Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes

Abstract

On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn.HCl) concentration required to denature 50% of disease-causing prion protein (PrP(Sc)) molecules, denoted as the [Gdn.HCl](1/2) value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn.HCl](1/2) values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn.HCl](1/2) values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrP(Sc) structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrP(Sc) conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrP(Sc) remains to be determined.

Fig. 1.

Neuropathologic analysis of FVB and Tg4053 mice inoculated with two isolates from Tg9949 mice that had received synthetic prions (see Table 1). FVB mice inoculated with the MK4977 isolate show moderate vacuolation in the thalamus (A) and diffuse PrP^Sc deposits in the CA1 region (B) and cerebellar cortex (C). In contrast, FVB mice inoculated with the MK4985 isolate show reduced vacuolation in the thalamus (D) and PrP^Sc clustering in the dentate gyrus (E) and deep cerebellar nuclei (F). Tg4053 mice inoculated with the MK4977 isolate show moderate vacuolation in the thalamus (G) but more in the hippocampus where it is associated with intense PrP^Sc deposits (H). Minimal vacuolation and PrP^Sc deposition in the cerebellar cortex (I). The MK4985 isolate caused more intense vacuolation of the thalamus (J) and other regions, except for the CA1 region, which contained sparse PrP^Sc deposits (K). In the cerebellar cortex, abundant vacuoles were associated with abundant plaque-like PrP^Sc deposits (L). Dt, dentate gyrus of the hippocampus; g, cerebellar granule cell layer; Pk, Purkinje cell; Py, pyramidal cell layer of the CA1 region. [Scale bar: J, 40 μm (and applies to A, G, and D); L, 30 μm (and applies to B, C, E, F, H, I, and K).]

Fig. 2.

Survival curves of Tg9949 mice after inoculation with synthetic prions. (A) First passage (filled triangles) of synthetic prions in Tg9949 mice. For the second passage (filled squares), homogenate from the brain of one Tg9949 mouse that died at 382 days (MK4977, indicated by the filled triangle symbol inside the square box) was inoculated into Tg9949 mice. (B) For the third passage of synthetic prions, homogenates were prepared from the brains of two ill Tg9949 mice, MM6055 (diamond in A) and MM6060 (circle in A), and inoculated into other Tg9949 mice. Tg9949 mice inoculated with the MM6055 homogenate developed neurologic signs with a mean incubation period of 172 ± 8 days (open diamonds). Tg9949 mice inoculated with the MM6060 homogenate exhibited a mean incubation of 335 ± 47 days (open circles).

Fig. 3.

Conformational-stability assays of synthetic prions on second passage in Tg9949 mice. Brain homogenates from one Tg9949 mouse (MK4977) that showed signs of neurologic dysfunction at 379 days after inoculation with synthetic prions were inoculated into two other Tg9949 mice. (A and C) Western blots of PrP^Sc in brain extracts of mouse MM6055 that was killed after developing signs of neurologic dysfunction at 204 days after inoculation (A) and mouse MM6060 that showed signs of neurologic dysfunction at 270 days after inoculation (C). Lanes 0–6, samples were exposed to increasing concentrations of GdnHCl (0–6 M, as indicated), then subjected to digestion with 20 μg/ml PK for 1 h at 37°C. Lane C, undigested control sample that was not exposed to Gdn·HCl. Molecular weight markers based on the migration of protein standards are indicated in kilodaltons. (B and D) Denaturation curves obtained by scanning the PrP 27–30 signals in the Western blots shown in A and C, respectively, show the [Gdn·HCl]_1/2 values.

Fig. 4.

The conformational stability of prions is directly proportional to the length of the incubation time in mice. The [Gdn·HCl]_1/2 values for prions were plotted as a function of the incubation times. (A) Prions in the brains of Tg9949 mice infected with synthetic prions showed an excellent correlation (R = 0.91) between these parameters. (B) Naturally occurring prions passaged in both non-Tg and Tg mice showed a strong correlation (R = 0.77). (C) Synthetic prions (circles) in the brains of Tg9949, Tg4053, and non-Tg mice were plotted with many naturally occurring prions passaged (squares) in both non-Tg and Tg mice. R > 0.93.