Figure 1.
Western blot for olfactory marker protein and prion protein in brainstem, olfactory bulb, and olfactory mucosa following HY TME infection of hamsters.
Tissue from age-matched, mock-infected (lanes 1, 4, 7) and HY TME-infected (lanes 2, 3, 5, 6, 8, 9) hamsters following intracerebral (lanes 2, 5, 8), and intra-olfactory bulb (lanes 1, 3, 4, 6, 7, 9) inoculation were homogenized and 50 (A, B) or 100 (C) micrograms of protein was analyzed per lane. In C, protein samples were digested with 10 ug/ml of proteinase K for 37°C for 1 hour prior to SDS-PAGE. This treatment removes PrPC in mock-infected samples and truncates PrPSc in HY TME lysates. Total prion (PrP) protein (B) and PrPSc (C) was immunodetected with anti-PrP 3F4 monoclonal antibody, while olfactory marker protein (A) was immunodetected with polyclonal goat serum to OMP. OMP is present in both the olfactory bulb, which contains the olfactory nerve and terminals, and in the olfactory mucosa that contain the soma, dendrites, and axons of ORNs. Marker (M) polypeptides correspond to 20, 30, and 40 kDa. The short vertical tick marks at the top and bottom of each panel are placed before lanes 4 and 7 in order to align lanes between each panel.
Figure 2.
Western blot for PrPSc in olfactory bulb and olfactory mucosa following HY TME infection of hamsters.
Olfactory bulb (lanes 1 to 4) and olfactory mucosa (lanes 5 to 8) lysates from clinical HY TME hamsters following intracerebral (lanes 1, 2, 5 and 6) and intra-olfactory bulb (lanes 3, 4, 7, and 8) inoculation were enriched for PrPSc by detergent extraction, ultracentrifugation, and proteinase K digestion. Western blot for prion protein was performed as described in Figure 1. Marker (M) polypeptides correspond to 20, 30, and 40 kDa.
Figure 3.
Distribution of PrPSc in main olfactory bulb following HY TME infection.
Hematoxylin and eosin staining of olfactory bulb (A) and PrPSc immunohistochemistry (B, brown chromagen and hematoxylin counterstain of nuclei) in adjacent tissue section revealed a strong PrPSc pattern throughout the olfactory bulb including the granule layer (GL), inner plexiform layer (IPL), mitral cell layer (M), outer plexiform layer (OPL), and glomeruli (G), but not in the outer nerve layer (ONL). Asteriks in panel B illustrate PrPSc deposition in a single glomerulus, which is enlarged in panel C (PG = periglomerular cells). In D to F, double immunofluorescence and laser scanning confocal microscopy was used to demonstrate the relationship of olfactory marker protein (OMP)(D) and PrPSc (E) in a single glomerulus of the olfactory bulb. OMP and PrPSc immunofluorescence are merged in panel F and nuclei were stained with ToPro-3 (blue). White arrow #1 illustrates PrPSc immunofluorescence that was surrounded by OMP immunofluorescence in the glomerulus, while white arrow #2 is an example of PrPSc and OMP overlap in the glomerulus. Immunofluorescence staining and LSCM were performed as described in materials and methods. Scale bar is 100 µm in A and C and 10 µm in D.
Figure 4.
Distribution of PrPSc in olfactory sensory epithelium following intra-olfactory bulb inoculation of the HY TME agent.
Laser scanning confocal microscopy of olfactory marker protein (OMP)(A, D, G), PrPSc (B, E, H), and for both OMP and PrPSc (Merge)(C, F, I). Panels A through C, D through F, and G through I are the same field of view. In A to C, the olfactory sensory epithelium (OSE or O) and nerve bundles (*) in the subepithelial layer (SE) express high levels of OMP and the airway lumen (AL) is observed between two opposing OSEs in the nasal turbinate. In D to F, the OSE is distinct from the nonsensory epithelium (NSE) by the presence of OMP and is separated by a dashed white line at the border between the two epithelial layers. The soma of an olfactory receptor neuron (ORN) in the OSE is identified by a white arrow and PrPSc was only observed in OMP-positive ORNs and not in the NSE. In G to I, the white arrow points to the soma of an OMP-positive ORN and the yellow arrow indicates the apical dendrite of an OMP-positive ORN, which sits adjacent to the airway lumen (G). PrPSc was present in both the soma and dendrites of ORNs (H, I). Immunofluorescence staining and LSCM were performed as described above. Scale bar is 100 µm in A and 10 µm in D and G.
Table 1.
Colocalization of PrPSc and olfactory marker protein in olfactory receptor neurons.
Figure 5.
PrPSc distribution at the border of airway lumen of the olfactory and vomeronasal sensory epithelium following intra-olfactory bulb inoculation of the HY TME agent.
Laser scanning confocal microscopy of adenylyl cyclase III (ACIII)(A), olfactory marker protein (OMP)(D), PrPSc (B, E), and for both ACIII or OMP and PrPSc (Merge)(C, F). Panels A through C, and D through F are the same field of view. PrPSc was prominent in the OSE including at the border with airway lumen (AL)(B) where it appears to overlap with ACIII (A), which is located on the sensory cilia that project from the terminal dendrites of ORNs into the AL (C). Boxed area is an inset corresponding to enlarged region indicated by arrow. In D to F, the soma, dendrites and dendritic knobs of vomeronasal receptor neurons (VRN, white arrows) in the vomeronasal sensory epithelium were OMP-positive. PrPSc was found associated with VRNs in the cell layer and at the border with the vomeronasal airway lumen (VL)(B). Inset illustrates enlarged area of PrPSc deposition in a location consistent with the terminal dendrites of OMP-positive dendritic knobs (C). PrPSc deposition was less frequently observed in the proximal dendrites as they transverse the support cells (sc, can visualize their elongated blue nuclei). Nuclei were stained with To-Pro 3. Scale bar is 10 µm.
Table 2.
Prion infectivity of olfactory bulb and olfactory mucosa lysates following HY TME inoculation.
Table 3.
Prion infectivity in nasal lavages of HY TME infected hamsters.
Figure 6.
Western blot detection of HY TME infection in nasal lavages using the QuIC assay.
Nasal lavages (2 µl) from mock- (M)(lanes 3, 4, 6, and 7 correspond to the four mock-infected samples from Table 3) and HY TME (H) -infected hamsters (lanes 9, 10, and 12 correspond to HY TME i.ob. #1 to #3 in Table 3) were subject to two serial rounds of the QuIC assay in order to detect low levels of PrPSc. PK-resistant PrP polypeptides between 11 and 17 kDa were observed in the HY TME nasal lavage samples indicating amplification of PrPSc. In mock-infected nasal lavage samples either no polypeptides or occassionally lower molecular weight polypeptides (≤12 kDa) were observed, but the latter were not observed in prion-positive control samples. Controls include mock-infected hamster brain homogenates at a 10−2 and 10−6 dilution (lanes 1 and 13, respectively), HY TME hamster brain at a 10−6, 10−8, and 10−10 dilution (lanes 5, 8, and 11, respectively), and 100 femtogram of purified PrPSc from 263K scrapie infected hamster brain (lane 2).
Table 4.
Primary antibodies used to investigate HY TME infection in olfactory mucosa by laser scanning confocal microscopy.