Accumulation of prion protein in the brain that is not associated with transmissible disease

Abstract

Prion diseases or transmissible spongiform encephalopathies are characterized histopathologically by the accumulation of prion protein (PrP) ranging from diffuse deposits to amyloid plaques. Moreover, pathologic PrP isoforms (PrP(Sc)) are detected by immunoblot analysis and used both as diagnostic markers of disease and as indicators of the presence of infectivity in tissues. It is not known which forms of PrP are associated with infectivity. To address this question, we performed bioassays using human brain extracts from two cases with phenotypically distinct forms of familial prion disease (Gerstmann-Sträussler-Scheinker P102L). Both cases had PrP accumulations in the brain, but each had different PrP(Sc) isoforms. Only one of the brains had spongiform degeneration. Tissue from this case transmitted disease efficiently to transgenic mice (Tg PrP101LL), resulting in spongiform encephalopathy. In contrast, inoculation of tissue from the case with no spongiform degeneration resulted in almost complete absence of disease transmission but elicited striking PrP-amyloid deposition in several recipient mouse brains. Brains of these mice failed to transmit any neurological disease on passage, but PrP-amyloid deposition was again observed in the brains of recipient mice. These data suggest the possible isolation of an infectious agent that promotes PrP amyloidogenesis in the absence of a spongiform encephalopathy. Alternatively, the infectious agent may be rendered nonpathogenic by sequestration in amyloid plaques, or PrP amyloid can seed amyloid accumulation in the brain, causing a proteinopathy that is unrelated to prion disease. Formation of PrP amyloid may therefore not necessarily be a reliable marker of transmissible spongiform encephalopathy infectivity.

Fig. 1.

Immunohistochemistry of the cerebrum of Tg 101LL mice showing PrP accumulation in the vicinity of the corpus callosum. (A and B) Mouse inoculated with PrP-21. (C and D) Mouse inoculated with PrP-8. Higher-power magnification of plaques in the corpus callosum are shown in B and D. (E and F) Serial sections from a mouse inoculated with PrP-8 stained with mAb 6H4 (E) and mAb 3F4 (F). (A and B) Brains from animals developing neurological signs were examined at the terminal stages of the disease (310 days postinoculation. (C–F) Animals without neurological disease were analyzed at the end of their lifespan 562 days postinoculation (C and D) and 731 days postinoculation (E and F). (A–E) PrP deposits were detected by immunohistochemical analysis using mAb 6H4 that recognizes mouse PrP. (F) No immunopositivity was observed with mAb 3F4 that recognizes human PrP. (Magnifications: ×4, A and C; ×20, B and D–F.)

Fig. 2.

Immunoblot analysis of brain homogenates from mice inoculated with PrP-21 and PrP-8. (A) Lanes 1 and 2, Tg 101LL-21 (mouse with neuropathologically confirmed prion disease, 277 days postinoculation); lanes 3 and 4, Tg 101LL-8s (mouse with neuropathologically confirmed prion disease, 622 days postinoculation); and lanes 5 and 6, Tg 101LL-8a (asymptomatic mouse, without spongiform degeneration showing multicentric amyloid plaques, 703 days postinoculation). Samples in lanes 2, 4, and 6 were treated with 20 μg/ml PK. The film was overexposed to show the low levels of PK-res PrP^Sc present in the samples shown in lanes 2 and 4. (B) Low levels of PrP^Sc were identified in one 101LL-8a mouse after treatment with 20 μg/ml PK. PrP was detected with mAb 7A12.

Fig. 3.

Cold-PK digestion of brain homogenate from Tg 101LL-8 mice and controls. Lane 1, Tg 101LL-8a (asymptomatic mouse with multiple PrP-amyloid plaques, 633 days postinoculation); lane 2, Tg 101LL-8a (asymptomatic mouse with multiple PrP-amyloid plaques, 716 days postinoculation); lane 3, uninfected Tg 101LL mouse; lane 4, uninfected 129/Ola control mouse; and lane 5, ME7-scrapie-infected 129/Ola control mouse. The 22- to 24-kDa PrP band (corresponding to “cold PK”-resistant fragment of PK-sensitive PrP^Sc) is present only in the ME7 scrapie-infected control mouse. All samples were treated with 250 μg/ml PK on ice for 1 h and deglycosylated with PNGase F. ME7 scrapie control was loaded at ≈25% of the concentration of lanes 1–4 to allow comparison. The blot was probed with mAb 7A12. The image was cropped from a single blot to remove lanes with irrelevant samples.