MicrobiologyBytes

Chronic wasting disease (CWD) is a prion disease affecting wild and captive deer. Like all mammalian prion diseases, which include Creutzfeldt-Jakob disease (CJD), kuru and variant CJD (vCJD) in humans and bovine spongiform encephalopathy (BSE) in cattle, the central event in CWD infection is the post-translational conversion of the host-encoded, cellular prion protein (PrPC), to an abnormal form called PrPSc. Progressive accumulation of PrPSc in the central nervous system is associated with clinical signs of CWD which includes weight loss, behavioural changes, excessive salivation, difficulty swallowing and ataxia prior to death. International concern over CWD is growing as infected cervids have now been reported in fourteen states in North America, two Canadian provinces and in South Korea. To date, CWD has not been reported in Europe, although surveillance has been limited.

The negative transmission data reported in this paper support the conclusion that the transmission barrier associated with the interaction of human PrP and these CWD prions is greater than that associated with interaction of human PrP and the prion strain causing epizootic BSE in cattle. This is good news from a human health perspective, but further studies will be required to evaluate the transmission properties of distinct deer prion strains as they are characterized.

Chronic wasting disease prions are not transmissible to transgenic mice over-expressing human prion protein. J Gen Virol. Jul 7 2010
Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk, and moose. CWD-infected cervids have been reported in fourteen US states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice over-expressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilised in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains; further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterised.

Prion diseases, also known as transmissible spongiform encephalopathies, are infectious fatal neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated form which may contribute to brain damage. In uninfected individuals, normal PrP is anchored to the outer cell membrane by a sugar-phosphate-lipid linker molecule. This report shows that prion infection of mice expressing PrP lacking the anchor can result in a new type of fatal neurodegenerative disease. The disease displays mechanisms of damage to brain cells and brain blood vessels found in Alzheimer’s disease and in familial amyloid brain diseases. In contrast, the typical sponge-like brain damage seen in prion diseases was not observed. These results suggest that presence or absence of PrP membrane anchoring can influence the type of neurodegeneration seen after prion infection. Understanding these interactiions could lead to new therapeutic approaches.

Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring. 2010 PLoS Pathog 6(3): e1000800. doi:10.1371/journal.ppat.1000800

Related:

• Researchers find new piece of BSE puzzle
• Novel mutation linked to mad cow disease
• What the heck are prions for?
• Drugs for treatment of prion infections

BSE, commonly known as mad cow disease, is caused by an infectious and abnormal form of the prion protein that is present on cells within the nervous system, but it is unclear as to what causes the abnormality to occur. Prion diseases are unique in that their infectious nature is not dependent on nucleic acid but is instead attributed to a misfolded protein, the prion protein. This misfolded prion protein is capable of inducing the misfolding of the normal form of the prion protein that is present on the surface of neurons and other cells in the body. However, the site in the cell at which this misfolding occurs and whether other proteins are involved remains controversial. Researchers have addressed these questions by investigating how the normal form of the prion protein is targeted to specialised domains on the plasma membrane termed cholesterol-rich lipid rafts. They showed that targeting is due, in part, to a particular heparin sulfate proteoglycan called glypican-1.

A new treatment route for bovine spongiform encephalopathy (BSE) and its human form Creutzfeldt Jakob disease (CJD) could be a step closer. This new works shows that the presence of Glypican-1 causes the numbers of abnormal prion proteins to rise. This suggests that Glypican-1 acts as a scaffold bringing the two forms of the prion protein together and that this contact causes normal prions to mutate into the infectious form. These findings may have implications for the treatment of both BSE and the human form of the disease, CJD. Although the scientists mainly conducted experiments using cells infected with prions, it is also possible that Glypican-1 is involved in other diseases of the nervous system, although experiments have not shown any link with other neurodegenerative diseases such as Alzheimer’s disease.

Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation. 2009 PLoS Pathog 5(11): e1000666. doi:10.1371/journal.ppat.1000666
In prion diseases, the cellular form of the prion protein, PrPC, undergoes a conformational conversion to the infectious isoform, PrPSc. PrPC associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrPC from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrPC. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrPC to rafts. Depletion of the major neuronal GPI anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrPC from rafts, promoting its endocytosis. Glypican-1 and PrPC colocalised on the cell surface and both PrPC and PrPSc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrPSc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrPC on the b-secretase cleavage of the Alzheimer’s amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrPC and PrPSc in lipid rafts.

