We have demonstrated in vivo that glycosylation of host PrP is not necessary to establish a TSE. Mice expressing only unglycosylated PrP (G3 mice) are capable of supporting a TSE, albeit in a strain-dependant manner, but disease susceptibility is reduced in response to the lack of glycans on host PrP. This differs from a previous study that reported that transgenic mice expressing unglycosylated PrP were resistant to two TSE strains [3
]. There are, however, considerable differences between these two studies that may account for the discrepancy including: (i) the expression of hamster rather than mouse PrP genes, (ii) different TSE strains, and (iii) the method of transgenic mouse production. Gene targeting was used to generate the mice described in this study, thus the altered PrP gene is in the normal chromosomal location and under the PrP gene (Prnp
) control elements. Whereas a previous study by De Armond et al. used transgenic mice generated by random integration of the PrP transgene [3
], which can result in disruption of genes at the site of integration, ectopic expression due to the influence of surrounding control elements, as well as high levels of transgene expression, all of which may affect the outcome of disease [14
]. Indeed PrP glycosylation mutant transgenic lines produced by random integration and expressing the same transgene have been reported to display different incubation periods with the same TSE strain, preventing direct comparisons between different lines [17
Our glycosylation-deficient PrP transgenic mice have both an alteration in amino acid sequence and glycosylation. Mutation of either asparagine or threonine in the NXT glycosylation consensus site of murine PrP will prevent the attachment of glycans to asparagine. We altered asparagines at both consensus sites to threonine, and this is regarded as a conservative amino acid alteration. The PrP expressed in our G1 and G2 transgenic lines is localised primarily at the cell surface in a manner comparable to that in wild-type mice and displays no detectable PrPSc
-like properties as determined by PK sensitivity, cleavability by phosphatidylinositol-specific phospholipase C (PIPLC), and detergent solubility assays [13
]. Moreover, remarkable similarity in the course of infection in parts of this study argues against the substituted amino acids affecting the course of disease but rather that the differences observed can be attributed to differences in glycosylation.
The observation that G3 mice are capable of supporting TSE disease is surprising given that the cellular location of the unglycosylated PrP appears primarily intracellular [13
]. This suggests that TSE disease can occur with perhaps complete absence or at least very low levels of PrP on the cell surface. 79A may be able to infect the cell via a mechanism independent of cell surface expression of host PrPC
, suggesting that transit of normal PrP to the surface of the cell [17
] may not be an absolute requirement for the development of TSE disease. PrPSc
accumulation in the TSE infected animal is considered to occur through the interaction of PrPSc
with host PrPC
, resulting in conversion of PrPC
. We have established that glycosylation of host PrP is not a requirement for the formation of de novo–generated PrPSc
in vivo, which is in agreement with in vitro studies [19
]. However, there is still uncertainty as to where PrPC
conversion occurs, with differing reports that it takes place either at the cell surface, extracellularly, in microsomes, along endocytic pathways, or in the cytosol [21
]. In our studies, the primarily intracellular location of the unglycosylated PrP in G3 mice suggests that either the very low levels of cell surface PrPC
expression is adequate to allow the conversion process to occur, or that conversion does not occur at the cell surface; in vitro studies will be necessary to resolve where conversion actually occurs in the G3 mice.
