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Background: Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE., Results: PrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification., Conclusions: PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

Prion diseases occur in many animal species, most notably in ruminants. While scrapie in sheep has been recognised for three centuries and goat scrapie has been recognised for decades, BSE in cattle is a relatively novel disease which was first diagnosed in the UK in the mid 1980s. BSE was most likely caused through dietary exposure to animal feed contaminated with prions and disease was subsequently transmitted to people. The BSE epidemic is almost at an end, but the recent identification of so called atypical forms of BSE and scrapie pose many questions about the possible spectrum of prion diseases in animals and their transmissibility to other species, including humans.The pathogenesis of animal prion diseases has been studied both in natural infections and in experimental animal models. Detection of infectivity is greatly helped by suitable rodent models, in particular transgenic mice. Clinically infected animals show characteristic neuropathology in the brain and spinal cord which is accompanied by the accumulation of a conformationally altered, protease-resistant host protein. The post-mortem diagnosis is based on the detection of this protein, PrP(Sc), but despite recent impressive developments a routine ante-mortem diagnostic test has proved elusive.There is no treatment for prion diseases in animals, but disease outbreaks are controlled through a mixture of movement restrictions on holdings, culling of affected animals and herds and, for classical scrapie in sheep, selective breeding for genetic resistance. Prions are very stable and can remain in the environment for prolonged periods. This poses serious practical questions with regard to the decontamination of infected premises. The control of BSE specifically through restrictions in animal feeding practises has been successful, but the changing spectrum of these diseases plus the economic pressures to relax feed bans and reduce levels of surveillance will require constant vigilance to safeguard animal and public health.

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

To identify the dynamic transcriptional alterations in CNS during the development of prion disease, brains of scrapie-infected mice and age-matched, mock-inoculated controls were analyzed immediately before inoculation and at different time points post-inoculation using Affymetrix microarray technique. A total of 449 probe sets, representing 430 genes, showed differential expression between scrapie- and mock-inoculated mice over the time course. These genes could be separated into two clusters according to expression patterns: the genes in cluster 1 demonstrated lower mRNA levels in scrapie-infected brains when compared with mock-inoculated brains, whereas genes in cluster 2 showed higher mRNA levels in scrapie-infected brains. Functional analysis of differentially expressed genes revealed the most severely affected biological process: cholesterol metabolism. The expression patterns of the cholesterol-related genes indicated an inhibited cholesterol synthesis in the diseased brains. Conspicuously, a number of cluster 1 genes, including some of cholesterol-related genes, showed not only decreasing mRNA levels in scrapie-infected brains but also increasing mRNA levels in mock-inoculated brains with increasing age. Quantitative RT-PCR analysis of some cholesterol-related genes in untreated mice suggested that changes of the examined genes observed in mock-inoculated brains are mainly age related. This finding indicated a link between age-related genes and scrapie-associated neurodegeneration.

A heterozygous T183A mutation in the prion protein (PrP) gene, PRNP, was identified in a patient with histopathologically confirmed spongiform encephalopathy. Clinically, this form of prion disease was characterized by early-onset dementia as the predominant sign, along with global cerebral atrophy and hypometabolism. The age at onset was 40 years and the disease duration was 4 years. Additional neurological signs including cerebellar ataxia and EEG abnormalities were absent until late stages of the disease. The T183A mutation was not found in non-affected family members. This mutation results in the removal of one of the two consensus sites for glycosylation of PrP. Neuropathological examination revealed severe spongiform degeneration and neuronal loss in the neocortex, putamen and claustrum, small plaque-like PrP-immunoreactive deposits in the molecular layer of the cerebellum, and faint intracellular cytoplasmic PrP immunoreactivity. Western blot analysis of the patient's brain tissue showed protease K-resistant PrP with a definite preponderance of the monoglycosylated form. The additional appearance of a band representing diglycosylated PrPSc strongly suggests that non-mutated PrP also acquires protease resistance in the present setting. Cell culture experiments confirmed previous reports on intracellular retention of the mutant protein in vitro. This is the second report of a disease-causing T183A mutation of PrP, and the clinical, histological and genetic observations strongly suggest that T183A is a disease-causing mutation.

The pathogenesis of prion diseases, a class of transmissible fatal neurodegenerative diseases in humans and animals, is still unclear. The aim of this study was to identify the differentially regulated genes that correlate with the development of prion diseases for a better understanding of their pathological mechanisms. We employed Affymetrix Mouse Expression Arrays 430A containing >22,000 transcripts and compared the global gene expression profiles from brains of mice who were intracerebrally inoculated with scrapie strains ME7 and RML with those from brains of uninfected and mock-infected mice. The microarray data were analyzed by Significance Analysis of Microarrays, revealing 121 genes whose expression increased at least twofold in both ME7- and RML-infected mouse brains, with an estimated false discovery rate of < or =5%. These genes encode proteins involved in proteolysis, protease inhibition, cell growth and maintenance, the immune response, signal transduction, cell adhesion, and molecular metabolism. The time course of expression generally showed up-regulation of these genes from 120 days postinoculation (dpi) for ME7-inoculated mouse brains and from 90 dpi for RML-inoculated mouse brains. The onset of elevated expression correlated temporally with the onset of PrP(Sc) accumulation and the activation of glia, which may have contributed to neuronal cell death. Among the differentially regulated genes reported in the present study, the emergence of genes for several cathepsins and S100 calcium binding proteins was conspicuous. These and other genes reported here may represent novel potential diagnostic and therapeutic targets for prion disease.

Abstract: The cellular prion protein (PrPC) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrPC expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrPC had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrPC was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrPC appeared to rely on copper binding to the N-terminal octapeptide of PrPC. Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrPC enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival. [Copyright &y& Elsevier]