Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein

Abstract

Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.

Fig. 1.

Spontaneous neurologic illness in Tg(BVPrP,I109) mice. (A) Kaplan–Meier survival curves for various lines of Tg(BVPrP,I109) mice. (B–G) Neuropathologic features of spontaneous neurologic disease in Tg(BVPrP,I109) mice. Spongiform degeneration (B), astrocytic gliosis (GFAP labeling, C), and punctate PrP deposition (PrP labeling, yellow arrows in corpus callosum; D) were present in the hippocampus, and activated microglia (Iba1 labeling, E) were observed in the cerebral cortex of a spontaneously ill Tg3581^+/− mouse. Plaque-like PrP deposits were present in the parietal cortex of spontaneously ill Tg3581^+/− (F) and Tg3615^+/− (G) mice. (Scale bar in B–E, 100 μm; in F and G, 50 μm.) cc, corpus callosum; CA1, CA1 layer of pyramidal neurons. (H) Immunoblots for PrP. The brains of spontaneously ill Tg3574^+/− mice contained increased levels of PTA-precipitable PrP compared with young asymptomatic mice, but no protease-resistant PrP was seen after digestion with 50 μg/mL of PK (“high”), as was observed in mice inoculated with meadow vole-passaged RML prions (RML→MV). Using milder digestion conditions (“low,” 3 μg/mL of PK), misfolded PrP could be detected in the brains of spontaneously ill Tg3574^+/− mice. (I) RT-QuIC analysis of brain homogenates from young or spontaneously ill Tg(BVPrP,I109) mice. Significantly greater seeding activity was observed in the brains of ill Tg(BVPrP,I109) mice compared with asymptomatic young Tg3574^+/− mice and wild-type FVB mice. Each data point represents the mean fluorescence value obtained from six replicates of an individual brain sample. **P < 0.01, *P < 0.05.

Fig. 2.

Rapid transmission of spontaneous disease to Tg(BVPrP,I109) mice. (A and B) Kaplan–Meier survival curves for Tg3581^+/− (A) or Tg3574^+/− (B) mice inoculated at ∼60 d of age with brain homogenate from the indicated spontaneously ill Tg(BVPrP,I109) mice. Uninoculated mice are shown for comparison. (C–H) Neuropathologic features of an inoculated Tg3574^+/− mouse that developed signs of neurologic dysfunction at 47 d postinoculation (dpi) with brain homogenate from a spontaneously ill Tg3581^+/− mouse (C–E) compared with an age-matched, uninoculated Tg3574^+/− mouse (F–H). Spongiform degeneration (C), astrocytic gliosis (D), and punctate PrP deposition (E) were present in the hippocampus. In contrast, no spongiform degeneration (F), minimal GFAP staining (G), and no PrP deposition (H) were observed in an uninoculated Tg3574^+/− mouse. (Scale bar in C–H, 100 μm.) cc, corpus callosum; CA1, CA1 layer of pyramidal neurons. (I) Immunoblotting for PrP in the brains of Tg3574^+/− mice inoculated with brain homogenate from spontaneously ill Tg(BVPrP,I109) mice; these Tg3574^+/− mice showed signs of neurologic dysfunction at 46–49 dpi and had increased levels of PTA-precipitable PrP and mildly PK-resistant PrP in their brains compared with age-matched, uninoculated mice. (J) Bioluminescence imaging (BLI) of the brains of uninoculated Tg(BVPrP:Gfap-luc)3581 mice (black, n = 9) and Tg(BVPrP:Gfap-luc)3581 mice inoculated with brain homogenate from a spontaneously ill Tg3581^+/− mouse (red, n = 8). An increase in the BLI signal was apparent by 26 dpi.

Fig. 3.

Transmission of spontaneous disease to Tg(MoPrP) and WT mice. (A) Kaplan–Meier survival curves for uninoculated Tg4053 mice and Tg4053 mice inoculated with brain homogenates from the indicated spontaneously ill Tg(BVPrP,I109) mice. (B) Immunoblots of PrP in ill Tg4053 mice inoculated with brain extracts of spontaneously ill Tg(BVPrP,I109) mice. Brain samples were left undigested (−) or digested with 50 μg/mL of PK (+). Tg4053 mice with incubation periods of <100 d showed PK-resistant PrP in their brains, whereas Tg4053 mice with incubation periods of >225 d showed no PK-resistant PrP. (C–K) Neuropathologic features of ill Tg4053 and FVB mice inoculated with spontaneously ill Tg(BVPrP,I109) brains. Spongiform degeneration (C, F, and I), astrocytic gliosis (D, G, and J), and punctate PrP deposition (E, H, and K) were observed in the brains of Tg4053 (C–H) or FVB (I–K) mice inoculated with brain homogenate from a spontaneously ill Tg3581^+/− mouse (C–E) or a spontaneously ill Tg3615^+/− mouse (F–K). Neuronal loss was apparent in the Tg4053 mouse inoculated with the Tg3615^+/− brain extract (yellow arrows in F). The hippocampus is shown in all sections. (Scale bar in C–K, 100 μm.) cc, corpus callosum; CA1, CA1 layer of pyramidal neurons. (L) Immunoblot of PrP in ill Tg4053 mice after second passage of Tg(BVPrP,I109) brain extract. The brain extract used for inoculation was from a Tg4053 mouse with an incubation period of 253 d and harbored no PK-resistant PrP in its brain. Upon second passage, PK-resistant PrP^Sc accumulated in the brains of ill Tg4053 mice. (M) Immunoblot of PrP in the brains of two ill WT FVB mice inoculated with brain homogenate from a spontaneously ill Tg3615^+/− mouse revealed PK-resistant PrP^Sc (50 μg/mL of PK).