Visualization of prion infection in transgenic mice expressing green fluorescent protein-tagged prion protein

Abstract

Tg(PrP-EGFP) mice express an enhanced green fluorescent protein (EGFP)-tagged version of the prion protein (PrP) that behaves like endogenous PrP in terms of its posttranslational processing, anatomical localization, and functional activity. In this study, we describe experiments in which Tg(PrP-EGFP) mice were inoculated intracerebrally with scrapie prions. Although PrP-EGFP was incapable of sustaining prion infection in Tg(PrP-EGFP)/Prn-p(0/0) mice, it acted as a dominant-negative inhibitor that bound to, and fluorescently marked, deposits of PrPSc generated from endogenous PrP in Tg(PrP-EGFP)/Prn-p(+/+) mice. Scrapie infection of these latter animals caused a progressive accumulation of fluorescent PrP-EGFP aggregates in neuropil, axons, and prominently in the Golgi apparatus of neurons. Our results provide an entirely new picture of PrPSc localization during the course of prion infection, and they identify for the first time intracellular sites of PrPSc formation that are not well visualized with conventional immunohistochemical techniques.

Figure 1.

PrP-EGFP does not sustain scrapie propagation in Prn-p^0/0 mice, and it delays the development of disease in Prn-p^+/+ and tga20 mice. Mice were inoculated with scrapie and monitored for the development of clinical illness.

Figure 2.

PrP-EGFP does not convert to PrP^Sc-EGFP. Brain homogenates from scrapie-infected mice and uninfected mice (Un) of the indicated genotypes were incubated with PK (+ lanes) or left untreated (- lanes) before analysis by SDS-PAGE and Western blotting to detect PrP. A, Blots were probed with the anti-PrP antibody 8H4, which recognizes both PrP-EGFP (arrowhead at 60-70 kDa, to the right of lane 18) and endogenous PrP (30-36 kDa). B, Blots were probed with anti-PrP antibody 3F4, which recognizes only PrP-EGFP (arrowhead at 60-70 kDa, to the left of lane 9). Lanes representing PK-digested samples contain 10-fold more protein than those corresponding to undigested samples. The bands at 25 kDa in lanes 10, 16, and 18 are nonspecific and are not related to infection, because they appear in samples from uninoculated mice (lane 10). Molecular size markers are given in kilodaltons.

Figure 3.

PrP-EGFP binds to PrP^Sc derived from endogenous PrP in the brains of infected mice. A, PrP-EGFP was immunoprecipitated from brain homogenates prepared from scrapie-infected mice of the indicated genotypes using anti-GFP antibodies (αGFP, lanes 1-4, 8-11) or nonspecific IgG antibodies (IgG, lanes 5-7). The immunoprecipitates (IP) were incubated with PK (+ lanes) or left untreated (- lanes) before analysis by SDS-PAGE and immunoblotting with 8H4 antibody. Lanes 1 and 8 represent input (In) samples before immunoprecipitation. Lanes marked 10x represent 10-fold more protein than lanes marked 1x. PrP-EGFP migrates at 60-70 kDa, and endogenous PrP migrates at 30-36 kDa (arrowhead and bracket, respectively, to the left of lane 1). The band at 55 kDa (asterisk to the left of lane 1) is nonspecific. The position of PrP 27-30 is indicated by the bracket to the right of lane 4. B, PrP^Sc was precipitated from the brains of uninfected and scrapie-infected Tg(PrP-EGFP^+/0)/Prn-p^+/+ mice using NaPTA, separated by SDS-PAGE, and probed with antibodies 8H4 (lanes 1 and 2), 3F4 (lanes 3 and 4), or anti-GFP (αGFP, lanes 5 and 6). The arrowhead to the right of lane 6 indicates the position of PrP-EGFP. C, Same as B, but NaPTA pellets were digested with PK before SDS-PAGE and Western blotting. The 30 kDa band in lanes 5 and 6 corresponds to an intrinsically protease-resistant fragment GFP (Cody et al., 1993). D, The supernatants left after NaPTA precipitation of brain homogenates from uninfected (Un) and infected (Inf) Tg (PrP-EGFP^+/0)/Prn-p^+/+ mice were probed with anti-actin antibodies to demonstrate equal protein content.