We have shown for the first time, to our knowledge, that glycosylation of PrPSc
is not necessary for the transmission of TSE infectivity to wild-type mice. This study also revealed that the de novo PrPSc
generated in the recipient animals consisted of di-, mono-, and unglycosylated PrPSc
, whereas the donor PrPSc
was fully unglycosylated. This is perhaps surprising, considering it is postulated that the donor PrPSc
acts as a template for its amplification [26
], yet our results suggest that the host dictates the glyscoylation status of the de novo–generated PrPSc
, rather than that of the donor as it has also been suggested in some in vitro experiments [27
]. The effect of host on PrPSc
glycosylation should therefore be considered when glycoform analysis, which determines the ratio of di- and monoglycosylated PrPSc
, as well as the relative mobility of unglycosylated PrPSc
, is used to identify TSE strains [29
] and classify newly emerging ones [12
In contrast to the results obtained using 79A, we found that ME7 was unable to cause clinical TSE disease in mice expressing only unglycosylated PrPC
. G1 mice (which have glycans only at N196) appear also resistant to TSE disease following ME7 inoculation, whereas G2 mice are fully susceptible to TSE disease with this strain. The fact that the cellular location of PrPC
in the G1 and G2 mice are comparable to each other and to wild-type mice [13
] indicates that the inability of ME7 to cause TSE disease in the G1 mice is not due to the cellular location of PrPC
but rather due to the absence of glycans at N180. Therefore, the inability of ME7 to cause clinical TSE disease in G3 mice may also be due to lack of carbohydrates at N180, rather than the low levels of PrPC
expressed at the cell surface.
G1 mice were found to be fully susceptible to TSE disease following 79A inoculation, but with a prolonged incubation time compared to wild-type mice. Similarly, the absence of glycans at N196 resulted in a prolongation of incubation period in 79A-inoculated G2 mice. With the cellular location of PrP in G1 and G2 mice being comparable to that in wild-type mice [13
], perhaps the prolongation of incubation period is due to the lack of glycans at N180 or N196, respectively, but whether this is due to a reduced efficiency of uptake, inefficient interaction of the infectious agent with PrPC
due to the lack of glycans at either site, or a slightly reduced efficiency of the disease process once the infectious agent has gained access to cells can not be determined from these studies. Experiments are underway to address this issue. In contrast to the observation that the lack of glycans at N196 has little or no effect on TSE disease resulting from inoculation with 79A or ME7, not all G2 mice inoculated with the TSE strain 301C showed clinical signs of TSE disease, and furthermore, those that did had a dramatic extension of the incubation period. However, clinical TSE disease was observed in the majority of 301C-inoculated mice, despite the increase in incubation period, which indicates that this strain does not have an absolute requirement for glycans at N196 but that the presence of them does allow for a more efficient infection.
Despite differences in susceptibility of G2 mice to infection with three different strains, the vacuolation profile of these mice and wild-type mice was similar, suggesting that glycosylation at the G2 site did not influence the strain targeting. Contrastingly, the 79A vacuolation profile in G1 was different when compared to wild type, indicating that glycosylation at the G1 site may have an effect on strain targeting. This finding will be further investigated in future experiments.
Clinical negative G3 mice infected with 79A or ME7 have shown the presence of PrP amyloid plaques in the brain. The formation of amyloid plaques is commonly associated with the pathological process of TSE disease as well as other neurodegenerative diseases [33
]. However an alternative view is that plaque formation may actually be a mechanism that the host uses to attempt to protect itself from the potentially harmful effects of oligomers [34
]. If this is the case in the TSE diseases, then the lack of clinical disease in the majority of the 79A-infected G3 mice (despite the presence of TSE infectivity as demonstrated by the transmission of TSE disease to wild-type mice) could indicate that the host is attempting to protect itself from the de novo generated abnormal PrP by sequestering it as amyloid. Interestingly, PrP-amyloid plaques have been detected previously in a transgenic mouse model expressing mutant PrP in the absence of clinical signs, spongiform degeneration, and infectivity [35
]. Thus a possible explanation for presence of plaques in clinical negative mice may be that amyloid formation can somehow block the propagation of disease within the brain. Our glycosylation mutant transgenic mice may therefore provide a model to investigate the involvement of PrP glycosylation on plaque formation in response to different TSE strains.
In conclusion, we have demonstrated that glycosylation of host PrP is not essential for the host to be susceptible to TSE disease. Moreover, glycosylation of PrPSc is not necessary for the transmission of TSE infectivity to a new host, and the glycotype of the host PrP has a major influence on the de novo generated PrPSc. Finally, we have shown that host PrP has a major role to play in TSE disease susceptibility and incubation period and that TSE strains differ dramatically in their requirements for host PrP glycosylation in order to allow TSE disease to occur.