Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

Abstract

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.

Keywords: Alzheimer disease; CAA; amyloid; basement membrane; brown fat; cerebral amyloid angiopathy; colon; follicular dendritic cells; glycosaminoglycan; glycosylphosphatidylinositol; heart; lymphoid tissues; macrophages; prion protein; spleen; transmissible spongiform encephalopathies; white fat.

Figure 1.

Detection of amyloid PrPSc in heart tissue of scrapie-infected tg anchorless mice. (A) IHC staining with Mab D13 shows PrPSc (brown) deposited around capillaries and in interstitial areas of myocardium (arrow). Most PrPSc appeared to be outside cardiomyocytes. (B) Low power electron micrograph shows PrPSc outside capillary endothelium (black arrow) and within interstitial areas between cardiomyocytes (yellow arrow) immunogold-labeled with anti-PrP antibody 1A8. (C) At high magnification aggregates of immunogold-labeled PrPSc amyloid fibrils (yellow arrows) were seen outside cardiomyocytes. (D) Using uranyl acetate-lead citrate (UALC) staining without etching for immunogold staining to better visualize membranes, PrPSc amyloid fibrils (yellow arrow) were observed outside myocytes adjacent to plasma membrane which had an irregular indented appearance (pink arrows) near fibrils. Occasional apparent inclusions were seen within cytoplasm near these areas (green arrowheads), however, these are likely to be extracellular invaginations sectioned across the direction of penetration. (E) Higher magnification of an area of disturbed plasma membrane (pink arrow) near extracellular amyloid fibrils (yellow arrows). Note undisturbed plasma membrane on the right half of photo. Myofibrils within cell are visible. (F) Uninfected heart stained with UALC has no amyloid fibrils in the extracellular space, and does not show plasma membrane invaginations or inclusions. Scale bars are as follows: A, 100 µm; B, 2 µm; C, D & E&F, 0.5 µm.

Figure 2.

Detection of amyloid PrPSc in brown fat tissue of scrapie-infected tg anchorless mice. (A) Interstitial extracellular area of brown fat tissue with 2 blood vessels (bv) and 2 adjacent adipocytes. Abundant immunogold-labeled amyloid PrPSc is visible as small fibrils (yellow arrow), and is located in the extracellular space (ecs) outside blood vessels and between adipocytes. Rounded fat globules are located within adipocytes (fat). Some areas with intact adipocyte plasma membrane are indicated by pink arrows, whereas in other areas the plasma membrane appears disrupted (see box). Immunogold staining used R30 antibody. (B) Enlarged region shown by box in panel A shows gold-labeled fibrils apparently disrupting and invaginating outer membrane of the adipocyte (yellow arrows). Vacuolar structures of various sizes are also seen in this area of the cell (turquoise arrows). (C) IHC staining with Mab D13 shows PrPSc amyloid along edges of brown fat lobules (yellow arrows) and surrounding a blood vessel (blue arrow). Red blood cells in lumen are labeled (rbc). Various-sized fat globules can be seen inside brown fat cells (pink arrow), but plasma membranes of fat cells are not well visualized in this section. (D) High magnification of ultrastructure of immunogold-labeled PrPSc amyloid fibrils (yellow arrow) invaginating a brown fat cell. Fat cell plasma membrane is shown by pink arrow. Immunogold staining used R30 antibody. (E) UALC- stained brown fat shows fibrils penetrating a fat cell in a narrow pocket (yellow arrow). (F) Uninfected brown fat stained with UALC shows collagen fibrils (black arrow) in extracellular space but no perturbations of the plasma membrane. Scale bars are as follows: A, 2 µm; B, 0.25 µm; C, 50 µm; D, E & F, 0.5 µm.

Figure 3.

Detection of amyloid PrPSc in white fat tissue of scrapie-infected tg anchorless mice. (A) Detection of PrPSc in interstitial areas of white fat by IHC using Mab D13. Blood vessel surrounded by PrPSc can also be seen (green arrow). (B) Ultrastructural view of interstitial region between 2 fat cells contains immunogold-labeled PrPSc fibrils (yellow arrows) in the extracellular space (ecs). The plasma membrane of the adipocytes is indicated by pink arrows, but is not seen as a sharp border in this section. Immunogold staining used R30 antibody. Scale bars are as follows: A, 50 µm; B, 0.5 µm.

Figure 4.

