Unusual cerebral vascular prion protein amyloid distribution in scrapie-infected transgenic mice expressing anchorless prion protein

Abstract

Background: In some prion diseases, misfolded aggregated protease-resistant prion protein (PrPres) is found in brain as amyloid, which can cause cerebral amyloid angiopathy. Small diffusible precursors of PrPres amyloid might flow with brain interstitial fluid (ISF), possibly accounting for the perivascular and intravascular distribution of PrPres amyloid. We previously reported that PrPres amyloid in scrapie-infected transgenic mice appeared to delay clearance of microinjected brain ISF tracer molecules.

Results: Here we studied distribution of PrPres amyloid on capillaries, arteries and veins to test whether vascular specificity of PrPres corresponded to distribution of ISF tracer molecules. To distinguish PrPres-positive arteries from veins and capillaries, scrapie-infected mouse brains were studied by immunodetection of alpha smooth muscle actin. ISF was studied using fluorescein-labeled ovalbumin microinjected into brain as a tracer. In infected preclinical or clinical mice, PrPres was found mostly on capillaries (73-78%). Lower levels were found on arteries (11-14%) and veins (11-13%). Compared to PrPres, ISF tracer was found at higher levels on capillaries (96-97%), and the remaining tracer was found at a skewed ratio of 4 to 1 on arteries and veins respectively.

Conclusions: PrPres association with blood vessels suggested that ISF flow might transport diffusible PrPres precursor molecules to perivascular sites. However, the different vascular specificity of PrPres and ISF tracer indicated that ISF flow did not alone control PrPres dissemination. Possibly blood vessel basement membrane (BM) components, such as glucosaminoglycans, might concentrate small PrPres aggregates and serve as scaffolds for PrP conversion on multiple vessel types.

Figure 1

Immunofluorescence detection of PrPres and ASMA in brain tissue of scrapie-infected Tg44+/+ mice. Mice were examined at 250–280 dpi which correlates with early clinical signs [10,27]. PrPres was detected with D13 monoclonal antibody (green) and ASMA was detected with rabbit anti-ASMA (red) as described in the Methods section. (a) Overview photo showing PrPres plaques (green) and ASMA detection (red). Several capillaries (yellow arrows), one venule (arrowhead) and one arteriole (pink arrow) are shown associated with PrPres plaques. (b) Three separate PrPres plaques (green) are seen. One has a central ASMA-positive arteriole (arrow) and the other two have a central vein (arrowheads). (c) Several capillaries (arrows) and one venule (arrowhead) are located in a group of small PrPres plaques. (d) PrPres plaques in meninges and adjacent parenchyma associated with 4 ASMA-positive arteries or arterioles (arrow) and two veins (arrowheads).(e) PrPres plaque with ASMA-negative vein (arrowhead). (f and g) PrPres plaques with associated ASMA-positive arterioles (arrows). Bars: 25 μm (b and f), 50 μm (a, c, d, e, and g).

Figure 2

Immunohistochemical detection of ASMA and PrPres in scrapie-infected Tg44+/+ transgenic mice at 308 dpi. (a) Overview showing perivascular PrPres amyloid plaques (brown) and ASMA (pink) in cerebral cortex. Note association of PrPres plaques with capillaries (green arrowheads), veins (black arrows) and arteries (green arrows). (b) Higher magnification of perivascular PrPres on a large ASMA-negative vein (black arrow) and a small capillary (green arrowhead). (c) PrPres surrounding an artery (green arrow) and a capillary (green arrowhead). (d) ASMA-positive leptomeningeal artery with thin ablumenal ring of PrPres (green arrow). (e) Leptomeningeal vein (black arrow) with adjacent PrPres. In lower area a small artery (green arrow) and a capillary (green arrowhead) have associated PrPres. Bars: 100 μm (a), 50 μm (b-e).

Figure 3

Immunofluorescence detection of ASMA and FITC-OVA in uninfected Tg44+/+ mice at 30 min after stereotaxic microinjection of tracer. ASMA is observed as red color and FITC-OVA tracer is green. (a) Epifluorescence, (b-e) Confocal optical sections of 0.38 μm thickness. (a) Most tracer is associated with capillaries (arrows), but some tracer is associated with a larger venule (arrowhead). (b) ASMA-positive artery with associated tracer. (c) Two ASMA-positive arteries with associated tracer. (d) ASMA-negative tracer-positive vein (arrowhead) with 25 μm diameter and two smaller tracer-positive capillaries (arrows) for comparison. (e) ASMA-negative tracer-positive vein (17 μm diameter). Bars: 100 μm (a), 40 μm (b) and 20 μm (c-e).

Figure 4

Cartoon depicting seeding of PrP conversion by fibrils of various sizes in injected scrapie brain homogenate. Following inoculation larger fibrils (green) might diffuse poorly in brain but would seed PrP conversion (orange) locally near inoculation site. Smaller oligomeric fibrils (blue) may be too small to seed PrP conversion [34], but can travel by diffusion and in brain interstitial fluid (ISF) flow towards blood vessel basement membranes where they might be bound by glucosaminoglycans (GAGs) and other molecules. These concentrated oligomers might form a scaffold capable of seeding new PrPres generation (orange) which in turn might self-scaffold further PrPres conversion (orange) extending both radially and linearly around blood vessel. Smaller oligomers generated at this new site might travel in the ISF flow to new blood vessels and might initiate seeding at these distant sites.