Related:

• Prions and Alzheimers disease
• Novel mutation linked to mad cow disease
• What the heck are prions for?
• Drugs for treatment of prion infections

Prions are transmissible, proteinaceous agents that cause fatal neurodegenerative diseases. In deer elk moose prions cause chronic wasting disease (CWD). The incidence of CWD can be remarkably high both in captive and wild herds and epidemiological data suggest that efficient horizontal transmission drives epidemic dynamics. Although deer can be infected orally and seem to be able to contract CWD from contaminated environments, precisely how and when CWD prions are shed into the environment have not been described. Previous studies have identified CWD prions in saliva, blood, urine, antler velvet, and muscle, lymphoid and other tissues of symptomatic cervids with late-stage disease. These sources of CWD prions may contribute to the spread of CWD, but none explains natural CWD transmission both within and between species in the deer family. To fit observed patterns, a natural CWD transmission mechanism must be effected within biologically realistic limits of the carrier medium, cannot require cannibalism and should be indirect to explain both environmental persistence and spread among multiple host species. Because empirical data and modelling suggested faecal excretion of prions throughout much of the disease course as potentially important to CWD transmission, researchers investigated whether prions are shed in faeces from mule deer during the course of CWD infection.

Asymptomatic deer excrete infectious prions in faeces. 2009 Nature 461: 529-532 doi:10.1038/nature08289
Infectious prion diseases – scrapie of sheep and chronic wasting disease (CWD) of several species in the deer family – are transmitted naturally within affected host populations. Although several possible sources of contagion have been identified in excretions and secretions from symptomatic animals, the biological importance of these sources in sustaining epidemics remains unclear. Here we show that asymptomatic CWD-infected mule deer (Odocoileus hemionus) excrete CWD prions in their faeces long before they develop clinical signs of prion disease. Intracerebral inoculation of irradiated deer faeces into transgenic mice overexpressing cervid prion protein (PrP) revealed infectivity in 14 of 15 faecal samples collected from five deer at 7–11 months before the onset of neurological disease. Although prion concentrations in deer faeces were considerably lower than in brain tissue from the same deer collected at the end of the disease, the estimated total infectious dose excreted in faeces by an infected deer over the disease course may approximate the total contained in a brain. Prolonged faecal prion excretion by infected deer provides a plausible natural mechanism that might explain the high incidence and efficient horizontal transmission of CWD within deer herds, as well as prion transmission among other susceptible cervids.

Why should we care?
Apart from the impact on wildlife, vCJD is a prion infection transmitted from infected cows to humans. People eat deer. Although there is no evidence that the CWN prion causes diseae in humans, this is definitely one to keep an eye on.

Related:

• A General Model of Prion Strains and Their Pathogenicity
• Prions, Proteasomes, and Mad Cows
• Oral transmission of prions is enhanced by binding to soil
• Prions and Cheetah Conservation

In PNAS, Warren Olanowa and Stanley Prusiner ask, Is Parkinson’s disease a prion disorder?
Parkinson’s disease (PD) is an age-related, neurodegenerative disease that affects approximately one million people in the United States. Pathologically, the disease is characterized by a loss of dopamine neurons in the substantia nigra coupled with proteinaceous inclusions in nerve cells and terminals, known as Lewy bodies and Lewy neurites, respectively. PD pathology is also known to affect nondopamine neurons in the upper and lower brainstem, olfactory system, cerebral hemisphere, spinal cord, and autonomic nervous system. The cause of cell death in PD is not known, but proteolytic stress with the accumulation of misfolded proteins has been implicated.

In the current issue of PNAS, Desplats et al demonstrate that nerve cells which overexpress tagged alpha-synuclein can transmit the protein to neural stem cells in both in vitro and in vivo models (Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. 2009 PNAS USA 106:13004–13005). This important study could explain the remarkable finding that human embryonic dopamine nerve cells implanted into the striatum of patients with PD develop PD pathology with loss of dopamine markers and classic Lewy bodies. It also provides insight into how alpha-synuclein pathology might sequentially spread throughout the nervous system in PD.

