Richard A. Bessen

Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte‐specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 ± 5 days and died 7 ± 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

Rapid Prion Neuroinvasion following Tongue Infection

ABSTRACT Food-borne transmission of prions can lead to infection of the gastrointestinal tract and neuroinvasion via the splanchnic and vagus nerves. Here we report that the transmission of transmissible mink encephalopathy (TME) is 100,000-fold more efficient by inoculation of prions into the tongues of hamsters than by oral ingestion. The incubation period following TME agent (hereinafter referred to as TME) inoculation into the lingual muscles was the shortest among the five nonneuronal routes of inoculation, including another intramuscular route. Deposition of the abnormal isoform of the prion protein, PrPSc, was first detected in the tongue and submandibular lymph node at 1 to 2 weeks following inoculation of the tongue with TME. PrPSc deposits in the tongue were associated with individual axons, and the initial appearance of TME in the brain stem was found in the hypoglossal nucleus at 2 weeks postinfection. At later time points, PrPSc was localized to brain cell groups that directly project to the hypoglossal nucleus, indicating the transneuronal spread of TME. TME PrPSc entry into the brain stem preceded PrPSc detection in the rostral cervical spinal cord. These results demonstrate that TME can replicate in both the tongue and regional lymph nodes but indicate that the faster route of brain invasion is via retrograde axonal transport within the hypoglossal nerve to the hypoglossal nucleus. Topical application of TME to a superficial wound on the surface of the tongue resulted in a higher incidence of disease and a shorter incubation period than with oral TME ingestion. Therefore, abrasions of the tongue in livestock and humans may predispose a host to oral prion infection of the tongue-associated cranial nerves. In a related study, PrPSc was detected in tongues following the intracerebral inoculation of six hamster-adapted prion strains, which demonstrates that prions can also travel from the brain to the tongue in the anterograde direction along the tongue-associated cranial nerves. These findings suggest that food products containing ruminant or cervid tongue may be a potential source of prion infection for humans.

Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent

Abstract To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellu, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scraple, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy

Epidemiological investigation of a new incident of transmissible mink encephalopathy (TME) in Stetsonville, Wisconsin, U.S.A. in 1985 revealed that the mink rancher had never fed sheep products to his mink but did feed them large amounts of products from fallen or sick dairy cattle. To investigate the possibility that this occurrence of TME may have resulted from exposure to infected cattle, two Holstein bull calves were injected intracerebrally with mink brain from the Stetsonville ranch. Each bull developed a fatal spongiform encephalopathy 18 and 19 months after inoculation, respectively, and both bovine brains passaged back into mink were highly pathogenic by either intracerebral or oral inoculation. These results suggest the presence of a previously unrecognized scrapie-like infection in cattle in the United States.

Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus)

ABSTRACT Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.

Adaptation and Selection of Prion Protein Strain Conformations following Interspecies Transmission of Transmissible Mink Encephalopathy

ABSTRACT Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrPSc). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrPSc following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrPSc strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10−5 dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrPSc strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrPScconformations that underwent selection until one type of PrPSc conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrPSc conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrPSc conformation that can determine the TSE strain phenotype.

Retrograde Transport of Transmissible Mink Encephalopathy within Descending Motor Tracts

ABSTRACT The spread of the abnormal conformation of the prion protein, PrPSc, within the spinal cord is central to the pathogenesis of transmissible prion diseases, but the mechanism of transport has not been determined. For this report, the route of transport of the HY strain of transmissible mink encephalopathy (TME), a prion disease of mink, in the central nervous system following unilateral inoculation into the sciatic nerves of Syrian hamsters was investigated. PrPSc was detected at 3 weeks postinfection in the lumbar spinal cord and ascended to the brain at a rate of approximately 3.3 mm per day. At 6 weeks postinfection, PrPSc was detected in the lateral vestibular nucleus and the interposed nucleus of the cerebellum ipsilateral to the site of sciatic nerve inoculation and in the red nucleus contralateral to HY TME inoculation. At 9 weeks postinfection, PrPSc was detected in the contralateral hind limb motor cortex and reticular thalamic nucleus. These patterns of PrPSc brain deposition at various times postinfection were consistent with that of HY TME spread from the sciatic nerve to the lumbar spinal cord followed by transsynaptic spread and retrograde transport to the brain and brain stem along descending spinal tracts (i.e., lateral vestibulospinal, rubrospinal, and corticospinal). The absence of PrPSc from the spleen suggested that the lymphoreticular system does not play a role in neuroinvasion following sciatic nerve infection. The rapid disease onset following sciatic nerve infection demonstrated that HY TME can spread by retrograde transport along specific descending motor pathways of the spinal cord and, as a result, can initially target brain regions that control vestibular and motor functions. The early clinical symptoms of HY TME infection such as head tremor and ataxia were consistent with neuronal damage to these brain areas.

Extraneural Prion Neuroinvasion without Lymphoreticular System Infection

ABSTRACT While prion infection of the lymphoreticular system (LRS) is necessary for neuroinvasion in many prion diseases, in bovine spongiform encephalopathy and atypical cases of sheep scrapie there is evidence to challenge that LRS infection is required for neuroinvasion. Here we investigated the role of prion infection of LRS tissues in neuroinvasion following extraneural inoculation with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. DY TME agent infectivity was not detected in spleen or lymph nodes following intraperitoneal inoculation and clinical disease was not observed following inoculation into the peritoneum or lymph nodes, or after oral ingestion. In contrast, inoculation of the HY TME agent by each of these peripheral routes resulted in replication in the spleen and lymph nodes and induced clinical disease. To clarify the role of the LRS in neuroinvasion, the HY and DY TME agents were also inoculated into the tongue because it is densely innervated and lesions on the tongue, which are common in ruminants, increase the susceptibility of hamsters to experimental prion disease. Following intratongue inoculation, the DY TME agent caused prion disease and was detected in both the tongue and brainstem nuclei that innervate the tongue, but the prion protein PrPSc was not detected in the spleen or lymph nodes. These findings indicate that the DY TME agent can spread from the tongue to the brain along cranial nerves and neuroinvasion does not require agent replication in the LRS. These studies provide support for prion neuroinvasion from highly innervated peripheral tissues in the absence of LRS infection in natural prion diseases of livestock.

Prion Infection of Skeletal Muscle Cells and Papillae in the Tongue

ABSTRACT The presence of the prion agent in skeletal muscle is thought to be due to the infection of nerve fibers located within the muscle. We report here that the pathological isoform of the prion protein, PrPSc, accumulates within skeletal muscle cells, in addition to axons, in the tongue of hamsters following intralingual and intracerebral inoculation of the HY strain of the transmissible mink encephalopathy agent. Localization of PrPSc to the neuromuscular junction suggests that this synapse is a site for prion agent spread between motor axon terminals and muscle cells. Following intracerebral inoculation, the majority of PrPSc in the tongue was found in the lamina propria, where it was associated with sensory nerve fibers in the core of the lingual papillae. PrPSc staining was also identified in the stratified squamous epithelium of the lingual mucosa. These findings indicate that prion infection of skeletal muscle cells and the epithelial layer in the tongue can be established following the spread of the prion agent from nerve terminals and/or axons that innervate the tongue. Our data suggest that ingestion of meat products containing prion-infected tongue could result in human exposure to the prion agent, while sloughing of prion-infected epithelial cells at the mucosal surface of the tongue could be a mechanism for prion agent shedding and subsequent prion transmission in animals.

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrPSc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrPSc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 103.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrPSc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants.