Non-amyloid and amyloid prion protein deposits in prion-infected mice differ in blockage of interstitial brain fluid

Abstract

Aims: Prion diseases are characterized by brain deposits of misfolded aggregated protease-resistant prion protein (PrP), termed PrPres. In humans and animals, PrPres is found as either disorganized non-amyloid aggregates or organized amyloid fibrils. Both PrPres forms are found in extracellular spaces of the brain. Thus, both might block drainage of brain interstitial fluid (ISF). The present experiments studied whether ISF blockage occurred during amyloid and/or non-amyloid prion diseases.

Methods: Various-sized fluorescein-labelled ISF tracers were stereotactically inoculated into the striatum of adult mice. At times from 5 min to 77 h, uninfected and scrapie-infected mice were compared. C57BL/10 mice expressing wild-type anchored PrP, which develop non-amyloid PrPres similar to humans with sporadic Creutzfeldt-Jakob disease, were compared with Tg44+/+ mice (transgenic mice secreting anchorless PrP) expressing anchorless PrP, which develop amyloid PrPres similar to certain human familial prion diseases.

Results: In C57BL/10 mice, extensive non-amyloid PrPres aggregate deposition was not associated with abnormal clearance kinetics of tracers. In contrast, scrapie-infected Tg44+/+ mice showed blockage of tracer clearance and colocalization of tracer with perivascular PrPres amyloid.

Conclusions: As tracer localization and clearance was normal in infected C57BL/10 mice, ISF blockage was not an important pathogenic mechanism in this model. Therefore, ISF blockage is unlikely to be a problem in non-amyloid human prion diseases such as sporadic Creutzfeldt-Jakob disease. In contrast, partial ISF blockage appeared to be a possible pathogenic mechanism in Tg44+/+ mice. Thus this mechanism might also influence human amyloid prion diseases where expression of anchorless or mutated PrP results in perivascular amyloid PrPres deposition and cerebral amyloid angiopathy.

Figure 1

Brain microinjection of FITC-OVA in uninfected and scrapie-infected C57BL/10 mice and uninfected and scrapie-infected Tg44+/+ mice. At 5 and 30 min post-injection in uninfected (A,E) and infected (B,F) C57BL/10 mice and in uninfected Tg44+/+ mice (C,G), FITC-OVA was associated with walls of capillaries (arrows) and larger vessels (arrowheads). At 5 and 30 min after injection in scrapie-infected Tg44+/+ mouse (D,H), FITC-OVA was mainly concentrated around large plaque-like perivascular structures (arrowheads), but association with walls of capillaries (arrows) could also be seen. Panel D: Scale bar 100 μm; same scale for all panels. (I,J,K) At 7 hours post-injection in uninfected and infected C57BL/10 mice and in uninfected Tg44+/+ mice FITC-OVA was barely visible associated with small capillaries (arrows). (L) At 7 hours post-injection in infected Tg44+/+ mice, FITC-OVA remained associated with perivascular plaque-like structures (arrowheads), but intensity of staining was decreased for most plaques, and normal capillaries were no longer visibly stained. (M,N,O) At 24 h after injection in both uninfected and infected C57BL/10 mice, and uninfected Tg44+/+ mice FITC-OVA was no longer visible. (P) At 24 hours post-injection in Tg44+/+ scrapie-infected mice, FITC-OVA was detectable at low intensity in occasional blood vessels in the center of perivascular plaque-like structures (arrow).

Figure 2

Immunohistochemical staining for PrPres in the striatum region of uninfected C57BL/10 (A) and scrapie-infected C57BL/10 mice with early clinical signs. Panel B shows abundant patchy diffuse brown staining of PrPres (arrows) in the infected mouse, whereas no staining of PrPres was seen in the uninfected mouse (A). Scale bar is 200μm. These sections were taken from the same mice studied at 30 min post-injection of FITC-OVA shown in Figure 1 E,F. PrPres amyloid with CAA in Tg44+/+ mice is not shown here because it was extensively documented in a previous paper [18].

