The mechanisms regulating peripheral propagation of the different TSE strains are not yet fully understood. Different organs, cells, and routes have been shown to be important according to which strain of agent is infecting a particular host. Some strains require amplification in the LRS before causing a productive infection in the CNS, whereas with other strains, such as naturally occurring BSE in cattle, the involvement of the LRS is limited (3
). A number of factors are likely to contribute to this diversity in neuroinvasion, including strain, host PrP genotype, and the route of entry. Our results now suggest that glycosylation status of the host PrP may be an important factor in this process and, in particular, in determining the timing of neuroinvasion and the final targeting in the CNS.
appears to be required at the early stages of the infectious process, which may involve uptake and transport from the site of infection to the spleen or amplification of infectivity in the spleen. Clinical disease was observed in each of the PrP glycosylation-deficient mice following intracerebral inoculation with at least one strain (45
), albeit with very long incubation periods and limited susceptibility in the G3 mice. In this study this was not the case as following intraperitoneal inoculation the G3 mice appeared to show complete resistance to infection with two different TSE strains.
This may be due to an inability of unglycosylated PrPC to sustain the transport of infectivity from the periphery to the brain and/or vice versa. Therefore, PrP glycosylation plays a central role in the spread of infectivity from the periphery to the CNS. Moreover, our results suggest that replication or transport in the LRS is a limiting factor during spreading of TSE infectivity.
The role played by PrPC
glycotypes in the pathogenesis of disease appears to be strain specific. G2 mice developed disease after ME7 i.p. inoculation with only a short delay of 21 days compared to wild-type mice. Similarly, when ME7 was injected directly into the CNS, G2 mice developed TSE disease with incubation periods similar to those of wild-type mice (45
). Thus, the monoglycosylated G2 PrP protein appears to be able to replicate and transport the ME7 agent almost as efficiently as the diglycosylated PrP. However, ME7 fails to establish disease following peripheral challenge of G1/G1 and G3/G3 mice, and PrPSc
is absent from spleens and brains of these mice. This is in accordance with what was observed following direct inoculation of ME7 into the brain; although these transgenic mice did not develop any clinical TSE disease, G3 mice presented some degree of PrP deposition in the form of amyloid plaques (45
). Thus, the first glycosylation site appears critical for the replication of ME7 in both the CNS and periphery. Failure to establish disease is therefore likely to be due to a failure of the host to replicate the agent.
G1 and G2 homozygous mice were susceptible to infection following peripheral challenge with 79A; however, the incubation time was more than 100 days longer than for wild-type animals. This delay after peripheral inoculation is much longer than that observed following inoculation of the same strain intracerebrally; in G1/G1 and G2/G2 mice the incubation of clinical prion disease was 40 and 20 days longer, respectively, than in wild-type mice (45
). This suggests either that there is a difference in efficiency of replication of the agents in the periphery and the CNS or that lack of diglycosylated protein in this case is delaying the transport to the CNS. However, the incubation time in G2 mice was rescued when a wild-type PrP was expressed alongside the monoglycosylated PrP. In heterozygous PrP mice a gene dosage effect on the length of TSE clinical disease onset was observed due to the contribution of both alleles. This effect was previously described in wild-type/knockout PrP heterozygous transgenic mice (PrP+/−
) in which the incubation time was almost doubled compared to incubation in PrP wild-type homozygous animals (32
). In Wt/G2 mice it was therefore surprising to observe the appearance of clinical disease at the same time as in Wt/Wt mice when it might have been expected that incubation periods in these mice would fall somewhere between those of wild-type and G2 homozygous mice. This suggests that the mono- and diglycosylated forms of PrP can act together to facilitate replication and transport of the agent. This suggestion is also supported by the observation that heterozygous animals presented a brain pathology that was an intermediate between that observed in wild-type and G2 homozygous mice, indicating that both alleles are also contributing to the final targeting in brain. Thus, the provision of diglycosylated PrP clearly provides an important function in the disease process and can overcome the incubation time delays observed with only monoglycosylated and unglycosylated PrP. The discrepancy between the hybrid pathology and the wild-type incubation times observed in the heterozygote mice demonstrates that these are an important model for defining the mechanisms regulating the neuroinvasion of TSE infectivity, and further studies are under way to address this issue.
Host PrP glycosylation appears to determine the targeting in the CNS following peripheral challenge. Absence of fully glycosylated PrP had a profound effect in determining the brain area targeted by PrPSc
deposition after infection with 79A. G1 mouse brains were characterized by PrPSc
accumulation in very restricted areas like the habenula and the thalamus. This deposition was remarkably different from the characteristic widespread distribution of PrPSc
in many brain areas observed in wild-type mice. G2 homozygous animals also presented changes in 79A targeting compared to wild-type animals, with cortex and midbrain heavily affected. This different targeting may be due to an effect of host PrP glycosylation in determining routes of propagation of infectivity from the periphery to the CNS or to the fact that the transgenic mice developed clinical disease with a much longer incubation time than the wild types. However, even in cases where there were modest alterations in the incubation times, in the glycosylation-deficient transgenic mice there were dramatic differences observed in PrPSc
deposition. Absence of a fully glycosylated PrP appears to facilitate the deposition of amyloid plaque formation in the brain, compared with fine punctuate staining observed in wild-type mice after ME7 inoculation. This contrasts with the targeting in the brain following intracerebral challenge, where no difference in targeting was observed in the different lines of mice (45
). After inoculation of 79A or ME7 directly into the brain, clinically positive G1/G1 and G2/G2 mice, indeed, did not present any differences in terms of spongiform degeneration or PrPSc
deposition compared to wild-type animals. Wild-type and glycosylation-deficient mice had the same vacuolation profiles and the same widespread, fine punctuate PrPSc
depositions throughout the brain (45
). These results may also suggest that the delay observed in the transgenic animals may be due to an initial targeting of infectivity in different brain areas that may delay the neurotoxic effect, as previously proposed (24
). Whether differences in routing to the CNS are responsible for the differences in targeting observed in the glycosylation-deficient mice remains to be established.
A difference in brain pathology between wild-type and glycosylation-deficient mice was also confirmed by the analysis of vacuolation damage in the CNS. Differences were observed in G1 and G2 mice after infection with both 79A and ME7 in several brain areas. This analysis also revealed a discrepancy between PrPSc
accumulation and spongiform degeneration. For example, the strong accumulation of PrPSc
observed in the cortex of G2 but not of wild-type animals was not linked with a difference in terms of spongiosis in the same areas. This observation may support previous observations suggesting that PrPSc
presence is not always associated with vacuolation (2
In summary, we have shown that PrP is a requirement for the peripheral events in TSE disease. In this process PrP glycosylation is a key component to determine the timing of neuroinvasion and the targeting in the CNS, and this effect varies according to TSE strain. Indeed, a fully glycosylated PrP molecule is required for the most efficient neuroinvasion by 79A. However, ME7, while less dependent on a fully glycosylated PrP, appears to be totally dependent on glycosylation at the first site. Finally, the results presented here indicating a role of N-linked glycans in the spread of TSE infectivity may have some important implications for the development of new therapeutic approaches. For example, destabilizing sugar-mediated interactions between host and the infectious agent would appear to provide an important approach to block propagation of infectivity.