Prion infection of oral and nasal mucosa

Abstract

Centrifugal spread of the prion agent to peripheral tissues is postulated to occur by axonal transport along nerve fibers. This study investigated the distribution of the pathological isoform of the protein (PrP(Sc)) in the tongues and nasal cavities of hamsters following intracerebral inoculation of the HY strain of the transmissible mink encephalopathy (TME) agent. We report that PrP(Sc) deposition was found in the lamina propria, taste buds, and stratified squamous epithelium of fungiform papillae in the tongue, as well as in skeletal muscle cells. Using laser scanning confocal microscopy, PrP(Sc) was localized to nerve fibers in each of these structures in the tongue, neuroepithelial taste cells of the taste bud, and, possibly, epithelial cells. This PrP(Sc) distribution was consistent with a spread of HY TME agent along both somatosensory and gustatory cranial nerves to the tongue and suggests subsequent synaptic spread to taste cells and epithelial cells via peripheral synapses. In the nasal cavity, PrP(Sc) accumulation was found in the olfactory and vomeronasal epithelium, where its location was consistent with a distribution in cell bodies and apical dendrites of the sensory neurons. Prion spread to these sites is consistent with transport via the olfactory nerve fibers that descend from the olfactory bulb. Our data suggest that epithelial cells, neuroepithelial taste cells, or olfactory sensory neurons at chemosensory mucosal surfaces, which undergo normal turnover, infected with the prion agent could be shed and play a role in the horizontal transmission of animal prion diseases.

FIG. 1.

PrP^Sc deposition in tongue following intracerebral inoculation of the HY TME agent. PrP^Sc immunohistochemistry in clinical HY TME-infected (B, D, E, and F) and asymptomatic mock-infected (C) hamsters is shown. PrP^Sc (red punctate staining) deposits (B and D) were present in taste bud, lamina propria, stratified squamous epithelium, and nerve fibers in the fungiform papillae of clinically ill hamsters but not in tongues of mock-infected hamsters (C). In the tongue parenchyma, PrP^Sc deposits were present in nerve fascicles (E) and skeletal muscle (F). Following PrP^Sc immunohistochemistry, tissue was counterstained with hematoxylin (B through F). The taste bud in the fungiform papilla is outlined with a dashed line (A through D) based on morphology. A filled arrowhead (B) indicates PrP^Sc deposits in the stratified squamous epithelium. Specific structures in fungiform papillae are identified in a hematoxylin-and-eosin-stained section (A). LP, lamina propria; N (in gray lettering), nerve fiber in lamina propria. Bar, 25 μm.

FIG. 2.

Laser scanning confocal microscopy for PGP 9.5 and PrP^Sc in the fungiform papillae of hamsters intracerebrally inoculated with the HY TME agent. Immunofluorescence images for PGP 9.5 (A, red) and PrP^Sc (B, green) on the same tissue section were merged to illustrate areas of overlap (C, yellow). ToPro-3 (blue) was used as a nuclear counterstain. During the clinical phase of TME disease, colocalization of PGP 9.5 and PrP^Sc was found in the taste bud and lamina propria, with a lesser amount in the stratified squamous epithelium. No PrP^Sc immunofluorescence was found in the fungiform papillae of mock-infected hamsters following dual immunofluorescence for PGP 9.5 and PrP^Sc (D). LP, lamina propria; TB, taste bud. Bar, 20 μm. Images have an optical thickness of 0.8 μm.

FIG. 3.

Laser scanning confocal microscopy of taste buds in the fungiform papillae of hamsters intracerebrally inoculated with the HY TME agent. All images are oriented with the apical end of the taste bud toward the bottom left quadrant. The dashed line delineates taste bud boundaries. Dual immunofluorescence for PrP^Sc (A through C) labeled with Alexa Fluor 488 (green) and either α-gustducin (A), synaptobrevin-2 (B), or SNAP-25 (C), which were visualized using an anti-rabbit Alexa Fluor 568-conjugated antibody (red) on the same tissues. ToPro-3 (blue) was used as a nuclear counterstain. In taste cells, colocalization (yellow) of PrP^Sc and synaptobrevin-2 (B) or SNAP-25 (C) was observed, but no colocalization was seen in α-gustducin-positive taste cells (A) during the clinical phase of TME disease. Synaptobrevin-2 and SNAP-25 can also identify intragemmal and perigemmal nerve fibers, and colocalization with PrP^Sc could be present in these fibers as well as in taste cells. Bar, 20 μm. Images have an optical thickness of 0.8 μm.

