Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system

Abstract

Mice devoid of PrPC (Prnp%) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnp% brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnp% mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnp% mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

Figure 1

Neural grafts overexpressing PrP^C in a Prnp-deficient host. (a) Schematic drawing of the transplantation procedure. (b–d) Coronal section of the thalamus of a PrP-overexpressing tg20 mouse 78 days after inoculation into the right eye. At time of analysis, the animal showed clinical symptoms of scrapie. (b) Pronounced gliosis in the visual pathway (optic tract and lateral geniculate nucleus, LGN) is visualized by immunocytochemistry for glial fibrillary acid protein (GFAP). (c and d) Asymmetric neurodegeneration of the LGN is visualized by synaptophysin immunostain. The affected left LGN displays coarse granular deposits and patchy staining that reflects significant synaptic loss, whereas the unaffected right LGN displays the fine granular synaptic stain typical of normal neural tissue. Because scrapie infection starts in the visual system and is followed by generalized disease in the CNS, the LGN and superior colliculus show a more prominent astrocytic reaction and severe loss of neuronal processes than other regions of the brain, e.g., the hippocampus. (e, g, and i) Graft (gr) and adjacent host brain (h) in hematoxylin and eosin stains. (f, h, and k) Immunohistochemical stain for GFAP. (e and f) Neural graft (tg20) in a Prnp^o/o host brain analyzed 232 days after grafting without scrapie inoculation. Cellular density and distribution of neurons and astrocytes within the graft are similar to that of normal host brain. The slightly enhanced GFAP stain results from the transplantation procedure. (g and h) Neural graft (tg20) in a Prnp^o/o host brain analyzed 231 days after grafting and 230 days after direct intracerebral infection with mouse prions. Spongiosis (g) and gliosis (h) are conspicuous within the graft and were not observed in uninoculated or mock-inoculated grafts (not shown). (i and k) Neural grafts (tg20) 217 days after transplantation in a Prnp^o/o host brain and 305 days after intraocular inoculation show no neuropathological changes. The asterisks label the border between graft and host tissue.

Figure 2

Histoblot analysis of PrP expression. Frozen sections from brains engrafted with PrP-overexpressing neuroectodermal tissue were mounted on nitrocellulose, and PrP was displayed immunochemically without (a, c, and e) or with (b, d, and f) previous protease digestion (25). Arrows pinpoint the position of grafts. Intracerebrally infected grafts (c) consistently show stronger total PrP immunoreactivity than uninfected (a) and intraocularly inoculated (e) grafts. Protease-resistant PrP was formed only in intracerebrally infected PrP^C-expressing grafts (d) and migrated into the surrounding structures as described earlier (19). Uninfected (b) and intraocularly inoculated (f) mice did not accumulate protease-resistant PrP (PrP^Sc). As previously reported (19), myelinated structures show faint homogeneous background staining even in noninoculated Prnp^o/o mice. no inf., No infection; d, days; i.c., intracerebrally infected; i.o., intraocularly inoculated.

Figure 3

Detection of immune response in sera of mock- and scrapie-inoculated grafted and control mice. Immunoblots containing purified recombinant PrP (lane r), brain homogenate derived from Prnp^o/o (lane o) and tg20 (lane +) mice were incubated with sera from mock- and scrapie-inoculated grafted and control mice (dilution 1:100), respectively, and visualized by enhanced chemiluminescence. Presence of PrP-specific antibodies in the serum is indicated by 27- to 30-kDa bands in lane r and by a cluster of bands present in lane + but absent from lane o. tg20-engrafted Prnp^o/o mice developed a strong humoral immune response to PrP both before (a) and after (b) inoculation with scrapie prions, whereas engrafted tg33 mice (Prnp^o/o, lck-PrP) did not develop antibodies to PrP even after intraocular inoculation (c and d). (e–g) As positive controls, we used both polyclonal antibody generated in Prnp^o/o mice (KO2) and polyclonal rabbit antiserum 1B3 to mouse PrP (24).