Bruce Chesebro

Getting a Grip on Prions: Oligomers, Amyloids, and Pathological Membrane Interactions*

The prion (infectious protein) concept has evolved with the discovery of new self-propagating protein states in organisms as diverse as mammals and fungi. The infectious agent of the mammalian transmissible spongiform encephalopathies (TSE) has long been considered the prototypical prion, and recent cell-free propagation and biophysical analyses of TSE infectivity have now firmly established its prion credentials. Other disease-associated protein aggregates, such as some amyloids, can also have prion-like characteristics under certain experimental conditions. However, most amyloids appear to lack the natural transmissibility of TSE prions. One feature that distinguishes the latter from the former is the glycophosphatidylinositol membrane anchor on prion protein, the molecule that is corrupted in TSE diseases. The presence of this anchor profoundly affects TSE pathogenesis, which involves major membrane distortions in the brain, and may be a key reason for the greater neurovirulence of TSE prions relative to many other autocatalytic protein aggregates.

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrPSc) into contiguous membranes

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol‐anchor that associates with detergent‐resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease‐sensitive PrP isoform (PrP‐sen) to the protease‐resistant isoform (PrP‐res). A cell‐free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP‐sen and brain microsomes from scrapie‐infected mice as a source of PrP‐res. Interestingly, DRM‐associated PrP‐sen was not converted to PrP‐res until the PrP‐sen was either released from DRMs by treatment with phosphatidylinositol‐specific phospholipase C (PI‐PLC), or the combined membrane fractions were treated with the membrane‐fusing agent polyethylene glycol (PEG). PEG‐assisted conversion was optimal at pH 6–7, and acid pre‐treating the DRMs was not sufficient to permit conversion without PI‐PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP‐res during TSE infection requires (i) removal of PrP‐sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP‐res into the raft domains of recipient cells.

Sulfated glycans and elevated temperature stimulate PrPSc-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease‐ sensitive prion protein (PrP‐sen or PrPC) to a protease‐resistant form (PrP‐res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP‐res deposits in vivo, suggesting that they may facilitate PrP‐res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP‐res accumulation and serve as prophylactic anti‐TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP‐res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell‐free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP‐res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP‐sen that may potentiate its PrP‐res‐induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

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.

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.

Specific Inhibition of in Vitro Formation of Protease-resistant Prion Protein by Synthetic Peptides

The transmissible spongiform encephalopathies are characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the neuropathogenic process in vivo. Recently, PrPres has been shown to be capable of directly inducing the conversion of PrPsen to PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to enhance or inhibit this cell-free conversion reaction. None of the synthetic peptides was able to confer protease-resistance to the labeled PrPsen molecules on their own. On the contrary, peptides from the central part of the hamster PrP sequence from 106 to 141 could completely inhibit the conversion induced by preformed PrPres. The presence of residues 119 and 120 from the highly hydrophobic sequence AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory effect. Fourier transform infrared spectroscopy analysis indicated that inhibitory peptides formed high β-sheet aggregates under the conditions of the conversion reaction, but this was also true of certain peptides that were not inhibitory. Thus, the potential to form β-sheeted aggregates may be necessary, but not sufficient, for peptides to act as inhibitors of PrPres formation. Clearly, the amino acid sequence of the peptide is also important for inhibition. The sequence specificity of the inhibition is consistent with the idea that residues in the vicinity of positions 106–141 of PrPres and/or PrPsen are critically involved in the intermolecular interactions that lead to PrPres formation.

Prion protein and the transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that occur in a wide variety of mammals. In humans, TSE diseases include kuru, sporadic and iatrogenic Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). So far, TSE diseases occur only rarely in humans; however, scrapie is a widespread problem in sheep, and the recent epidemic of bovine spongiform encephalopathy (BSE or mad cow disease) has seriously affected the British cattle industry. Of special concern is the recent appearance of a new variant of CJD in humans that is suspected of being caused by infections from BSE-infected cattle products. In all these diseases, an abnormal form of a host protein, prion protein (PrP), is essential for the pathogenic process. The relationship of this protein to the transmissible agent is currently the subject of great interest and controversy and is the subject of this review.

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.