Mutability of prions

Abstract

Murine prions transferred from brain to cultured cells gradually adapt to the new environment. Brain-derived 22L prions can infect neuroblastoma-derived PK1 cells in the presence of swainsonine (swa); that is, they are 'swa resistant'. PK1 cell-adapted 22L prions are swa sensitive; however, propagation in swa results in selection of swa-resistant substrains. Cloned, PK1 cell-adapted 22L prions were initially unable to develop swa resistance ('swa incompetent'); however, after serial propagation for 30-90 doublings, four of nine clones became swa competent, showing that swa-resistant 'mutants' arose during replication. Mutations in the case of prions are attributed to heritable changes in PrP(Sc) conformation. One clone remained swa incompetent even after 10(35)-fold expansion; surprisingly, after propagation in brain, it yielded swa-resistant prions, indistinguishable from the original 22L population. Thus, cell-adapted 22L prions assumed either mutable or virtually immutable conformations; however, when passaged through the brain all became mutable. Mutability is thus a substrain-specific attribute.

Figure 1

Mutations in cloned prion populations. (A) Scheme of the experimental strategy. ‘a’ represents a mouse brain containing swainsonine (swa)-resistant (blue filled circles) and swa-sensitive (orange filled circles) cells. The ovals represent PK1 cells. Pathway I represents the behaviour of clones #1 and #2, pathway II that of #3 and #4 and pathway III that of #9. The events are explained in the text. Part of the scheme is based on data from Li et al (2010). (B) Conjectural mechanism of prion mutation. (a) A large aggregate of PrP^Sc is cleaved to smaller fragments (Orgel, 1996). (b) Small aggregates are susceptible to thermally induced conformational fluctuations. (c) A conformationally altered small aggregate is stabilized by accretion of PrP (prion protein).

Figure 2

Swa competence of various 22L prion clones. PK1 cells containing clones #4–8 after altogether 86 doublings, and #9 after 116 doublings, were propagated for five 1:20 splits in the presence or absence of 2 μg/ml swa, and the 100 × concentrated conditioned medium was subjected to the SSCA (standard scrapie cell assay) on PK1 cells in the absence (blue) or presence (red) of 2 μg/ml swa. The prions of clones #7, #8 and #9 were wiped out after propagation in swa, and were therefore swa incompetent. Clone #4 (3B12) prions became fully swa resistant, whereas clone #5 (8H6) prions persisted at low levels in swa and were swa resistant. Clone #6 (8B4) prions survived at a low level but showed only marginal swa resistance. The analysis of clones #1–3 is reported in Li et al (2010). Each point is the average of triplicate measurements; the bars indicate standard deviations. swa, swainsonine.

Figure 3

SSCA of homogenates of brains infected with various PK1[22L] prion clones. Concentrated conditioned medium from PK1[22L]#1, -#4, -#7 and -#8 or lysate from PK1[22L]#9 was inoculated intracerebrally into C57BL/6 mice, as detailed in Table 1, to yield brain{PK1[22L]#1}, brain{PK1[22L]#4}, brain{PK1[22L]#7}, brain{PK1[22L]#8} and brain{PK1[22L]#9}. The brain homogenates were subjected to the SSCA in the absence (blue) or presence (red) of 2 μg/ml swa. A and B were assayed in separate experiments, in parallel with brain[22L] homogenate as reference. All samples were equally swa resistant and R33-2H11 competent. Each point is the average of triplicate measurements; the bars indicate standard deviations. SSCA, standard scrapie cell assay; swa, swainsonine.

Figure 4

SSCA assay of 22L prions cloned in PK1 cells, passaged through brain and returned to PK1 cells. PK1 cells were infected with the homogenates of brain{PK1[22L]#7}, brain{PK1[22L]#9} and brain[22L]; the conditioned medium of these cells was analysed at passage 0 (P0), after nine 1:20 splits in the absence of swa (P9) or after five 1:20 splits in the presence of 2 μg/ml swa (P5+swa). In all cases, at P0, the prions secreted by the freshly infected cells were largely swa resistant, after nine splits they had become swa sensitive, but after five splits in the presence of swa they retained swa resistance. Samples were assayed in the absence (blue) or presence (red) of 2 μg/ml swa. Each point is the average of triplicate measurements; the bars indicate standard deviations. SSCA, standard scrapie cell assay; swa, swainsonine.