It is thus possible that PD is a prion disorder resulting from increased production and/or impaired clearance of proteins such as alpha-synuclein, leading to misfolding and the formation of toxic oligomers, aggregates, and cell death. Further, it is possible that alpha-synuclein is a prion protein that can self-aggregate and be transmitted to unaffected cells, thus extending the disease process. While genetic causes represent an obvious source of increased levels of aberrantly folded alpha-synuclein in familial PD cases, a combination of aging, oxidative stress, inflammation, environmental toxins, hereditary factors, and impaired clearance may all feature in varying ways in causing altered metabolism of alpha-synuclein, resulting in the pathogenesis of sporadic PD. This concept suggests that drugs directed toward reducing the formation and/or facilitating the clearance of misfolded alpha-synuclein, so as to arrest or reverse the self-propagation process, might represent a novel therapeutic interventions for the treatment of PD.

Related:

• Stanley Prusiner at the SGM: Prion Biology and Diseases
• What the heck are prions for?
• Drugs for treatment of prion infections
• Alzheimer’s Disease and Prions

All scientific papers are important, but some are more important than others. Aside from its scientific importance, this paper is particularly important to me in purely personal terms. It comes from my own department. Ben Maddison was a PhD student in my laboratory many years ago and now heads up his own research group within the department. It’s also one of the final papers to come from Gary Whitelam, my former head of department, who died tragically last year. And as if all that wasn’t enough, as the UK starts to forget about how close we came to disaster with BSE, we’re still not completely sure that it’s all over.

Using the cutting-edge research technique of serial protein misfolding cyclic amplification (sPMCA), my colleagues show that prions are secreted in the milk from scrapie-exposed sheep. The sPMCA method involves incubating a small amount of abnormal prion with an excess of normal prion protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into misfolded prion.

Since scrapie is not transmissible to humans, these findings do not indicate the likely introduction of zoonotic prions from sheep into the human food chain. Nevertheless, the data do indicate caution in the risk assessment associated with such foods. Although it is unknown if analogous shedding of prions into milk occurs with bovine BSE, evidence from previous epidemiological and bioassay studies suggests that such a scenario seems unlikely to cause clinical disease. However, the present report strongly suggests that given the importance of cow’s milk in the human diet the potential presence of low levels of prions within milk warrants further investigation. Analyzing milk samples by sPMCA offers a methodology with clear potential for the identification of clinically sick animals and those with preclinical/subclinical prion disease. Such a non-invasive, live animal assay has the potential to contribute to the epidemiological study, management and control of prion diseases within farmed animals.

Prions are secreted in milk from clinically normal scrapie-exposed sheep. J Virol. Jun 3 2009. doi:10.1128/JVI.00051-09
The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically-affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and CNS tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a non-invasive methodology to detect scrapie during preclinical/subclinical disease.

Related:

• Prions in Milk
• The Origin of BSE
• What the heck are prions for?
• Fears over new wave of vCJD deaths

Stanley Prusiner was awarded the first ever SGM Prize Medal (to a microbiologist of international standing whose work has had a far-reaching impact beyond microbiology) at the SGM Spring meeting at Harrogate on 1st April 2009. MicrobiologyBytes was there and this is a summary of his Prize lecture.

Prions are infectious proteins which multiply by binding to a host cell protein and converting it into insolubile fibrils (“amyloid“). Prions are associated with infectious, inherited and sporadic diseases – a feature unique to these entities. Tikvah Alper was the first person to identify prions in the 1960s, but when Prusiner started working on them in 1974, at first he didn’t believe the protein-only hypothesis. After eight years of failing to be able to identify any nucleic acid associated with them, in 1982 he changed his mind and invented the name prion (“pree-on”).

In prion diseases, the cellular form of the protein, PrP^c, is converted into a disease-associated form, PrP^Sc. If prions really are infectious proteins, PrP^Sc produced in bacteria should be able to cause disease – and it does. It is also possible to produce synthetic amyloids with different biological properties – essentially strains of the protein.

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Quinacrine cures cultured cells of prions. In mice, the drug increases survival time of infected animals by up to 20%, but recently concluded clinical trials in humans have shown little effect. Prusiner’s group have found that quinacrine does work in stationary phase cells – such as those in the brain. Future trials of anti-prion (or amyloid) drugs need to be carried out in stationary cells. The latest assay uses genetically-modified mice which express luciferase when glial cells are disturbed. The resulting luminescence can be detected in the brains of live mice, and signs of disease can be recorded even before any neurological symptoms appear. This is up to eight times faster than waiting for the mice to die and examining their brains, and only requires one tenth of the animals. Prusiner hopes to use this approach to study Alzheimer’s and Parkinson’s disease, which also involve brain injury and amyloid deposits.

