Progress 12/01/08 to 11/30/09

Outputs
The long-term goal of the proposed research is to define the route and mechanism of prion agent infection of skeletal muscle during prion diseases of livestock. The hypothesis to be tested is that prion neuroinvasion of the brainstem following oral ingestion of the prion agent leads to infection of one or more of the tongue-associated cranial nerves. In the past year we focused on the following areas: 1) to determine if the prion agent can infect epithelial cells at the mucosa and the route(s) of prion spread to epithelial mucosa, and 2) to investigate prion infection of tongue and nasal mucosa in ruminants with prion disease. We continued our studies on the presence of prion infection in tissues from the oral and nasal mucosa from ruminants with prion disease. Greater than 80% of these mucosa-associated tissues were positive for PrP-res in sheep with scrapie and deer and elk with chronic wasting disease. In cattle that were infected with chronic wasting disease and transmissible mink encephalopathy, PrP-res was not detected in tongue and nasal septal tissues. These findings indicate that prion infection is present in mucosal tissues in ruminant species in which horizontal transmission of prions (i.e., sheep and cervids) is a common pathway of spread, but is not in these mucosal tissues of cattle in which there is not an endemic prion disease. To measure prion infection in epithelial cells at the oral mucosa, PrP-res deposition was analyzed in the keratin layer of the stratified squamous epithelium (SSE) of fungiform papillae and in the SSE of filiform papillae, which rarely contains nerve fibers. Following infection of cranial nerves, we were able to detect PrP-res in both the keratin layer of the SSE and the SSE of filiform papillae. We were also able to demonstrate that PrP-res does colocalize with markers for nerve fibers in these locations in some instances, but is also deposited in areas without clear evidence of nerve fiber innervation. These studies strongly suggest that epithelial cells in the oral mucosa can support prion infection and shedding of the mucosa may be a source of prion infection in saliva.

Impacts
The implication of these findings is that prion infection of muscle and epithelium represents a potential source of prion transmission via either consumption of meat products or from bodily fluids in contact with mucosal epithelium, respectively. Nerve fibers that transverse almost all food products are also a potential source of prion contamination of food. The prion distribution into these sites in the tongue is likely via centrifugal spread along motor and sensory fibers with subsequent transynatpic spread into muscle cells or epithelial cells, respectively. These findings suggest that the prion agent can use nerve fibers to spread to peripheral tissues and spread across peripheral synapses to infect mucosal epithelium. This is the first study to demonstrate prion spread via peripheral synapses into tissues. This conclusion is also supported by in vitro studies that demonstrate the prion infection of muscle cells in vitro required contact with neuronal cell lines and not non-neuronal cells lines suggesting that a NMJ is required for prion infection of muscle cells. Since the SSE of the tongue undergoes continual turnover, renewal, and shedding, these findings suggest that the prion agent could establish infection in epithelial cells and could be shed as these cells mature in the SSE, become the keratinized layer, and are sloughed into oral bodily fluids. Therefore, saliva may be one source of prions that is shed from a host and can infect a naive host through direct or indirect contact. The spread of prions into skeletal muscle cells via nerve fibers suggest that muscle tissue, and not just nerves that transverse muscle, are a potential source of prion exposure upon ingestion of food products containing meat.

Publications

• Bessen, R.A., Martinka, S., Kelly, J., & Gonzalez, D. 2009. The role of the lymphoreticular system in prion neuroinvasion from the oral and nasal mucosa. Journal of Virology 83:6435-6445.

Progress 12/01/05 to 11/30/09

Outputs
Outputs: The long-term goal of the proposed research is to define the route and mechanism of prion agent infection of skeletal muscle during prion diseases of livestock. The goals of this project were 1) to investigate the role of peripheral synapses (e.g., neuromuscular junction) in prion infection of skeletal muscle cells; 2) to establish prion infection in muscle cells in vitro; 3) to determine if the prion agent can infect epithelial cells at the mucosa and the route(s) of prion spread to epithelial mucosa; and 4) to investigate prion infection of tongue and nasal mucosa in ruminants with prion disease. To investigate the role of the neuromuscular junction (NMJ) in prion entry into skeletal muscle, a spatiotemporal analysis was performed on the deposition of the disease-specific prion protein, PrP-res, in tongue following prion infection. Our findings indicate that PrP-res colocalized with the NMJ at the time of entry into skeletal muscle, but not later in the disease course. These findings indicate that at the initial time of prion entry into cells that PrP-res was linked to the NMJ, and suggests that this peripheral synapse acts as a route for prion agent entry into muscle clls. To determine if skeletal muscle cells can directly support prion infection, we attempted to establish prion infection in a muscle cell line (C2C12 cells) in vitro. Scrapie infection of murine C2C12 myoblasts and myotubes in vitro was established following co-culture with a scrapie-infected murine neuroblastoma cell line, but not following incubation with a non-neuronal cell line or a scrapie brain homogenate. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals. To determine if the prion agent can directly infect epithelial cells at the tongue mucosa we analyzed the keratin layer of the stratified squamous epithelium (SSE). PrP-res was detected in both the keratin layer of the SSE and the SSE of filiform papillae. PrP-res did not always colocalize with markers for nerve fibers in these locations suggesting infection was present in epithelial cells. These studies strongly suggest that epithelial cells in the oral mucosa can support prion infection and shedding of the mucosa may be a source of prion infection in saliva. We investigated infection of tongue and nasal turbinates in over 80 ruminants infected with prion diseases. In prion-infected sheep, deer, and elk greater than 85% of tongue and nasal turbinates were PrP-res positive. Tongue and nasal turbinates from CWD or TME-infected cattle were examined from over 25 animals, but we were unable to detect PrP-res in any of these samples. These findings indicate that prion infection is present in mucosal tissues in ruminant species in which there is horizontal transmission of prions, but not in cattle in which there is not an endemic prion infection.