FIG. 4.

Laser scanning confocal microscopy for PrP^Sc and cytokeratin in fungiform papillae of hamsters intracerebrally inoculated with the HY TME agent. Immunofluorescence images (optical thickness, 0.8 μm) for cytokeratin (A, red) and PrP^Sc (B, green) were merged in order to investigate areas of overlap (C, yellow). Colocalization of PrP^Sc and cytokeratin was found in the stratified squamous epithelium during the clinical phase of TME disease. Deconvolution and colocalization analysis of a z stack (i.e., a compiled stack of images acquired at 0.25-μm intervals through the thickness of the tissue) through the area shown in panels A through C illustrates only areas of colocalization (yellow) between cytokeratin and PrP^Sc (D). LP, lamina propria. Bar, 20 μm.

FIG. 5.

PrP^Sc distribution in the nasal cavity following intracerebral inoculation of the HY TME agent. Mock-infected (C) and clinical HY TME-infected (A, B, and D) hamsters were analyzed for prion infection at time of clinical disease using PrP^Sc immunohistochemistry as described in Materials and Methods. (A) PrP^Sc deposition was found in the olfactory sensory epithelium (OSE) where it was associated with the cell body layer, dendrites (open arrow), and dendritic terminals (filled arrow). In the vomeronasal organ (B), PrP^Sc was present in the vomeronasal sensory epithelium (VSE) but not the nonsensory epithelium. There was a demarcation (filled arrowhead) between the PrP^Sc deposits at the dendritic terminals near the mucosal surface of the VSE (filled arrow) and the ciliated nonsensory epithelium. PrP^Sc was also observed in the nasal-associated lymphoid tissue (NALT) in the nasal cavity (D). PrP^Sc immunostaining was not apparent in the nasal epithelium or vomeronasal organ from mock-infected hamsters (C). S, septum; Na, nasal airway. Bars: panel A, 200 μm; panel B, 20 μm; panel C, 200 μm; panel D, 20 μm.

FIG. 6.

Model for centrifugal spread of the HY TME agent from the brain stem to the tongue. The deposition of PrP^Sc in taste cells and the stratified squamous epithelium following intracerebral inoculation with the HY TME agent is consistent with centrifugal spread from the brain stem to the tongue via the chorda tympani branch of the facial nerve (CN VII) and the lingual branch of the mandibular division of the trigeminal nerve (CN V), respectively. The cell bodies for these two cranial nerves are located in the geniculate ganglion (GN) and the trigeminal ganglion (TGG), respectively. The central processes of the axons that convey taste information (i.e., in CN VII) terminate in the NST. The central processes of the axons that convey the general sensory information (i.e., in CN V) from the epithelium and muscles terminate in the principal and spinal trigeminal nucleus (V). Centrifugal spread of the HY TME agent to the tongue along these cranial nerves would likely require prion agent infection of NST or V nucleus and subsequent transsynaptic spread to nerve terminals in CN V and CN VII. The inset diagram illustrates a taste bud within the stratified squamous epithelium of a fungiform papilla. CN VII innervates a subset of taste cells within the taste bud, while branches of CN V ascend apically in the stratified squamous epithelium of the fungiform papilla. In addition, HY TME agent deposition in skeletal muscle cells is consistent with infection of motor neurons in the hypoglossal nucleus (XII) and retrograde transport within the hypoglossal nerve (CN XII). HY TME infection of either skeletal muscle cells or taste cells could be due to transsynaptic spread from CN XII or CN VII, respectively.