Figure 4.

Scrapie infection causes accumulation of fluorescent aggregates of PrP-EGFP. Brain sections from terminally ill Tg(PrP-EGFP^+/0)/Prn-p^+/+ mice (202-284 dpi; A-D) and from age-matched, uninoculated control mice (E-G) were viewed using confocal fluorescence microscopy. Sections are from the granule cell layer of the cerebellum (A, E), layers II-III of the cerebral cortex (B, F), the thalamus (C, G), and the corpus callosum (D, H). The red boxes over the anatomical models shown along the top indicate the brain regions from which sections were taken. The arrowheads in C indicate plaque-like PrP-EGFP aggregates in the neuropil, and the arrow in B indicates a perinuclear deposit of PrP-EGFP in a neuronal cell body. Scale bar: (in A) A-H, 20 μm.

Figure 5.

Aggregation of PrP-EGFP occurs gradually during the course of scrapie infection. Brain sections from uninoculated Tg(PrP-EGFP^+/0)Prn-p^+/+ mice (A, F) or from scrapie-infected Tg(PrP-EGFP^+/0)Prn-p^+/+ mice at 100 dpi (B, G), 150 dpi (C, H), 200 dpi (D, I), 284 dpi (E), or 202 dpi (J) were viewed using confocal fluorescence microscopy. Sections were from the interposed nuclei of the cerebellum (A-E) and the thalamus (F-J). Scale bar: (in A) A-J, 20 μm.

Figure 6.

PrP-EGFP does not colocalize with Thy1 during scrapie infection. Sections from the cerebral cortex of terminally ill (267 dpi; A-C) and age-matched, uninfected (D, E) Tg(PrP-EGFP^+/0)/Prn-p^+/+ mice were immunostained for Thy1 and examined using confocal fluorescent microscopy. The intrinsic fluorescence of PrP-EGFP appears in green (A, D), and Thy1 immunofluorescence appears in red (B, E). Merged red and green images are shown in C and F. A, C, Arrowheads and arrows indicate, respectively, neuropil and intracellular aggregates of PrP-EGFP. Scale bar: (in A) A-F, 20 μm.

Figure 7.

PrP-EGFP accumulates in the Golgi apparatus of neurons beginning early in the course of scrapie infection. Brain sections from the hilus of the dentate gyrus (A-C, G, H) and layers II and III of the cerebral cortex (D-F) of Tg(PrP-EGFP^+/0)/Prn-p^+/+ mice were immunostained for giantin (A-F), TRAPα (G), or LAMP1 (H) using Alexa 594-coupled secondary antibodies. Sections were then viewed by confocal fluorescence microscopy to reveal the intrinsic green fluorescence of PrP-EGFP and the red fluorescence from the immunostained marker proteins. All panels show merged green and red images. Mice were either uninoculated (A, D) or inoculated with scrapie (B, C, E-H; dpi are indicated on each panel). Scale bar: (in A) A-H, 20 μm.

Figure 8.

Comparison of PrP-EGFP fluorescence with conventional immunohistochemistry for visualization of PrP^Sc. Sections were taken from the cerebral cortex (layers II-III) of a terminally ill Tg(PrP-EGFP^+/0)/Prn-p^+/+ mouse (284 dpi; A, B), a terminally ill, nontransgenic Prn-p^+/+ mouse (218 dpi; C), an uninfected Tg(PrP-EGFP^+/0)/Prn-p^+/+ mouse (D, E), and an uninfected Prn-p^+/+ mouse (F). A, D, The intrinsic fluorescence of PrP-EGFP. B, C, E, F, PrP^Sc immunostaining using 8H4 antibody (with a red-coupled secondary antibody) after formic acid treatment of the sections. PrP immunostaining was relatively weak in sections from uninfected mice (E, F), resulting from loss of PrP^C signal caused by formic acid pretreatment; staining was slightly more intense in transgenic animals (E) because of the presence of PrP-EGFP. Scale bar: (in A) A-F, 20 μm.