Impacts
The implication from these findings is that the prion agent can spread away from the brain into mucosa tissues in the head, specifically the tongue and nasal cavity. Since these tissues have a mucosal surface, it may be possible that the prion agent is shed from the tongue or nasal cavity. In the tongue, the epithelium is continually undergoing terminal differentiation and shedding, and is then sloughed into saliva. In the nasal mucosa, olfactory neurons continually mature and turnover during adult life and prions may be shed from this mucosa into nasal secretions. Therefore, saliva and nasal secretions may be a potential source of prions that are shed from a host and can infect a naive host through direct or indirect contact. Another implication of these findings is that prion infection of ruminant muscle is a potential threat to animal and human food safety. The head of ruminants is banned for human or ruminant consumption with the exception of the tongue. Our findings indicate that prions undergo centrifugal spread from the brainstem to the tongue and can enter skeletal muscle cells via the neuromuscular junction and further replicate in muscle cells. These studies suggest that tongue should also be included in the specified risk materials in order to minimize exposure to tongue products containing prion agent.

Publications

• DeJoia, C., Moreaux, B., O Connell, K. & Bessen, R.A. 2006. Prion infection of oral and nasal mucosa. Journal of Virology 80:4546-4556.
• Dlakic, W.M., Grigg, E. & Bessen, R.A. 2007. Prion infection of muscle cells in vitro. Journal of Virology 81:4615-4624. (Faculty of 1000 Biology selection.)
• Bessen, R.A., Martinka, S., Kelly, J., & Gonzalez, D. 2009. The role of the lymphoreticular system in prion neuroinvasion from the oral and nasal mucosa. Journal of Virology 83:6435-6445.
• Bessen, R.A., Shearin, H., Martinka, S., Boharski, R., Lowe, D., Wilham, J., Caughey, & Wiley, J. 2010. Prion shedding from olfactory neurons into nasal secretions. PLoS Pathogens 6:e1000837.

Progress 12/01/07 to 11/30/08

Outputs
The long-term goal of the proposed research is to define the route and mechanism of prion agent infection of skeletal muscle during prion diseases of livestock. Prion infection of skeletal muscle is a potential threat to animal and human food products. The hypothesis to be tested is that prion neuroinvasion of the brainstem following oral ingestion of the prion agent leads to infection of one or more of the tongue-associated cranial nerves. In the past year we focused on the following areas: 1) to investigate the role of peripheral synapses (i.e., neuromuscular junction) in prion infection of skeletal muscle cells, 2) to determine if the prion agent can infect epithelial cells at the mucosa and the route(s) of prion spread to epithelial mucosa, and 3) to investigate prion infection of tongue and nasal mucosa in ruminants with prion disease. To investigate prion infection of oronasal tissues, we investigated infection of tongue and nasal turbinates in >80 ruminant animals that were experimentally infected with prion diseases. In prion-infected sheep, deer, and elk greater than 85% of tongue and nasal turbinates were PrP-res positive. These findings indicate that prion infection is present in mucosal tissues in ruminant species in which there is horizontal transmission of prions (i.e., sheep and cervids). To measure prion infection in epithelial cells at the oral mucosa, PrP-res deposition was analyzed in tongue, specifically in the keratin layer of the stratified squamous epithelium (SSE) of fungiform papillae and in the SSE of filiform papillae, which rarely contains nerve fibers. Following infection of cranial nerves, we were able to detect PrP-res in both the keratin layer of the SSE and the SSE of filiform papillae. We demonstrated that PrP-res does colocalize with markers for nerve fibers in these locations in most instances, but is also located in the SSE in areas without clear evidence of nerve fiber innervation. These studies strongly suggest that prions can invade epithelial tissues via nerve fibers and may be able to enter or replicate in epithelial cells in the oral mucosa. Prion localization to the oral epithelium has the potential to result in the shedding of prions from the mucosa, which may be a source of prion infection in saliva. We also continued to define the pathway by which prions spread along cranial nerves of the tongue and the role of the neuromuscular junction (NMJ) in prion entry into skeletal muscle. We initiated a series of studies that examined prion infection of the hypoglossal nerve and performed a spatiotemporal analysis of prion agent entry into the brainstem and muscles of the tongue. Besides demonstating that prions likely enter the tongue via the NMJ, we established that prions likely spread in axons will being transported in organelles such as late endosomes, and can accumulate to high levels in skeletal muscle. We are further investigating the location of prions in axons in order to better understand how they move in the nervous system since axonal spreading is one of the main pathways of prion dissemination throughout the body of the host. These findings indicate that at the initial time of prion entry into cells that PrP-res is linked to the NMJ and suggests that this peripheral synapse acts as a route or prion agent entry into muscle cells. They also suggest that prions can spread along axons into peripheral tissues.

