Richard E. Race

The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimers, Parkinsons and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrPres, a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrPsen) into PrPres (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrPres (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrPres-containing aggregates, PrPres was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17–27-nm (300–600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of ≤5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14–28 PrP molecules, are the most efficient initiators of TSE disease.

Potent Inhibition of Scrapie‐Associated PrP Accumulation by Congo Red

Abstract: Transmissible spongiform encephalopathies (prion diseases), Alzheimers disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using serapieinfected neuroblastoma cells as a model system, we found that Congo red, an amyloid‐binding dye, potently inhibits the accumulation of the scrapie‐associated, protease‐resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease‐resistant PrP detected in the cultures by >90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease‐resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.

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.

Scrapie Infectivity and Proteinase K—Resistant Prion Protein in Sheep Placenta, Brain, Spleen, and Lymph Node: Implications for Transmission and Antemortem Diagnosis

Probable transmission of bovine spongiform encephalopathy to humans has focused intense interest on all of the transmissible spongiform encephalopathies (TSEs) and how they spread. In all TSEs, an abnormal disease-associated, proteinase K-resistant protein referred to as PrP-res or PrPsc accumulates in brain. In some species, PrP-res accumulates in other tissues as well. Sheep placenta, brain, spleen, and lymph node were analyzed in detail for PrP-res and infectivity. Both were detected in all brain and spleen samples and in placenta and lymph nodes of 80% of the scrapie-infected sheep. A perfect correlation was observed between infectivity and PrP-res detection. These results substantiate the probability that placenta plays an important role in natural transmission of scrapie, suggest that analysis of placenta for PrP-res could be the basis for an antemortem test for sheep scrapie, and show that PrP-res, scrapie infectivity, and scrapie disease are closely associated.

Characterization of scrapie infection in mouse neuroblastoma cells.

A mouse neuroblastoma cell line was successfully infected with scrapie agent. Agent derived from infected mouse brain or spleen infected cultures. However, agent from infected hamsters did not infect mouse cell cultures, suggesting that species specificity influenced the infection process in vitro. Positive cultures supported scrapie replication for as many as 47 passages in vitro. Agent was shown to be cell-associated and between 631 and 7943 unselected culture cells constituted 1 mouse LD50. However, fluctuation analysis indicated that only one of 144 cells in unselected cultures was actually infected. Thus, agent was confined to a small percentage of cells and only 4.4 to 55.1 positive cells were needed to confer a mouse LD50.

Long-Term Subclinical Carrier State Precedes Scrapie Replication and Adaptation in a Resistant Species: Analogies to Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease in Humans

ABSTRACT Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.

Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen.

ABSTRACT Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.

Mice devoid of prion protein have cognitive deficits that are rescued by reconstitution of PrP in neurons

Prion protein (PrP(C)) is a constituent of most normal mammalian cells and plays an essential role in the pathogenesis of transmissible spongiform encephalopathies (TSE). However, the normal cellular function of PrP(C) remains unclear. Here, we document that mice with a selective deletion of PrP(C) exhibited deficits in hippocampal-dependent spatial learning, but non-spatial learning remained intact. mPrP-/- mice also showed reduction in paired-pulse facilitation and long-term potentiation in the dentate gyrus in vivo. These deficits were rescued in transgenic mPrP-/- mice expressing PrP(C) in neurons under control of the neuron-specific enolase (NSE) promoter indicating that they were due to lack of PrP(C) function in neurons. The deficits were seen in mPrP-/- mice with a homogeneous 129/Ola background and in mPrP-/- mice in the mixed (129/Ola x C57BL/10) background indicating that these abnormalities were unlikely due to variability of background genes or alteration of the nearby Prnd (doppel) gene.

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.

Virologic and neurohistologic findings in dairy goats affected with natural scrapie.

Virologic and neurohistologic findings in three dairy goats that became affected with scrapie while living with naturally infected Suffolk sheep were essentially like those in affected sheep. Virus, detected by mouse inoculation, was widespread in non-neural sites, particularly in lymphatic tissues and intestine. In most sites, titers of virus ranged from 3.0 to 3.5 log10 mouse intracerebral LD50/30 mg of tissue. Virus was in nervous tissue in much higher titer. Ranging from 5.1 to 5.8 log10, the highest mean titers were in the diencephalon, midbrain, medulla oblongata and cerebellar cortex—sites of the most severe histologic changes. Although these changes were like those in naturally affected Suffolk sheep, they differed somewhat from those in goats affected with the experimental disease. Spongiform alteration of neuropil was minimal, and the more rostral parts of the brain, such as corpus striatum, globus pallidus and septal area, had few changes. Concentrations and distribution of virus in non-neural tissues were consistent with the conclusion that scrapie virus no doubt can be maintained by contagion in a herd of goats living apart from infected sheep.