Transmissible spongiform encephalopathies (TSEs) are a group of fatal
neurodegenerative diseases. They can be readily transmitted within a host species but
are rarely transmitted between different species. This "species barrier''' effect is
characterised by reduced susceptibility of exposed novel hosts and an increase in the
length and variability of the incubation period. The mechanisms that underlie this
barrier are yet to be fully understood; hence it is currently not possible to accurately
predict the host range of a novel TSE disease. During TSE pathogenesis the host
protein, PrP, misfolds and accumulates within the central nervous system. This
misfolded form is denoted PrPˢᶜ to discriminate it from the normal cellular form PrPᶜ.
The prion hypothesis proposes that PrPˢᶜ is the TSE infectious agent and propagates
its aberrant conformation by inducing PrPᶜ to misfold. PrP is variably glycosylated at
two sites in vivo, such that di-, mono- and un-glycosylated glycotypes are observed;
all of these PrP glycotypes can form PrPˢᶜ. However the role of each glycotype in
cross species transmission is unclear. In vitro conversion experiments have suggested
that PrPᶜ's N-glycans specifically retard the cross species PrPˢᶜ seeded conversion of
PrPᶜ. Thus glycosylation of PrPᶜ may inhibit cross-species TSE transmission.Previous studies have not examined the role of glycosylation of PrPᶜ in the crossspecies transmission of TSE disease. In this study glycosylation deficient transgenic
mice [NPU] were challenged with three non-murine TSE agents. A barrier to crossspecies transmission was observed in normally glycosylated control mice, although a
proportion of animals did develop signs of TSE disease after challenge with hamster
scrapie (263K) and variant CJD (vCJD). Transgenic mice that lack both N-glycan
attachment sites were resistant to cross-species TSE challenge (263K, vCJD and
sporadic CJD). Moreover, absence of N-glycans at the first site only, resulted in a
significant decrease in disease incidence after challenge with 263K. These data
suggest that glycosylation of the first site mediates the transmission of TSE between
species. However, this effect is not unique to cross-species transmission as previous
reports have demonstrated that the within-species transmission of TSE is also delayed
by the absence of the first N-glycan attachment site. Transgenic mice which lack
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glycosylation at the second site were shown to have a higher disease incidence than
controls after cross-species challenge with 263K or sporadic CJD. Therefore,
glycosylation of the second site inhibits the cross-species transmission of these
strains. This effect is specific to cross-species transmission as previous murine TSE
transmissions to these mice have not demonstrated an acceleration of disease. Thus
the site of glycosylation determines the role of the N-glycan in cross-species
transmission. Moreover, here it is shown that mice which lack the second N-glycan
attachment site exhibit a reduced disease incidence after challenge with vCJD
compared to control mice. Therefore, TSE strain determines the role of N-glycans in
disease transmissionTo further interpret these transmission studies, the localisation of PrPᶜ in the
glycosylation deficient transgenics was investigated using confocal microscopy. No
significant difference in the cellular localisation of PrPᶜ was detected in mice which
lack the first or second N-glycosylation sites. Therefore, the effect of N-glycan
attachment at these sites on TSE transmission occurs independently of PrPᶜ's cellular
location. Lower levels of anti-PrP signal were detected in the neuropil of mice that
lack both N-glycan attachment sites than in control animals, suggesting a possible
mechanism for the enhanced resistance of these transgenic mice to TSE challenge.Normally glycosylated PrP can be induced to adopt a misfolded conformation in
vitro, by exposure to infected brain homogenate, mimicking the in vivo formation of
PrPˢᶜ. Using an in vitro conversion assay, the ability of PrP derived from the
glycosylation deficient transgenic to misfold was studied. PrP that lacks the first Nglycan attachment site did not misfold, suggesting a potential cause of the enhanced
resistance of the transgenic mice that lack this site to TSE transmission. PrP that
lacks the second N-glycan attachment site adopted a misfolded form in vitro, with
efficiency equal to that of normally glycosylated PrP. Therefore the higher disease
incidence observed in the transgenic mice, which lack the second PrP glycosylation
site, did not occur because PrP in these animals is more readily misfolded. This
suggests that other aspects of PrP biology, such as PrPˢᶜ toxicity or clearance, are
influenced by glycosylation of PrP's second site and that these can alter TSE disease