Impacts
The implication of these findings is that prion infection of muscle and epithelium represents a potential source of prion transmission via either consumption of meat products or from bodily fluids in contact with mucosal epithelium, respectively. Nerve fibers transverse all bodily tissue and therefore, almost all food products from an animal with subclinical prion disease are a potential source of prion contamination of food. The prion distribution into these sites in the tongue is likely via centrifugal spread along motor and sensory fibers with subsequent transynatpic spread into muscle cells or epithelial cells, respectively. These findings suggest that the prion agent can use nerve fibers to spread to peripheral tissues and spread across peripheral synapses to infect mucosal epithelium. This is the first study to demonstrate prion spread via peripheral synapses into tissues. This conclusion is also supported by in vitro studies that demonstrate the prion infection of muscle cells in vitro required contact with neuronal cell lines and not non-neuronal cells lines suggesting that a NMJ is required for prion infection of muscle cells. Since the SSE of the tongue undergoes continual turnover, renewal, and shedding, these findings suggest that the prion agent could establish infection in epithelial cells and could be shed as these cells mature in the SSE, become the keratinized layer, and are sloughed into oral bodily fluids. Therefore, saliva may be one source of prions that is shed from a host and can infect a naive host through direct or indirect contact. The spread of prions into skeletal muscle cells via nerve fibers suggest that muscle tissue, and not just nerves that transverse muscle, are a potential source of prion exposure upon ingestion of food products containing meat.

Publications

Progress 12/01/06 to 11/30/07

Outputs
The long-term goal of the proposed research is to define the route and mechanism of prion agent infection of skeletal muscle during prion diseases of livestock. Prion infection of skeletal muscle is a potential threat to animal and human food products. The hypothesis to be tested is that prion neuroinvasion of the brainstem following oral ingestion of the prion agent leads to infection of one or more of the tongue-associated cranial nerves. In the past year we focused on the following areas: 1) to investigate the role of peripheral synapses (i.e., neuromuscular junction) in prion infection of skeletal muscle cells, 2) to determine if the prion agent can infect epithelial cells at the mucosa and the route(s) of prion spread to epithelial mucosa, and 3) to establish prion infection in muscle cells in vitro. To investigate the role of the neuromuscular junction (NMJ) in prion entry into skeletal muscle, the hypoglossal motor nerve was inoculated with prions and a spatiotemporal analysis was performed on the deposition of the disease-specific prion protein, PrP-res. To determine the role of the NMJ in prion agent entry into muscle cells, laser scanning confocal microscopy (LSCM) was used to determine the co-localization of PrP-res with synaptophysin (a marker for the NMJ) at the initial time of entry into muscle versus at the time of onset of clinical symptoms. We have now demonstrated that PrP-res is statistically colocalized with the NMJ at the time of entry into muscle cells, but not at later times in the disease course when infection was widely distributed through muscle cells. These findings indicate that at the initial time of prion entry into cells that PrP-res is linked to the NMJ and suggests that this peripheral synapse acts as a route or prion agent entry into muscle cells. Currently, we are investigating prion movement in nerve bundles prior to entry into muscle cells to determine if this site is also important for prion replication. To determine if skeletal muscles cells can directly support prion infection, we have been able to establish prion infection in a muscle cell line (i.e., C2C12 cells) in vitro. Scrapie infection of murine C2C12 myoblasts and myotubes in vitro was established following co-culture with a scrapie-infected murine neuroblastoma (N2a) cell line but not following incubation with a non-neuronal cell line or a scrapie brain homogenate. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals. This study has now been published. To further determine if the prion agent can directly infect epithelial cells at the tongue mucosa via cranial nerves, the prion agent was inoculated into the sensory nerves of the tongue. This resulted in early targeting of prion infection to the nerve fibers of the tongue. A high level of prion infection was found in the sensory fibers and taste cells in fungiform papillae of the tongue. We are currently investigating whether prions can infect the epithelium of the oral mucosa and preliminary findings are promising. These studies may suggest that epithelial cells in the oral mucosa can support prion infection and shedding of the mucosa may be a source of prion infection in saliva.

