Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen

Abstract

Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.

FIG. 1

HaPrP detection in various tissues of Tg7-HaPrP/MoPrP(−/−) and NSE-HaPrP/MoPrP(−/−) transgenic mice. (A) Western blots containing 2 mg of tissue extracts were developed with PrP-specific monoclonal antibody 3F4 (23). To verify the PrP specificity of protein bands, duplicate Western blots were developed with monoclonal antibody preabsorbed by incubation of 50 ml of diluted ascites with 3.4 μg of synthetic HaPrP peptide, p106–126, containing the epitope recognized by 3F4. HaPrP bands are specifically eliminated by the peptide competition. These results were consistent with previous findings where other tissues, including lung and muscle, from NSE-HaPrP mice were analyzed and found to be negative (37). (B) Immunoblot detection of HaPrP in the brain and sciatic nerve of NSE-HaPrP/MoPrP(−/−) mice. Brain (0.15 mg) or pooled sciatic nerves (0.5 mg) were loaded per lane, and Western blots were developed as above. (C) RT-PCR detection of HaPrP mRNA from the brain (CNS) and various lymphoreticular tissues of transgenic mice. Insufficient RNA was recovered from the sciatic nerve for testing by RT-PCR. All tissues shown were positive for detection of β-actin mRNA by RT-PCR (data not shown).

FIG. 2

(Top) Time from inoculation until severe clinical disease in NSE-HaPrP/MoPrP(−/−) and Tg7-HaPrP/MoPrP(−/−) transgenic mice infected with hamster scrapie strain 263K by the i.c., oral, and i.p. routes. The doses used were 1 × 10⁷ i.c. LD₅₀ for the i.c. and i.p. routes and 2 × 10⁸ i.c. LD₅₀ for the oral route. (Bottom) Effect of splenectomy on the susceptibility of transgenic mice to i.p. infection with 10⁷ i.c. LD₅₀ of hamster scrapie strain 263K. Splenectomy was done 10 to 14 days prior to scrapie infection.

FIG. 3

Western blot detection of PrP-res in the brains and spleens of NSE-HaPrP/MoPrP(−/−) mice 195 days after i.p. inoculation with 10⁷ i.c. LD₅₀ of hamster scrapie strain 263K. The milligram equivalents loaded in each lane are shown above the lane itself. The blot was developed with monoclonal antibody 3F4 (23). The faint band in lane Spleen (arrow), which was more intense after longer exposures, had a lower molecular mass than brain-derived PrP-res; however, it is well documented that the PrP-res apparent molecular mass can vary in different organs depending on differences in glycosylation (36).

FIG. 4

Effect of MoPrP expression [(+/+) versus (−/−)] on the incidence of hamster scrapie in NSE-HaPrP and Tg7-HaPrP mice inoculated i.p. or orally with hamster scrapie agent, strain 263K. Mice inoculated i.p. received 1 × 10⁷ i.c. LD₅₀, and those inoculated orally received 2 × 10⁸ i.c. LD₅₀. The vertical axis shows cumulative deaths due to scrapie. The horizontal axis indicates days postinoculation. ●, NSE-HaPrP/MoPrP(−/−) mice (n = 21 for i.p., n = 17 for oral); ○, NSE-HaPrP/MoPrP(+/+) mice (n = 20 for i.p., n = 19 for oral); ■, Tg7-HaPrP/MoPrP(−/−) mice (n = 13 for i.p., n = 19 for oral); □, Tg7-HaPrP/MoPrP(+/+) mice (n = 16 for i.p., n = 17 for oral).

FIG. 5

Possible pathways of neuroinvasion after peripheral scrapie infection. Direct invasion of the CNS by nerves may occur after peripheral infection with high doses of agent or exposure to highly neuroinvasive agent strains. Alternatively, after infection with lower doses of agent or with less neuroinvasive agent strains, amplification of infectivity in lymphoreticular organs such as spleen or lymph nodes (LN) might be necessary prior to neuroinvasion via PrP-positive peripheral nerves. Additional experiments with lower doses and different strains of hamster scrapie will be required to confirm this possibility. Neuroinvasion by the hematogenous route may occur only rarely (3). IP, intraperitoneal.