In the present experiments the influence of a normal human prion protein gene allelic variation, N171S, on prion disease susceptibility was studied in a mouse system using transgenic mice expressing the mouse homolog, N170S. Susceptibility to prion disease induced by mouse scrapie strains, 22L and ME7, was identical in control mice expressing PrP-170N versus transgenic mice expressing PrP-170S. In contrast, after inoculation with scrapie strains, RML or 79A, mice expressing PrP-170S were markedly resistant compared to control mice expressing PrP-170N. Of the 43 transgenic mice inoculated with these two scrapie strains, disease occurred only after 439 days and this was seen in only 4 mice. An additional 10 mice had subclinical infection after 545–603 days, as shown by detection of PrPres by IHC, however the remaining 29 mice had no evidence of clinical signs or PrPres accumulation in brain after observation up to 603 days. These results demonstrated that mice expressing PrP-170S were highly resistant to infection by strains RML and 79A, but this resistance was strain-specific since there was no resistance to two other strains (ME7 and 22L).
One explanation for the scrapie strain-specific differences seen in our experiments may lie in the origins of the scrapie strains used (). Strain ME7 was the result of a passage of natural scrapie in Suffolk sheep directly to mice and therefore was not related to other scrapie strains
. On the other hand, 22L, RML and 79A share a common origin in that they were all derived from the Moredun Institute's sheep scrapie brain pool 1(SSBP/1)
. However, there were major differences in the passage history of each of these 3 strains, and strains 79A and RML were more closely related to each other than to strain 22L 
(). This may explain the similar resistance pattern of strains 79A and RML in PrP-170S mice.
History of scrapie strains used.
Although the biochemical explanation for scrapie strains in general remains a mystery at this time, it has been hypothesized that PrPres from each strain adopts a slightly different conformation which is conferred on successive PrPsen molecules as they are converted to PrPres 
. Accordingly, our current data might be explained by the conformational selection model of prion strains and species barriers 
which was first developed to explain prion strain differences involving the Sup35 protein in yeast 
. This model suggests that only a subset of all possible PrPres conformations is compatible with any individual PrP primary structure. Thus incompatibility between the infecting prion strain and the host PrP would result in a transmission barrier 
. For example, mouse PrP-170S might easily assume the conformations required by strains ME7 or 22L, but might be less adept at assuming the conformations associated with strains RML or 79A. In practice most mutations studied lead to altered incubation periods 
, but when strong species barriers exist, either no transmission or low level subclinical cross-species transmission has been observed 
Another possible explanation for our results is that in vivo conversion of PrP-170S by RML or 79A strains might generate PrPres with a lower than usual stability. This might be due to either high dissociation of PrPres into smaller oligomers or increased susceptibility of PrPres to catabolic degradation. However, we were not able to detect any evidence for the presence of PrPres with increased susceptibility to proteinase K in PrP-170S transgenic mice infected by either RML or 79A scrapie strains (data not shown).
Effects of PrP amino acid variations on prion disease species and strain transmission barriers in some cases appear to correlate with differences in PrP conversion. For example, sheep homozygous for PrP with V136
show opposite patterns of susceptibility to scrapie strains SSBP1 and CH1641 
, and in vitro generation of PrPres in PMCA reactions agreed with the in vivo resistance observed 
. However, when we tested PrP conversion in vitro using PMCA, both 22L and RML PrPres were able to seed the generation of PrPres derived from PrP-170S (). This conversion of PrP-170S by RML scrapie was in contrast to the ineffective production of disease and slow and low PrPres generation in PrP-170S mice infected with RML scrapie. Biological and biochemical differences between conditions in brain tissue of live mice and PMCA test tube reactions might account for the discrepancies between our in vivo and in vitro results. Similar discrepancies between PrPres generation by PMCA in vitro and clinical susceptibility have been noted previously, and in some cases strong in vivo transmission barriers between species have been easily overcome by using PMCA with minor alterations in conditions 
. Thus PrP conversion by PMCA would appear to be less selective than in vivo infection by TSE agents.
Interestingly, the N171S polymorphism in humans (homologous to the N170S change in our Tg330 and Tg340 mice) occurs near other PrP residues implicated in influencing PrP structure and folding as well as susceptibility to prion diseases in animals. For example, alterations in PrP folding in vitro have been noted after mutations homologous to human residues 168 
and 170 
. At the structural level, PrP mutation at human residue 170 (S170N) appears to create a stabilized loop structure located near residues 165-175 
, and this change may influence susceptibility to CWD and other prion agents 
. However, others have reported no effect of S170N on species-specific PrP conversion in vitro in a mouse-hamster system 
. In contrast, resistance of rabbits to prion disease appears to be associated with a serine at human PrP residue 174 
. Similarly, the sheep polymorphism at residue 171 (human residue 168) is important in the resistance of sheep to classical scrapie strains 
. Possibly the N171S polymorphism examined in the present study might be able to modulate prion disease because of its location near the PrP loop structure and other nearby influential residues.
In humans, familial prion diseases have been associated with PrP mutations in the near vicinity of the PrP loop structure, i.e. Q160X 
, Y163X 
, D178N 
and V180I 
. The N171S polymorphism is not by itself associated with familial prion disease 
. However, a previous study identified a family with an unusual psychiatric disorder associated with PrP N171S 
. More recently, in an African-American family with unusual psychiatric signs and sleep abnormalities preceding onset of familial CJD, disease was linked to expression of a PrP molecule containing both PrP N171S and D178N mutations 
. Interestingly this is the first family with African ancestry where the D178N mutation has been detected, as the 12 previously reported families were of European or Japanese descent 
. This family might be an example where the N171S polymorphism altered the clinical disease signs when expressed in combination with a known pathogenic PrP mutation. It remains unclear whether this effect is mediated by a direct influence of these mutations on PrP misfolding or whether indirect effects involving other non-PrP molecules might also play a role. Nevertheless, the fact that the N171S polymorphism is present in healthy populations of humans 
, suggests that N171S is likely non-pathogenic by itself and that there may even be a selective advantage for maintaining its presence in human genomes.