Figure 3

Comparison of relative fluorescence signal of tracers associated with blood vessels or PrPres plaques at various times after microinjection in replicate mice. (A) FITC-OVA-injected C57BL/10-U (uninfected) and C57BL/10-S (scrapie-infected) mice. (B) FITC-OVA-injected Tg44-U (uninfected) and Tg44-S (scrapie-infected) mice. (C) FITC-Cadaverine injected Tg44-U (uninfected) and Tg44-S (scrapie-infected) mice. Scale for relative fluorescence signal is defined in the Methods section. Points and error bars are means and standard errors. Each point has data from 2–6 mice. Data was analyzed statistically by a 2-way ANOVA with Bonferroni’s correction using Graph Pad software. For experiments studying FITC-OVA, Tg44-S in panel B was significantly different (P<0.001) from Tg44-U in same panel and from both C57BL/10-S and C57BL/10-U in panel A. In panel C showing data for FITC-Cadaverine, Tg44-S was significantly different (P<0.001) from Tg44-U.

Figure 4

Brain microinjection of uninfected and scrapie-infected Tg44+/+ mice with FITC-cadaverine tracer (FITC-CDV) (green). (A) 30 min post-injection of uninfected Tg44+/+ mice showing tracer associated with capillaries (arrow) and a larger vessel (arrowhead). Scale bar = 100 μm; panels (B–F) are same scale. (B) 30 min post-injection of infected mouse showing tracer associated with large perivascular plaque-like structures (arrowhead), as well as small capillaries lacking plaques (arrow). (C) Clearance of tracer at 2 h post-injection in uninfected mouse. (D) At 2 h post-injection in infected mouse tracer is still visible associated with some perivascular plaques (arrowheads). Some faint staining of capillaries lacking plaques is also still visible (arrow). (E) and (F) At 7 h post-injection tracer is no longer visible in uninfected and infected mice.

Figure 5

Detection of FITC-OVA (green) and PrPres (red) at 30 min post-microinjection in scrapie-infected Tg44+/+ mice by confocal (A–I) and epifluorescent (J–L) microscopy. Panels show optical sections of 0.5 micron thickness. (A) FITC-OVA tracer in both perivascular (arrowhead) and plaque-like (arrows) distributions. (B) PrPres immunostaining with monoclonal antibody D13 in same section shows perivascular PrPres and a large PrPres plaque. (C) Merge of panels (A) and (B) shows partial co-localization of FITC-OVA with PrPres (yellow). (D) FITC-OVA distributed along the basement membrane of a blood vessel (arrow). Fine deposits of tracer are also seen outside the BM (arrowheads). Smaller capillaries associated with FITC-OVA are also visible in the background. (E) Immunodetection of perivascular PrPres in same area. The blood vessel itself appears as a dark shape surrounded by red PrPres staining. In this section the plaque is less dense than in panel (B). (F) Merge shows minimal co-localization of FITC-OVA and PrPres. DAPI channel was included in the merge, and this blue nuclear staining reveals FITC-OVA (green) ablumenal to the blue stained endothelial cell nuclei in the vessel (arrows). (G) In a different optical section of the same field, FITC-OVA accumulation is seen around vessels (arrowhead) and along BM (arrow). (H) PrPres in perivascular distribution around the same vessels (arrowheads). (I) Merge shows partial co-localization (yellow) of PrPres and FITC-OVA in perivascular and BM areas (arrows). (J,K,L) Detection of FITC-CDV (green) and PrPres (red) on vessels within plaques of infected Tg44+/+ mouse at 30 min post-injection. (J) Tracer is concentrated on vessel walls (arrows), but faint staining of larger plaques is also visible (arrowhead). (K) Immunostaining of PrPres plaques in same section. (L) Merge of previous panels shows FITC-CDV on vessels (green arrows) within PrPres plaques in same section. Scale bars: panels (A–C), 50 μm; panels (D–I), 30 μm; panels (J–L) 100 μm.

Figure 6

Detection of laminin and FITC-OVA by confocal microscopy in uninfected (A,B,C) and infected (D,E,F) Tg44+/+ mice at 30 min post-microinjection of FITC-OVA. Panels show optical sections of 0.25 μm. (A) FITC-OVA (green) associated with large 40 μm diameter blood vessel (arrow) in uninfected mouse. Scale bar = 20 μm; panels B and C have same scale. (B) Detection of laminin (red) in double basement membrane pattern in same section as panel (A). (C) Merge shows partial co-localization of laminin and FITC-OVA (orange) in same section (arrow). (D) Infected Tg44+/+ mouse showing FITC-OVA (green) in perivascular distribution around two large vessels (arrows) and adjacent PrPres plaques (arrowheads). Scale bar = 20 μm; panels (E and F) have same scale. (E) Laminin (red) around blood vessels in same section (arrowheads). (F) Merge of same section shows partial co-localization of FITC-OVA and laminin (orange) in basement membrane areas (arrowheads).