Differences in scrapie-induced pathology of the retina and brain in transgenic mice that express hamster prion protein in neurons, astrocytes, or multiple cell types

Abstract

Prion protein (PrP) is expressed in many tissues and is required for susceptibility to scrapie and other prion diseases. To investigate the role of PrP expression in different cell types on pathology in retina and brain after scrapie infection, we examined transgenic mice expressing hamster PrP from the PrP promoter (tg7), the neuron-specific enolase promoter (tgNSE), or the astrocyte-specific glial fibrillary acidic protein promoter (tgGFAP). After intraocular inoculation with hamster scrapie, clinical disease developed in tg7 and tgNSE mice by 100 days and in tgGFAP mice by 350 days. Astrogliosis and scrapie-associated protease-resistant PrP (PrP-res) were detected in retina and brain before clinical onset. Retinal PrP-res was present in high amounts in both tg7 and tgNSE mice, however only tg7 mice developed retinal degeneration and extensive apoptosis. In contrast, in all three lines of mice high levels of brain PrP-res accompanied by neurodegeneration were observed. Thus, PrP expression on neurons or astrocytes was sufficient for development of scrapie-induced degeneration in brain but not in retina. The combined effects of PrP-res production in multiple cell types was required to produce retinal degeneration, whereas in brain PrP-res production by neurons or astrocytes alone was sufficient to cause neuronal damage via direct or indirect mechanisms.

Figure 1

Kinetics of death of tg7, tgNSE, and tgGFAP mice after intraocular (i.o.), intracerebral (i.c.), intraperitoneal (i.p.), and oral inoculation of hamster scrapie strain 263K. The doses used for each type of inoculation are described in the Materials and Methods. The data are represented as percentage of animals dead versus days after infection, which were pooled from at least two independent experiments (tg7 mice: n = 6 intraocular, n = 15 intracranial, n = 13 intraperitoneal, and n = 19 oral; tgNSE mice: n = 5 intraocular, n = 10 intracranial, n = 10 intraperitoneal, and n = 12 oral; tgGFAP mice: n = 6 intraocular, n = 8 intracranial, n = 12 intraperitoneal, and n = 6 oral). The results of the intracranial inoculation were similar to what was published previously.

Figure 2

Western blot of PrP-sen from transgenic mice. Eye (A) and brain (B) tissue from hamster, tg7, tgNSE, tgGFAP, and nontransgenic (wt) mice were blotted and hamster PrP was detected with 3F4 monoclonal antibody and ECF as described in Materials and Methods. PrP-sen bands representing multiple glycosylated forms are indicated by the approximate molecular weights of 26 to 38 kd. A (right): Methanol-precipitated total protein from a whole tgGFAP eye to demonstrate detection of PrP-sen. tgNSE and wt mouse eyes are shown for comparison of positive and negative tissues, respectively. To determine amounts of PrP-sen each homogenate was adjusted to 1 mg of total protein/10 μl (tissue equivalents), then serial twofold dilutions were analyzed starting with (A) undiluted (hamster), 1:4 (tg); or (B) 1:2 (hamster), 1:32 (tg). C: At least three mice per group were analyzed for densitometric analysis. The relative amounts of PrP-sen from tg7, tgNSE, and tgGFAP were calculated by densitometric analysis after comparison with standard curves from hamster eye (undiluted to 1:16) or hamster brain (undiluted to 1:256). The values are expressed as percentage ± SD of PrP-sen in eye or brain compared to the level of PrP in hamster eye or brain, which was set at 100%.

