Mechanism of PrP-amyloid formation in mice without transmissible spongiform encephalopathy

Abstract

Gerstmann-Sträussler-Scheinker (GSS) P102L disease is a familial form of a transmissible spongiform encephalopathy (TSE) that can present with or without vacuolation of neuropil. Inefficient disease transmission into 101LL transgenic mice was previously observed from GSS P102L without vacuolation. However, several aged, healthy mice had large plaques composed of abnormal prion protein (PrP(d)). Here we perform the ultrastructural characterization of such plaques and compare them with PrP(d) aggregates found in TSE caused by an infectious mechanism. PrP(d) plaques in 101LL mice varied in maturity, with some being composed of deposits without visible amyloid fibrils. PrP(d) was present on cell membranes in the vicinity of all types of plaques. In contrast to the unicentric plaques seen in infectious murine scrapie, the plaques seen in the current model were multicentric and were initiated by protofibrillar forms of PrP(d) situated on oligodendroglia, astrocytes and neuritic cell membranes. We speculate that the initial conversion process leading to plaque formation begins with membrane-bound PrP(C) but that subsequent fibrillization does not require membrane attachment. We also observed that the membrane alterations consistently seen in murine scrapie and other infectious TSEs were not present in 101LL mice with plaques, suggesting differences in the pathogenesis of these conditions.

Figure 1

Plaques in the corpus callosum and adjacent hippocampus of 101LL‐8a mice and VM mice with 87V scrapie. A. Mature plaques with an irregular contour and showing central unstained core. Plastic‐embedded 1‐µm‐thick section labeled with Saf 84. Bar = 16 µm. B. PrP^d labeling partly surrounding a reactive astrocyte within the corpus callosum. No amyloid was visible in a serial 1‐µm‐thick section stained with toluidine blue. Plastic embedded 1‐µm‐thick section labeled with Saf 84. Bar = 14 µm. C,D. Semiserial sections of a 101LL‐8a mouse showing strong labeling of the periphery of amyloid plaques with R20 (C) and R24 (D) recognizing C‐ and N‐terminal sequences of the PrP protein, respectively. Bar = 64 µm. E,F. There is no such separate labeling of periphery and core plaques in 87V scrapie with R20 (E) and R24 (F). Bar = 32 µm.

Figure 2

Electron microscopy of plaques of 101LL‐8a mice. A. The center of a large plaque showing dense uniform amyloid fibrils arranged in interweaving bundles that virtually exclude all other cellular elements. Bar = 225 nm. B. The periphery of a large plaque showing infiltration of groups of amyloid fibrils between neuritic and glial processes. Many of the processes belong to astrocytes, identified by the presence of glycogen granules (arrowheads) and the paucity of other subcellular organelles. Bar = 470 nm. C. Two sub‐units of a multicentric plaque that was mainly located in gray matter of the stratum oriens and which consisted of more than five sub‐units. Each sub‐unit is of similar dimensions and is surrounded by microglial processes (m). Bar = 430 nm. D. A small sub‐unit of a multicentric plaque. This plaque consists of only a few amyloid fibers within the extracellular space. These fibers are already isolated from neural elements within the adjacent gray matter by astrocytic processes, characterized by their glycogen granules and the paucity of other organelles. Bar = 1000 nm. E. Detail of D showing the sparse number of amyloid fibers in tangential and transverse section (arrowheads). a, Astrocytic processes. Bar = 165 nm. F. Dystrophic neurites within myelinated processes adjacent to a large plaque. One dystrophic neurite occupies one side of a paranode (p) while the opposite side appears unaffected. Bar = 920 nm.

Figure 3

Immunogold labeling for PrP^d in 101LL‐8a mice. A. Despite the paucity of labeling of plaque cores by light microscopy, the core of a mature plaque shows good immunogold labeling for PrP^d. Bar = 138 nm. B. The periphery of a large plaque with one of several smaller adjacent satellite plaques is shown. Both parent and satellite plaques are labeled for PrP^d. Bar = 1600 nm. C. PrP^d labeling is present around an oligodendrocyte located at the extreme periphery of a large plaque, the edge of which is seen at the bottom right. [Box indicates the area shown in the inset (inset: showing the localization of PrP^d on the membranes of the oligodendrocyte]. Bar = 1600 nm. D. Extreme periphery of a plaque. No amyloid fibrils are visible but PrP^d labels the plasma‐membrane membranes of several processes including that of a dendrite (d) and an astrocyte (a). Bar = 336 nm. E. A plaque identified by immunolabeling in the plastic section (inset) shows no morphological change by uranyl acetate/lead citrate staining but in the corresponding location, there is membrane PrP^d labeling on several neuritic processes or in the associated extracellular space. Bar = 290 nm. Immunogold for PrP^d using 1A8 antibody.

Figure 4

Features of plaques in 87V infected VM mice. A. PrP^d labeling of small numbers of amyloid fibrils in longitudinal, transverse and tangential section in the extracellular space at the periphery of a large unicentric plaque. Bar = 275 nm. B. PrP^d labeling on the plasma lemma of neurites and glial processes at the extreme periphery of a unicentric plaque. All processes have abnormal irregular contours. No fibrils are evident. Bar = 160 nm. C. PrP^d labeling of a lysosome within a microglial cell body near the edge of a plaque shown only by sparse PrP^d labeling on cell membranes of adjacent processes. Bar = 200 nm. D,E. PrP^d labeling of small membrane protrusions or microfolds in transverse (arrow in D) and longitudinal (arrows in E) sections. (D) and (E). Bars = 200 nm. F. PrP^d labeling associated with a coated spiral membrane inclusion in an axon terminal. The continuity of the inclusion with the axonal plasma membrane is not in the plane of section. The abnormal coated vesicles that are associated with these invaginations (arrow) are also clearly evident. The inset shows a further axon terminal membrane invagination. In this non‐immunolabeled section, the continuity with the external plasma membrane and the spiral nature of the membrane invagination is evident as are intact synaptic vesicles confirming the process as an axon terminal. Bar = 180 nm (Bar in inset = 250 nm). G. Part of an abnormal dendrite lacking organelles with an uncoated spiral plasma‐membrane invagination. The section is not immunolabeled. Bar = 280 nm.