility profile of Ki-ChM mouse is characterized by the rather short incubation periods, 150 days, for some types of prions including MM1-sCJD. The reason for the rather short incubation periods of Ki-ChM mouse is not obvious. In the above-mentioned Tg mice expressing MHu2M(M165V, E167Q) dramatically short incubation periods for MM1-sCJD were achieved by replacing the two amino acid residues at the C-terminal end of the central human sequence part to mouse residues.13
Because these residues are postulated to be involved in the interface with the third component in the conversion reaction, protein X, the abbreviated incubation periods could be presumably attributed to the improved interaction with the third component.24
On the other hand, in the chimeric PrP of Ki-ChM mouse the corresponding two residues remain as human residues, instead this chimeric PrP is different from MHu2M PrP in that the N-terminal part including the octapeptide repeat region is all human sequence after posttranslational modification. The role of the octapeptide repeat region in the conversion of PrPC to PrPSc and its propagation has not been defined. But given the facts that amplified repeats are associated with familial prion diseases,1
and the experimental fact that Tg mice expressing N-terminus-truncated PrP are relatively resistant to transmission of prions,25
it seems possible that the octapeptide repeat region plays some role in prion propagation, and N-terminal human sequence has contributed to the abbreviated incubation periods of Ki-ChM mouse. In combination with other mutations that are known to shorten incubation periods,13
chimeric PrP with all human sequence in the N-terminal part may further shorten the incubation.
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. Whether a mouse more susceptible to some types of prions becomes unsusceptible all of the more to other types of prions is a problem of practical importance; if so, the versatility of genetically modified mice designed to have short incubation periods may be limited to some specific prions. In the already reported transmission studies with Tg mice expressing other kinds of chimeric PrP, similar tendency was observed, too; they showed shorter incubation periods for MM1-sCJD prions than Tg mice expressing human PrP, however the former were less susceptible to other types of prion diseases than the latter (Table 3)
.8-13 The importance of the PrPSc conformation in the transmission is demonstrated in the presented results. Among the human prion diseases subjected to transmission to Ki-ChM mice in this study, the five prions, MM1-sCJD, np-dCJD, MM2-T-sCJD, p-dCJD, and vCJD, had the identical primary structure of PrP, however, their transmissibility varied greatly: MM1-sCJD and np-dCJD transmitted quite efficiently, MM2-T-sCJD and vCJD transmitted inefficiently, and p-dCJD did not transmit at all. Given that the PrPres sizes reflect the PrPSc conformation to some degree,26,27 the efficiently transmitting two prions and the inefficiently transmitting two could have rather different PrPSc conformation because MM1-sCJD and np-dCJD prions have PrPres of 21 kd, whereas MM2-T-sCJD and vCJD have PrPres of 19 kd. This indicates that the PrPSc conformation has more influence on the transmission efficiency of prions than the primary structure, being consistent with recently reported experiments.28,29 In addition to the identical primary structure, the PrPres of the three prions, MM1-sCJD, np-dCJD, and p-dCJD have the identical PrPres size of 21 kd, being practically indistinguishable from one another;16 despite that, p-dCJD never transmitted to the Ki-ChM mouse, whereas the other two prions transmitted very efficiently. Such heterogeneity in transmission properties among prions with the identical type of PrPres and identical etiology is also observed in other transmission studies;11,13 this suggests a considerable heterogeneity among the prions categorized into the same group based on the properties of PrPres and etiology. According to protein-only hypothesis, this may stem from a subtle structural variation of PrPSc that does not manifest in the currently recognized properties of PrPres, but transmission experiments could detect the difference.
Although much remains to be elucidated about the mechanism of prion propagation, it seems true that the infectivity titer of a prion varies among different hosts depending on the host-encoded PrP, for instance a vCJD case, 96/02, exhibited quite different transmissibility to Ki-ChM and Tg(MHu2M, M165V,E167Q)Prnp0/0, and that each of the many human prion strains has its optimal host-encoded PrP, which makes the host animal very susceptible to the prion and develop illness in rather short incubation periods. Assessing the transmissibility profile of a given human prion to a panel of Tg or knock-in mice with different primary structures of PrP, and specifying the optimal host-encoded PrP would facilitate in developing more accurate classification of human prions. On specifying the optimal PrP for a prion, quantitation of the infectivity of the prion for the mouse by end-point titration should be taken into account as well as the length of the incubation periods. The present study sets an example; as for Ki-ChM mouse, the infectivity titer of a case of MM1-sCJD, H-3, which transmitted to this line of mice in 150 days, was determined to be 7.8 logLD50 U/g. This parameter is important because such titrated infectivity would not only allow objective comparison between different lineages of mice, but also would guarantee the reliability of the mouse as a bioassay tool for the type of prion. In addition, the accumulation of such systematic data would help develop genetically engineered mice suitable for bioassay for various human prions, and, moreover, advance our understanding of the molecular mechanism of prion propagation.
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Footnotes |
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Supported by a grant from the Organization for Pharmaceutical Safety and Research (to S. M. and T. K.); a grant from the Ministry of Health, Labor, and Welfare, Japan (to S. M. and T. K.); and grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to T. M. and T. K.).
Accepted for publication August 14, 2003.
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