Figure 3

Immunohistochemical analysis of PrP-res in hamster PrP transgenic mouse retinas. Shown are representative sections of mock-infected retinas age-matched from the midway or clinical time of disease (A–C), and retinas from early (28 days after infection) (D–F), midway (45 days after infection for tg7 and tgNSE; 140 days after infection for tgGFAP) (G–I), and clinical (90 to 100 days after infection for tg7 and tgNSE; 350 to 355 days after infection for tgGFAP) (J–L) time points of disease after intraocular infection of tg7, tgNSE, and tgGFAP mice with hamster scrapie. A–F: No specific PrP-res immunoreactivity was detected in mock-infected retinas or retinas from early time points of infection. The small amount of staining seen in the OPL of tgNSE mice at the early time point was also seen in mock-infected mice and was therefore not PrP-res-specific. Specific PrP-res immunoreactivity was detected midway in infection in the plexiform layers of all three transgenic retinas tested (G–I) and accumulated primarily in the inner retinal layers of the retinas of tgNSE and tgGFAP mice (J and K, IPL and OPL). J: In contrast with the tgNSE and tgGFAP retinas, the retinas from tg7 mice were completely degenerated by the time of clinical disease. GL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; PL, photoreceptor layer; RPE, retinal pigment epithelium. Scale bar, 20 μm. Original magnifications: ×20 (A, I, K–L); ×40 (J).

Figure 4

Histopathological analysis and Müller cell astrocytosis in PrP-transgenic mouse retinas. Shown are representative retinal sections midway (A–F) and at the clinical time (G–L) of disease (see Figure 3 legend for precise times). Retinal sections were stained with H&E for histopathological analysis (A–C and G–I) and with anti-GFAP antibody for astrocytosis (D–F and J–L). A (arrow): Vitritis in tg7 mouse at the midway point of infection. Scale bar, 20 μm. Original magnifications, ×20.

Figure 5

Apoptosis in retinas of scrapie-infected transgenic mice. Shown are representative retinal sections stained by TUNEL of mock-infected tg7 mice (A) and scrapie-infected tg7 (B), tgNSE (C), and tgGFAP (D) mice. tgGFAP and tgNSE retinas were stained at the clinical time of disease, and tg7 retinas were stained midway through disease (see Figure 3 legend for precise times). B: Characteristic pyknotic nuclei from apoptotic cells in tg7 retinas are indicated by white arrowheads (inset). E: Staining with anti-cleaved caspase-3 at a higher magnification shows typical cytoplasmic staining (white arrows). F: In a different mouse with more severe retinal degeneration, H&E staining shows retinal atrophy and cell loss in outer nuclear layer with typical pyknotic nuclei and apoptotic bodies (white arrows). Original magnifications, ×40.

Figure 6

Western blot of PrP-res from clinically sick transgenic mice. Shown are representative blots from tg7 (A), tgNSE (B), and tgGFAP (C) eye and brain tissue at the clinical time of infection after intraocular inoculation with hamster scrapie. Each 10% (w/v) homogenate was treated with proteinase K as described in the Materials and Methods, and PrP-res bands representing multiple glycosylation forms are indicated by approximate molecular weight markers (16 to 30 kd). M (mock), uninfected brain or eye homogenate. Lanes 1 to 3 represent protein from up to three individual animals. All blots were loaded with an equivalent amount of protein (5 mg/10 μl tissue equivalents). All exposure times were equivalent except for tgGFAP eye, which was 100-fold longer because of low levels of detectable PrP-res. This very long exposure also resulted in visualization of a band in the lane with mock-infected eye. It is unclear whether this is a non-PrP protein or actual PrP-res from the adjacent lane.

Figure 7

Immunohistochemical analysis of PrP-res and spongiosis in hamster PrP transgenic mouse brains. Shown are representative sections of brains from clinical time points of disease after intraocular infection with hamster scrapie of aged-matched mock-infected tg7 mice (A and E) and scrapie-infected tg7 (B and F), tgNSE (C and G), and tgGFAP (D and H) mice. A–D: Thalamus (Th) and hippocampal (Hc) region immunohistochemically stained with monoclonal antibody 3F4, showing diffuse and punctate staining for PrP-res. D, inset: The hippocampal region of the tgGFAP mice. E–H: Representative sections of thalamus stained with H&E to show characteristic spongy lesions (arrows) at the time of clinical disease. Scale bar, 20 μm. Original magnifications: ×4 (A–D); ×20 (E–H).