Judyth Sassoon

Elevated manganese levels in blood and CNS in human prion disease

Prion disease or transmissible spongiform encephalopathies are neurodegenerative disorders of humans and other mammals. They are fatal and difficult to diagnose. Previous studies have suggested that some prion diseases cause elevation of manganese in the blood and brain. In the current study we analysed blood and brain samples from humans to determine whether elevation in manganese is a specific characteristic of Creutzfeldt-Jakob disease, the most common form of human prion disease. Analysis of manganese in the blood of normal humans showed that concentrations vary little with age or sex. Analysis of other diseases, including other neurodegenerative disease showed that only CJD showed an elevation in manganese and copper. Other diseases that showed elevated manganese included blood-brain barrier disorders and haemochromatosis. However, CJD could be easily distinguished from these diseases. This implies that increased blood manganese in prion disease is a highly specific characteristic of the disease.

Copper-dependent functions for the prion protein

Prion diseases such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease are fatal neurodegenerative diseases. These diseases are characterized by the conversion of a normal cellular protein, the prion protein, to an abnormal isoform that is thought to be responsible for both pathogenesis in the disease and the infectious nature of the disease agent. Understanding the biology and metabolism of the normal prion protein is therefore important for understanding the nature of these diseases. This review presents evidence for the normal function of the cellular prion protein, which appears to depend on its ability to bind copper (Cu). There is now considerable evidence that the prion protein is an antioxidant. Once the prion protein binds Cu, it may have an activity like that of a superoxide dismutase. Conversion of the prion protein to an abnormal isoform might lead to a loss of antioxidant protection that could be responsible for neurodegeneration in the disease.

Analysis of doppel protein toxicity

The recently described doppel protein (Dpl) is a homologue of the prion protein (PrP(c)). This protein, expressed in the brains of mice that lack the expression of PrP(c), causes neuronal death as the mice age. Previous studies have suggested this neuronal damage is caused by oxidative assault and changes in the activity of NOS proteins. We investigated the toxicity of Dpl in cell culture models and showed that Dpl was toxic to neurons. This toxicity was inhibited by the expression of PrP(c) and possibly involved direct interaction between the two proteins. The mechanism of toxicity involved stimulation of nitric oxide production via activation of the nitric oxide synthases, nNOS and iNOS. This mechanism of toxicity is quite different from that of PrP(Sc) and does not require the protein to change conformation. These results provide the first evidence for the mechanism of Dpl toxicity.

A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation.

The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp°/° cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP‐knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.

Astrocytic regulation of NMDA receptor subunit composition modulates the toxicity of prion peptide PrP106-126.

Prion diseases are neurodegenerative conditions. The main pathological alterations common to these diseases include the loss of neurones, gliosis and the deposition of an abnormal isoform of the prion protein in aggregates in the nervous tissue. Prevention of the devastating effects of prion disease requires prevention of neuronal death. Therefore, understanding the mechanism by which this occurs is essential. Cell culture studies using the synthetic peptide PrP106-126 have been central to developing a model of this mechanism. Using a coculture system, we have shown that PrP106-126 caused neuronal death mediated by glutamate. This neuronal death resulted from modification of the expression of NMDA receptor subtypes stimulated by the exposure of neurones to the combination of astrocytic factors, elevated Cu and PrP106-126. The results of these experiments suggest neuronal death in prion disease might be reduced by the use of NMDA receptor antagonists such as MK801 or inhibitors of the arachidonic acid metabolism pathway.

BSE and vCJD cause disturbance to uric acid levels

Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) are two new members of the family of neurodegenerative conditions termed prion diseases. Oxidative damage has been shown to occur in prion diseases and is potentially responsible for the rapid neurodegeneration that is central to the pathogenesis of these diseases. An important nonenzymatic antioxidant in the brain is uric acid. Analysis of uric acid in the brain and cerebrospinal fluid (CSF) of cases of BSE and CJD showed a specific reduction in CSF levels for both BSE and variant CJD, but not sporadic CJD. Further studies based on cell culture experiments suggested that uric acid in the brain was produced by microglia. Uric acid was also shown to inhibit neurotoxicity of a prion protein peptide, production of the abnormal prion protein isoform (PrP(Sc)) by infected cells, and polymerization of recombinant prion protein. These findings suggest that changes in uric acid may aid differential diagnosis of vCJD. Uric acid could be used to inhibit cell death or PrP(Sc) formation in prion disease.

Mapping the antigenicity of copper-treated cellular prion protein with the scrapie isoform

Abstract: When recombinant and cellular prion protein (PrPC) binds copper, it acquires properties resembling the scrapie isoform (PrPSc), namely protease resistance, detergent insolubility and increased β sheet content. However, whether the conformations of PrPC induced by copper and PrPSc are similar has not been studied in great detail. Here, we use a panel of seven monoclonal antibodies to decipher the epitopes on full-length mouse PrPC that are affected by exogenous copper, and to compare the antigenicity of the copper-treated full-length PrPC with the full-length PrPSc present in scrapie-infected mouse brains. In the presence of copper, we found that epitopes along residues 115–130 and 153–165 become more accessible on PrPC. These regions correspond to the two β sheet strands in recombinant PrP and they were proposed to be important for prion conversion. However, when we compared the antibody-binding patterns between full-length PrPC with full-length PrPSc and between copper-treated full-length PrPC with full-length PrPSc, antibody binding to residues 143–155 and 175–185 was consistently increased on PrPSc. Collectively, our results suggest that copper-treated full-length PrPC does not resemble full-length PrPSc, despite acquiring PrPSc-like properties. In addition, since each full-length protein reacts distinctively to some of the antibodies, this binding pattern could discriminate between PrPC and PrPSc.

Therapeutics and prion disease: Can immunisation or drugs be effective?

Prion diseases are of considerable importance because of the threat of a variant form of Creutzfeldt Jakob disease that has emerged in recent years. Pre-clinical diagnosis of prion diseases still remains poor and effective therapies also do not exist at present. This review examines research on possible therapeutic strategies that might have potential benefits if applied before neurodegeneration has occurred.

Role of glia in prion disease.

Publisher Summary This chapter explains that prion diseases are also called transmissible spongiform encephalopathies. It discusses that their transmission has been linked to an abnormal isoform of the prion protein. Deposition of this abnormal protein isoform in the central nervous system is also associated with the onset of pathological changes and disease progression. The second most significant change to the brain other than neurodegeneration is gliosis involving both microglia and astrocytes. The possible association between gliosis and neuronal death has been investigated using cell culture models. These models, using peptide mimics of the abnormal prion protein isoform, indicate a role for both microglia and astrocytes in the neurotoxic action of the peptide. The chapter reviews that microglia activated by the prion protein peptide release both cytokines and toxic substances, such as superoxide. The cytokines released by the microglia stimulate astroglial proliferation. Alterations in the interactions between astrocytes and neurones expose neurones to the toxicity of glutamate. Thus, a model of neuronal death in prion disease has emerged, indicating that both microglia and astrocytes can either induce or exacerbate neuronal death induced by the abnormal prion protein isoform.

Neurotoxicity and Prion Disease

Prion diseases are characterised by the conversion of a normal glycoprotein, the prion protein, to an abnormal protease resistant form, which is suggested to be both the infectious agent and the cause of neuronal cell death in the disease. Death of the patient results from neurodegeneration. In this review, we present data arguing that neurodegeneration in prion diseases is a complex process, involving the action of other factors in addition to the abnormal prion protein. Clearly, an understanding of the mechanisms by which prion mediated neuronal death occurs might lead to treatments that would prevent the life threatening nature of these diseases.