Detection of amyloid PrPSc in colon of scrapie-infected tg anchorless mice. (A) PrPSc detected by IHC using Mab D13 in lamina propria (lp) of colon. Blood vessel with red blood cells (rbc) in lumen has abundant PrPSc in a perivascular distribution. A small amount of PrPSc staining can also be seen at inner edge of epithelial cells (pink arrow). (B) Thioflavin S staining of amyloid PrPSc in colon of tg anchorless mouse similar to mouse shown in panel A. Most amyloid was located in the lamina propria (arrow) surrounding the villi. (C) Large macrophage-like cell appears to surround a clump of immunogold-labeled PrPSc amyloid with visible fibrils (yellow arrow). Immunogold staining in panels C, D, F, and G used 1A8 antibody. (D) Capillary in lamina propria is surrounded by a thick ring of immunogold-labeled PrPSc amyloid starting near the outer surface of the endothelial cells (green arrow). (E) At higher power UALC-stained arteriole shows thick ring of PrPSc amyloid fibrils between smooth muscle basement membrane and outer smooth muscle cells (pink arrow). Note that the endothelial cell body borders the lumen and separates the lumen from the smooth muscle. (F) Immunogold-labeled PrPSc accumulated in phagolysosomes (yellow arrows) of a macrophage-like cell in the lamina propria. (G) Immunogold-labeled PrPSc amyloid (yellow arrow) partially surrounds neuronal processes in a ganglion of the enteric nervous system (ens). Scale bars are as follows: A,&B 50 µm; C, 2 µm; D, 0.5 μm; E & G, 1 µm; F, 0.5 µm.

Figure 5.

Detection of PrPSc by immunohistochemistry using monoclonal anti-PrP antibody D13 in lymphoid tissues of scrapie-infected C57BL and tg anchorless mice. (A) In a C57BL mouse, PrPSc is mostly located in multiple secondary follicles (arrow) in spleen. There is a small amount of staining outside of the follicles (arrowhead) and almost no detectable sub-capsular staining. (B) In a tg anchorless mouse, in spleen PrPSc staining is mostly in secondary follicles (arrow) with a small amount in red pulp (arrowhead). (C) Mesenteric lymph node in a tg anchorless mouse shows pronounced sub-capsular PrPSc (arrow). (D) At a higher magnification, a single follicle from a C57BL mouse shows dense PrPSc staining (yellow arrow) concentrated in a rounded region consistent with FDC networks found in light zones of follicles. Single dense punctate PrPSc consistent with accumulation in macrophages was also noted (black arrows). (E) In spleen follicle from another scrapie-infected C57BL mouse, PrPSc staining occurred both in small punctate accumulations (black arrows), probably macrophages, and in patchy less dense accumulations suggestive of FDCs (yellow arrows). (F) In a follicle from the scrapie-infected tg anchorless mouse shown in panel B, punctate PrPSc staining (black arrows) characteristic of macrophages was seen mostly near the edge of the follicle. However, the clustered patchy PrPSc staining characteristic of FDC was not noted. Asterisks in panels D and F indicate the approximate location of the light zone of the follicles. Scale bars: Panels A and B, 100 μm; C, 50 μm; D, E and F, 20 μm.

Figure 6.

Ultrastructural detection of PrPSc by immunogold labeling in lymphoid tissues of scrapie-infected C57BL and tg anchorless mice. (A) Large immunogold-labeled PrPSc plaque in red pulp area of spleen from a tg anchorless mouse. (B) Higher magnification view of PrPSc plaque in a tg anchorless spleen shows immuno-gold labeling associated with amyloid fibrils (arrow). (C) Tingible body macrophage in Peyer's patch of tg anchorless mouse shows several cytoplasmic phagolysosomes (arrows) containing immunogold-labeled PrPSc (arrowhead). Two adjacent lymphocytes with round nuclei are partially visible on lower border. (D) Ultrastructural view of a follicular dendritic cell within a lymph node secondary follicle of a scrapie-infected tg anchorless mouse seen with UALC staining. FDC dendrites (den) surround the nucleus (N), however, these dendrites are not extended, and the extracellular space between dendrites is not expanded nor do they accumulate excess electron dense deposits as is typical in scrapie affected lymphoreticular tissues. Arrows point to the apposed membranes of adjacent dendrites. At the magnification shown, the extracellular space lying between apposed membranes of adjacent dendrites is not visible. No PrPSc was detected in associated with these FDC in infected tg anchorless mice by immunogold labeling (not shown), and this result was consistent with light microscopy results (Fig. 5E). (E) In a C57BL mouse, immunogold-labeled PrPSc is located within the extracellular space surrounding hypertrophic FDC dendrites. In some places dendrite membranes are visible (arrows). Immunogold labeling for PrPSc is associated with amorphous electron dense material which lies in an expanded extracellular space between dendrites (arrowhead). Additional uninfected mice were also examined as controls. These mice did not show any of the features described here for the scrapie-infected mice. Scale bars: A, 2 μm; B, 0.5 μm; C, 1 μm; D, 1 μm, E, 0.3 μm.