Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Abstract

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP(Sc)(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4 degrees C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP(Sc)(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a beta-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP(Sc)(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP(Sc) in the brains of Tg196 mice. Our results argue that MoPrP(Sc)(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP(C)(P101L) as well as in Tg196 mice expressing low levels of MoPrP(C)(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a beta-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.

FIG. 1.

Selective immunoreactivity of rabbit polyclonal antiserum 5449 raised against the MoPrP(89-143,P101L) peptide with PrPs carrying the P101L mutation. Brain homogenates (20 μg of total protein per lane) from mice expressing wt MoPrP-A (FVB), wt MoPrP-B (ILn/J), MoPrP(P101L) [Tg(MoPrP,P101L)196/Prnp^0/0], HuPrP(M129) [Tg(HuPrP,M129)440/Prnp^0/0], HuPrP(V129) [Tg(HuPrP,V129)152/Prnp^0/0], HuPrP(P102L) [Tg(HuPrP,P102L)7/Prnp^0/0], and MHu2M(P102L) [Tg(MHu2M,P102L)69/Prnp^0/0] were processed. Western blots were developed using the anti-PrP(P101L) 5449 polyclonal antiserum, and for comparison, with the anti-PrP RO73 polyclonal antiserum and the 3F4 monoclonal antibody. The apparent molecular weights of the migrated fragments are shown in thousands.

FIG.2.

GSS-like neuropathological features in the absence of PK-resistant PrP^Sc in spontaneous and synthetic peptide-induced prion disease in Tg mice expressing the MoPrP(P101L) allele. The analyzed brains are from Tg(MoPrP,P101L)196/Prnp^0/0 mice inoculated with prions from spontaneously ill Tg(MoPrP,P101L)2866/Prnp^0/0 mice (A, B, and C), β-rich MoPrP(89-143,P101L) peptide (D, E, and F), or for comparison, with RML (G, H, and I), 139A (J, K, and L), or 301V (M, N, and O) prions. Distribution of rPrP^Sc was determined by histoblotting (A, D, G, J, and M), and PrP-immunoreactive plaques were detected by hydrolytic autoclaving in the hippocampus (B, E, H, K, and N) and cerebellum (C, F, I, L, and O). Abbreviations: cc, corpus callosum; g, granule cell layer; hp, stratum radiatum of the CA1 region of the hippocampus; m, molecular layer; p, Purkinje cell layer. The bar in panel N represents 100 μm and applies to panels B, E, H, and K; The bar in panel O represents 30 μm and applies to panels C, F, I, and L.

FIG. 3.

Serial passage of aberrant PrP conformers in Tg(MoPrP,P101L) mice, detected by selective PTA precipitation and cold PK treatment. The 22- to 24-kDa PrP fragment (arrowheads) was found exclusively in Tg196 mice following inoculation with mouse GSS(P101L) prions or the β-rich MoPrP(89-143,P101L) peptide after both first and second passages. (A to D) Brain homogenates from uninoculated Prnp^0/0 mice (lane 1), Tg(MoPrP-A)4053/Prnp^0/0 mice (lane 2), uninoculated Tg(MoPrP,P101L)196/Prnp^0/0 mice (lane 3), Tg196 mice inoculated with brain homogenates from ill Tg2866 mice (lane 4), spontaneously ill Tg2866 mice (lane 5), Tg196 mice inoculated with RML prions (lane 6), or Tg196 mice inoculated with 301V prions (lane 7) are shown. Brain homogenates were untreated (A), precipitated with PTA (B), PK digested and subjected to ultracentrifugation (C), or subjected to cold PK digestion and precipitated with PTA (D). (E) Brain homogenates subjected to cold PK digestion and PTA precipitation from spontaneously ill Tg2866 mice (lanes 1 to 3), Tg196 mice inoculated with brain homogenates of diseased Tg2866 mice (lanes 4 to 6), or second passage of Tg2866 brain homogenates in Tg196 mice (lanes 7 to 9). (F) Brain homogenates from Tg196 mice inoculated with brain homogenates from ill Tg2866 mice (lanes 1, 4, and 7), β-rich MoPrP(89-134,P101L) peptide (lanes 2, 5, and 8), or second passage of MoPrP(89-134,P101L) peptide into Tg196 mice (lanes 3, 6, and 9). Samples were untreated (lanes 1 to 3), PK digested and ultracentrifuged (lanes 4 to 6), or subjected to cold PK digestion and PTA precipitation (lanes 7 to 9). Blots were developed using anti-PrP RO73 (A to D) or anti-PrP(P101L) 5449 polyclonal antisera raised against a random-coil MoPrP(89-143,P101L) peptide (E and F). The apparent molecular weights of the migrated fragments are shown in thousands.

FIG. 4.

