Brains Of Prion-infected Mice Research Articles

Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC.

Effect of Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells on Mice Infected with Prions

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of beta-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.

Induced Neuroprotection Independently From PrPSc Accumulation in a Mouse Model for Prion Disease Treated With Simvastatin

The misfolding and aggregation of specific proteins has emerged as a key feature of several neurodegenerative diseases. In prion diseases, progressive disease and neuronal loss are associated with the accumulation of PrP(Sc), the misfolded isoform of PrP(C). Previous in vitro studies suggest that cholesterol-lowering drugs inhibit the conversion of PrP(C) to PrP(Sc) and the accumulation of the latter, possibly through the disturbance of cholesterol-rich membrane domains (lipid rafts). To examine the effect of simvastatin, a cholesterol-lowering drug, on prion disease progression and survival. Controlled animal study. University medical center research laboratory. Female mice from the FVB/N strain. Peripheral and central nervous system inoculations with scrapie Rocky Mountain Laboratory inoculum. Clinical, immunological, pathological, and molecular assays were performed. Simvastatin delayed disease progression, leading to increased survival in peripheral as well as central nervous system inoculations. Simvastatin's beneficial effect is mediated through the l-mevalonate pathway; however, it is independent of brain cholesterol levels. Interestingly, simvastatin treatment induced PrP(Sc) accumulation in parallel with an induced neuroprotective effect. In accordance, we found that simvastatin induced immunomodulatory mechanisms in the brains of infected mice, affecting expression levels of specific microglial chemokines and cytokines. Simvastatin delays prion disease progression and increases survival in vivo, independently of the pathogenic conversion of PrP(C) to PrP(Sc). We show that simvastatin's effects on neuroprotection are correlated with downregulation of Cox2 levels and induction of microglial activation in prion-infected mouse brains.

Development of an Enzymatic Detergent Treatment Protocol That Significantly Reduces Protease Resistant Prion Protein Load and Infectivity from Prion-Contaminated Surgical-Steel Monofilaments

BACKGROUND/OBJECTIVES: The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. In this study PrP^Sc was used as a biochemical marker of prion infectivity to identify enzymatic detergent preparations and conditions that could be used to degrade PrP^Sc present in homogenate and surface bound preparations of prion-infected brain tissue. METHODS: The ability of four enzymatic detergent preparations including new formulations of 3M™ Rapid Multi-Enzyme Cleaner (RMEC) trial formulation A and trial formulation B; Ruhof Endozime AW Plus; Steris Klenzyme, to degrade PrP^Sc associated with prion-infected mouse brain homogenates and prion-contaminated surgical-steel wires was analysed using a high sensitivity, quantitative western blot method. Prion infectivity was determined by mice bioassay of prion-contaminated surgical-steel wires. RESULTS: All products tested failed to completely degrade PrP^Sc under conditions experienced in automated washing systems. Further optimisation of RMEC trial formulation A and RMEC trial formulation B indicated that an extended treatment time (30 minutes) and higher temperatures (50-60°C) decreased detectable levels of PrP^Sc from prion-infected brain homogenates by more than 2 logs and completely removed detectable levels of PrP^Sc from prion-contaminated surgical-steel monofilaments. Bioassay indicated that incubation period was extended when wires were treated under optimised conditions with RMEC trial formulation A and trial formulation B versus wires treated with 1N NaOH. CONCLUSIONS: Optimisation of treatment protocols indicates that with extended treatment times and higher temperatures enzymatic detergents can be used to reduce the PrP^Sc load and prion infectivity from surgical-steel instruments. Direct research support provided by a commercial organization. 3M

Disease-related Prion Protein Forms Aggresomes in Neuronal Cells Leading to Caspase Activation and Apoptosis

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrP(Sc)) and accumulated cytosolic PrP(C) have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrP(Sc). Whereas cells overexpressing PrP(C) developed cytosolic PrP(C) aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrP(Sc), heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrP(Sc) aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrP(Sc) aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.

A perforin-like protein from a marine mollusk

Abalone (gastropod mollusks) express a protein, abMpeg1, which is a homolog of two mammalian proteins that share homology with mammalian perforin, a cytolytic and immune-regulatory protein of lymphocytes. One of the mammalian proteins, Mpeg1, is expressed in mature macrophage and prion-infected mouse brains, while the other, Epcs50, is expressed in ectoplacental cone cells of the invading placenta. Although the functions of these three proteins remain unknown, their structural similarity to mammalian perforin suggests that they may be involved in cell killing, the inflammatory response or tissue invasion. Consistent with these proposed functions, the Mpeg1 gene family shows the signature of positive Darwinian selection (adaptive evolution). The perforin-homology domain of abMpeg1 contains the cytolytic “helix-turn-helix” domain of perforin, supporting the idea that abMpeg1 is a cytolytic protein of the abalone innate immune system. The α-helices of abMpeg1 are amphipathic as are those of perforin. The conservation among abMpeg1, mammalian Mpeg1, and Epcs50 shows that Mpeg1 proteins represent a novel, ancient protein family of probable immunological function.

A Differentially Expressed Prion Gene mRNA Is Found in Prion-Infected Mouse Brains and in N2A Cells but Not in Uninfected Mice

The transmissible spongiform encephalopathies comprise a group of fatal neurodegenerative diseases that are characterized by the conversion of the normal host cellular prion protein (PrPC) to the abnormal protease-resistant isoform PrPSC. Distinct alterations of transcriptional regulation during disease progression could not be observed. The regulation of transcription 5′ and 3′ to the previously described cap sites of the prion gene mRNA as well as usage of internal short ORF's was investigated. We identified a mRNA species which is expressed differentially in prion-infected mice and in N2A cells. This mRNA is detectable neither in uninfected mice nor in early stages of the disease. The novel mRNA contains two short open reading frames which encode two small peptides with a calculated molecular weight of 2.1 kDA and 0.7 kDa. These peptides were also found to be expressedin vitroandin vivo.