Stanley Prusiner’s take home message to all the students present was: it’s important to be lucky! But as Robin Weiss, SGM President, pointed out, Pasteur said: Fortune favours the prepared mind!

Related:

• Alzheimer’s Disease and Prions
• The Origin of BSE
• What the heck are prions for?
• Drugs for treatment of prion infections
• What drove the cow mad? Lessons from a fish

For over twenty years scientists have known that a normal protein in the brain, PrP, or prion protein, can become harmful and cause deadly illnesses like Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. What they could not explain is why large amounts of this normal protein are produced by our bodies in the first place. In a new study, researchers reveal that PrP indeed plays a beneficial role for the organism – PrP helps cells communicate with one another during embryonic development.

In prion diseases, what transforms the normal PrP protein into a life-threatening substance is the abnormal alteration of its chemical structure. Moreover, prions have the treacherous ability to replicate by imprinting their abnormal structure into healthy PrPs, thereby generating new pathogenic particles. While this conversion process explains how prions are disseminated, an abnormal function of the prion protein is considered to be one of the reasons for neuronal degeneration. However, the normal function of PrP has remained an unsolved mystery for many years. All previous experiments in genetically modified mice had failed to provide conclusive evidence, as these animals lacking PrP seemed perfectly healthy. The scientists were able to show that the lack of PrP can cause clear physiological abnormalities in a living animal by using the tiny zebrafish as a model.

When the researchers microinjected zebrafish eggs with morpholinos, DNA-like molecules that prevent the normal production of PrP, the treated zebrafish embryos were unable to develop normally and eventually died. The proteins in the fish embryos normally found at cell-to-cell contact sites disappeared, rendering these cells unable to communicate and carry out the differentiation program that shapes the major structures of the body, including the nervous system. PrP serves as a glue element, bringing cells together and keeping them in contact. When two neighboring cells make contact, they become able to exchange important signals that affect the function of a tissue in the body. Although this work does not offer an immediate cure for CJD or BSE, it widens our understanding of prion diseases and provides hope for effective treatments.

Regulation of embryonic cell adhesion by the prion protein. 2009 PLoS Biol 7(3): e1000055
Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Caþ2-independent homophilic cell adhesion and signaling; and (2) modulates Caþ2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

Related:

• The Origin of BSE
• A novel approach in the molecular differentiation of prion strains
• What the heck are prions for?
• Alzheimer’s Disease and Prions

Prion diseases are a closely related group of fatal neurodegenerative disorders affecting the central nervous system of humans and animals. They include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru in humans; bovine spongiform encephalopathy (BSE) in cattle; and scrapie in sheep. The identification of variant CJD (vCJD) in the U.K. in 1996 and the subsequent experimental confirmation that BSE in cattle and vCJD in humans are caused by the same prion strain has led to a variety of concerns relating to public health. Iatrogenic transmission of classical (sporadic) CJD by a contaminated neurosurgical instrument has been reported, and epidemiological evidence suggests a fraction of apparently sporadic CJD may be caused by unrecognized iatrogenic infection during general surgery. The unknown but potentially substantial prevalence of clinically silent infection with vCJD prions in populations exposed to dietary BSE prions, together with the much wider tissue distribution of infectivity in vCJD, highlights the concerns of risk of infection through contact with surgical instruments. Secondary vCJD arising from blood transfusion has now been documented, indicating significant prionemia in asymptomatic donors during the incubation period. This wide distribution of infectivity makes common surgical and endoscopic procedures, in addition to neurosurgery and eye surgery, a potential risk factor for iatrogenic transmission of vCJD. Further, it is established that tissue prions withstand many forms of sterilization techniques and that the metal-adsorbed agent is even more resistant to both thermal and chemical treatments.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces. PNAS USA February 9, 2009
Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100X higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Related:

• What the heck are prions for?
• Prion infectivity found in fat tissues of mice
• Prions in Milk
• Novel mutation linked to mad cow disease
• A novel approach in the molecular differentiation of prion strains