Impacts
The implication of these findings is that prion infection of muscle and epithelium represents a potential source of prion transmission via either consumption of meat products or from bodily fluids in contact with mucosal epithelium, respectively. The prion distribution into these sites in the tongue is likely via centrifugal spread along motor and sensory fibers with subsequent transynatpic spread into muscle cells or epithelial cells, respectively. These findings suggest that the prion agent can use nerve fibers to spread to peripheral tissues and spread across peripheral synapses to infect mucosal epithelium. This is the first study to demonstrate prion spread via peripheral synapses into tissues. This conclusion is also supported by in vitro studies that demonstrate the prion infection of muscle cells in vitro required contact with neuronal cell lines and not non-neuronal cells lines suggesting that a NMJ is required for prion infection of muscle cells. Since the SSE of the tongue undergoes continual turnover, renewal, and shedding, these findings suggest that the prion agent could establish infection in epithelial cells and could be shed as these cells mature in the SSE, become the keratinized layer, and are sloughed into oral bodily fluids. Therefore, saliva may be one source of prions that is shed from a host and can infect a naive host through direct or indirect contact. The spread of prions into skeletal muscle cells via nerve fibers suggest that muscle tissue, and not just nerves that transverse muscle, are a potential source of prion exposure upon ingestion of food products containing meat.

Publications

• Dlakic, W.M., Grigg, E. & Bessen, R.A. 2007. Prion infection of muscle cells in vitro. Journal of Virology 81:4615-4624.

Progress 12/01/05 to 11/30/06

Outputs
The long-term goal of the proposed research is to define the route and mechanism of prion agent infection of skeletal muscle during prion diseases of livestock. Prion infection of skeletal muscle is a potential threat to animal and human food products. The hypothesis to be tested is that prion neuroinvasion of the brainstem following oral ingestion of the prion agent leads to infection of one or more of the tongue-associated cranial nerves. In the past year we focused on the following aims: 1) to investigate the role of peripheral synapses (i.e., neuromuscular junction) in prion infection of skeletal muscle cells and 2) to establish prion infection in muscle cells in vitro. To investigate the role of the neuromuscular junction (NMJ) in prion entry into skeletal muscle, a spatiotemporal analysis was performed on the deposition of the disease-specific prion protein, PrP-res, in nerve fibers and muscle of the tongue, which we use as a model for prion infection of skeletal muscle. To determine the role of the NMJ in prion agent entry into muscle cells, laser scanning confocal microscopy (LSCM) was used to determine the co-localization of PrP-res with synaptophysin (a marker for the NMJ) at the initial time of entry into muscle versus at the time of onset of clinical symptoms. Our early findings suggest that PrP-res is colocalized with the NMJ at the time of entry into muscle cells but not later in the disease course when PrP-res is widely distributed in skeletal muscle. These preliminary findings suggest that prion agent entry into cells is linked to the NMJ and suggests that this peripheral synapse acts as a route or prion agent entry into muscle cells. To determine if skeletal muscles cells can directly support prion infection, we attempted to establish prion infection in a muscle cell line (i.e., C2C12 cells) in vitro. Scrapie infection of murine C2C12 myoblasts and myotubes in vitro was established following co-culture with a scrapie-infected murine neuroblastoma (N2a) cell line but not following incubation with a non-neuronal cell line or a scrapie brain homogenate. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

Impacts
The implication of these findings is that prion infection of muscle represents a potential source of prion transmission via either consumption of meat products. The prion distribution into these sites in the tongue is likely via centrifugal spread along motor and sensory fibers with subsequent transynatpic spread into muscle cells or epithelial cells, respectively. These findings suggest that the prion agent can use nerve fibers to spread to peripheral tissues. Our conclusion is also supported by in vitro studies that demonstrate the prion infection of muscle cells in vitro required contact with neuronal cell lines and not non-neuronal cells lines suggesting that a NMJ is required for prion infection of muscle cells. The spread of prions into skeletal muscle cells via nerve fibers suggest that muscle tissue, and not just nerves that transverse muscle, are a potential source of prion exposure upon ingestion of food products containing meat.

Publications