A standardized comparison of commercially available prion decontamination reagents using the Standard Steel-Binding Assay.

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of mammals, such as variant Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Prions pose significant public health concerns through contamination of blood products and surgical instruments, and can resist conventional hospital sterilization methods. Prion infectivity binds avidly to surgical steel and can efficiently transfer infectivity to a suitable host, and much research has been performed to achieve effective prion decontamination of metal surfaces. Here, we exploit the highly sensitive Standard Steel-Binding Assay (SSBA) to perform a direct comparison of a variety of commercially available decontamination reagents marketed for the removal of prions, alongside conventional sterilization methods. We demonstrate that the efficacy of marketed prion decontamination reagents is highly variable and that the SSBA is able to rapidly evaluate current and future decontamination reagents.

Spontaneous generation of mammalian prions.

Prions are transmissible agents that cause lethal neurodegeneration in humans and other mammals. Prions bind avidly to metal surfaces such as steel wires and, when surface-bound, can initiate infection of brain or cultured cells with remarkable efficiency. While investigating the properties of metal-bound prions by using the scrapie cell assay to measure infectivity, we observed, at low frequency, positive assay results in control groups in which metal wires had been coated with uninfected mouse brain homogenate. This phenomenon proved to be reproducible in rigorous and exhaustive control experiments designed to exclude prion contamination. The infectivity generated in cell culture could be readily transferred to mice and had strain characteristics distinct from the mouse-adapted prion strains used in the laboratory. The apparent "spontaneous generation" of prions from normal brain tissue could result if the metal surface, possibly with bound cofactors, catalyzed de novo formation of prions from normal cellular prion protein. Alternatively, if prions were naturally present in the brain at levels not detectable by conventional methods, metal surfaces might concentrate them to the extent that they become quantifiable by the scrapie cell assay.

Investigation of mcp1 as a quantitative trait gene for prion disease incubation time in mouse.

The genetic basis of prion disease incubation time is principally determined by polymorphisms in the prion protein gene, Prnp. However, it is now known that other genetic factors are important. Several quantitative trait loci (QTL) have been identified across the genome including a broad region of linkage on Mmu11. Monocyte chemoattractant protein 1 (MCP-1) maps to this region and has been associated with microglial activation and reduced survival in the ME7 mouse scrapie model of prion disease. We have identified 10 polymorphisms, 3 of which are nonsynonomous, in Mcp1 between "long" (CAST) and "short" (SJL or NZW) incubation-time mouse strains. Crosses between these strains and Mcp1(-/-) mice inoculated with the Chandler/RML mouse scrapie prion strain formed the basis of a quantitative complementation test. In these models loss of Mcp1 did not show an increase in incubation time suggesting that the effects of Mcp1 may be specific to the ME7 prion strain and that Mcp1 does not contribute to the QTL described on Mmu11.

An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model.

BACKGROUND: Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown. RESULTS: We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes. CONCLUSION: We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal DIfferentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect.

Hyperphosphorylation of tau and neurofilaments and activation of CDK5 and ERK1/2 in PTEN-deficient cerebella.

Inherited mutations to the tumor suppressor PTEN sporadically lead to cerebellar gangliocytoma characterized by migration defects. This has been modeled by CNS-specific PTEN ablation in mice, but the underlying mechanism cannot be explained by the known role of PTEN in Akt/PKB inactivation. Here we show that the loss of PTEN in mouse cerebellar neurons causes neurodegeneration by hyperphosphorylation of tau and neurofilaments, and activation of Cdk5 and pERK1/2, suggesting that dysregulation of the PTEN/pAkt pathway can mediate neurodegeneration.

An enzyme-detergent method for effective prion decontamination of surgical steel.

Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP(Sc) from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel. Seven proteolytic enzymes were evaluated individually and in pairs at a range of temperatures and pH values and the additional effects of detergents, lipases and metal ions were assessed. A combination of proteinase K and Pronase, in conjunction with SDS, was shown to degrade PrP(Sc) material from highly concentrated vCJD-infected brain preparations to a level below detection. When RML prion-infected wires were exposed to the same enzymic treatment, intracerebral bioassay in highly susceptible hosts showed virtually no infectivity. The prion-degrading reagents identified in this study are readily available, inexpensive, non-corrosive to instruments, non-hazardous to staff and compatible with current equipment and procedures used in hospital sterilization units.

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation.

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.

Transgene-driven expression of the Doppel protein in Purkinje cells causes Purkinje cell degeneration and motor impairment.

The Doppel (Dpl) and Prion (PrP) proteins show 25% sequence identity and share several structural features with only minor differences. Dpl shows a PrP-like fold of its C-terminal globular domain and lacks the flexible N-terminal tail. The physiological functions of both proteins are unknown. However, ubiquitous Dpl overexpression in the brain of PrP knockout mice correlated with ataxia and Purkinje cell degeneration in the cerebellum. Interestingly, a similar phenotype was reported in transgenic mice expressing an N-terminally truncated PrP (DeltaPrP) in Purkinje cells by the L7 promoter (TgL7-DeltaPrP). Coexpression of full-length PrP rescued both the neurological syndromes caused by either Dpl or DeltaPrP. To evaluate whether the two proteins caused cerebellar neurodegeneration by the same mechanism, we generated transgenic mice selectively expressing Dpl in Purkinje cells by the same L7 promoter. Such mice showed ataxia and Purkinje cell loss that depended on the level of Dpl expression. Interestingly, the effects of high levels of Dpl were not counterbalanced by the presence of two Prnp alleles. By contrast, PrP coexpression was sufficient to abrogate motor impairment and to delay the neurodegenerative process caused by moderate level of Dpl. A similar situation was reported for the corresponding TgL7-DeltaPrP mice supporting the concept that Dpl and DeltaPrP cause cell death, possibly by interfering with a common signaling cascade essential for cell survival.

Monoclonal antibodies inhibit prion replication and delay the development of prion disease.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are fatal, neuro-degenerative disorders with no known therapy. A proportion of the UK population has been exposed to a bovine spongiform encephalopathy-like prion strain and are at risk of developing variant CJD. A hallmark of prion disease is the transformation of normal cellular prion protein (PrP(C)) into an infectious disease-associated isoform, PrP(Sc). Recent in vitro studies indicate that anti-PrP monoclonal antibodies with little or no affinity for PrP(Sc) can prevent the incorporation of PrP(C) into propagating prions. We therefore investigated in a murine scrapie model whether anti-PrP monoclonal antibodies show similar inhibitory effects on prion replication in vivo. We found that peripheral PrP(Sc) levels and prion infectivity were markedly reduced, even when the antibodies were first administered at the point of near maximal accumulation of PrP(Sc) in the spleen. Furthermore, animals in which the treatment was continued remained healthy for over 300 days after equivalent untreated animals had succumbed to the disease. These findings indicate that immunotherapeutic strategies for human prion diseases are worth pursuing.