Progressive accumulation of abnormal MoPrP(P101L) conformers (A to D) correlates with the accumulation of infectious prions (E) as a function of age in Tg(MoPrP,P101L)2866/Prnp^0/0 mice. Presymptomatic animals were sacrificed at birth, at 14 days, 28 days, 56 days, 84 days, or 112 days of age or at ∼132 days (when diagnosed with CNS dysfunction). Blots were developed using the RO73 polyclonal antibody. The apparent molecular weights of the migrated fragments are shown in thousands. (A) Untreated brain homogenates from Prnp^0/0 mice (lane 1), Tg4053 mice (lane 2), or Tg2866 mice (lanes 3 to 9) sacrificed at birth (lane 3), 14 days (lane 4), 28 days (lane 5), 56 days (lane 6), 84 days (lane 7), 112 days (lane 8), or ∼132 days (lane 9). (B) Brain homogenates from Tg4053 mice (lane 1), Tg2866 mice sacrificed at 14 days (lane 2), 28 days (lane 3), 56 days (lane 4), 84 days (lane 5), 112 days (lane 6), or ∼132 days (lane 7). Samples were precipitated with PTA prior to immunoblotting. (C) Brain homogenates subjected to cold PK digestion followed by PTA precipitation. Lane assignments are as described for panel B. The presence of a 22-kDa to 24-kDa PrP fragment specific to infectious GSS prions is indicated by the arrowhead. (D) The accumulation of abnormal PrP conformers in the brains of aging Tg(MoPrP,P101L)2866/Prnp^0/0 mice was quantified using the CDI with the RO73 antiserum and a secondary Eu-labeled anti-rabbit polyclonal antibody. (E) The same brain homogenates from aging Tg(MoPrP,P101L)2866/Prnp^0/0 mice used in the CDI were inoculated into Tg(MoPrP,P101L)196/Prnp^0/0 mice to measure incubation times, which are inversely proportional to the prion titer (38). Animals that did not display signs of neurological dysfunction were sacrificed after 500 days.

FIG. 5.

Aberrant HuPrP(P102L) conformers present in the brains of GSS patients and GSS-inoculated Tg mice (A to D) are indistinguishable from those that progressively accumulate in spontaneously ill chimeric Tg(MHu2M,P102L) mice (E to H). Samples were developed using the 3F4 monoclonal antibody. The apparent molecular weights of migrated fragments are shown in thousands. The presence of a 22-kDa to 24-kDa PrP fragment specific to GSS prions is indicated by the arrowheads. (A to D) Brain homogenates from spontaneously ill Tg(MHu2M,P102L)69/Prnp^0/0 mice (lane 2), Tg(MHu2M,P102L) mice inoculated with homogenates from GSS patients expressing either Val (lane 3) or Met (lane 4) at codon 129, and a GSS(P102L,M129) patient (lane 5). As controls, homogenates from an age-matched, healthy Tg(MHu2M)Prnp^0/0 mouse (lane 1) and normal human brain (lane 6) are shown. (E to H) Brain homogenates from spontaneously ill Tg69 mice sacrificed at 50 days (lane 2), 100 days (lane 3), 200 days (lane 4), ∼360 days (lane 5), when the mice became ill, demonstrate the accumulation of PrP^Sc conformers that are similar to those observed in GSS patients. As a control, homogenate from a healthy, age-matched Tg(MHu2M)Prnp^0/0 mouse (lane 1) is shown. Samples were untreated (A and E), precipitated with PTA (B and F), digested with PK followed by ultracentrifugation (C and G), or subjected to cold PK digestion followed by PTA precipitation (D and H).

FIG. 6.

Differential proteolytic resistance of aberrant, distinct PrP conformers in the brains of Tg(MoPrP,P101L) mice detected by the CDI. (A) Brain homogenates from spontaneously ill Tg(MoPrP,P101L)2866/Prnp^0/0 mice and Tg(MoPrP,P101L)196/Prnp^0/0 mice inoculated with RML prions, 301V prions, or GSS(P101L) prions from ill Tg2866 mice. Controls were age matched, uninoculated Tg mice. Samples were either left untreated (total PrP content), PTA precipitated, digested with cold PK followed by PTA precipitation (cPK/PTA), or digested with PK followed by ultracentrifugation (PK/Ucent). Results are shown as the fraction of total PrP recovered after each treatment. (B) Amount of rPrP^Sc recovered after ultracentrifugation plotted as a function of the amount of antibody bound to the denatured and native forms of PrP (D:N ratio). Samples were obtained from the same mice depicted in panel A, in addition to Tg(MoPrP,P101L)196/Prnp^0/0 mice inoculated with the β-rich MoPrP(89-143,P101L) peptide on first [Tg196(pep)] and second [Tg196(196(pep))] passage. Data points and bars represent the means ± standard errors of the mean obtained from three independent measurements.