Prion disease induced alterations in gene expression in spleen and brain prior to clinical symptoms.

Prion diseases are fatal neurodegenerative disorders that affect animals and humans. There is a need to gain understanding of prion disease pathogenesis and to develop diagnostic assays to detect prion diseases prior to the onset of clinical symptoms. The goal of this study was to identify genes that show altered expression early in the disease process in the spleen and brain of prion disease-infected mice. Using Affymetrix microarrays, we identified 67 genes that showed increased expression in the brains of prion disease-infected mice prior to the onset of clinical symptoms. These genes function in many cellular processes including immunity, the endosome/lysosome system, hormone activity, and the cytoskeleton. We confirmed a subset of these gene expression alterations using other methods and determined the time course in which these changes occur. We also identified 14 genes showing altered expression prior to the onset of clinical symptoms in spleens of prion disease infected mice. Interestingly, four genes, Atp1b1, Gh, Anp32a, and Grn, were altered at the very early time of 46 days post-infection. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as surrogate markers for the early detection and diagnosis of prion disease.

Levels of abnormal prion protein in deer and elk with chronic wasting disease.

Chronic wasting disease (CWD) of deer and elk is a widespread health concern because its potential for crossspecies transmission is undetermined. CWD prevalence in wild elk is much lower than its prevalence in wild deer, and whether CWD-infected deer and elk differ in ability to infect other species is unknown. Because lymphoid tissues are important in the pathogenesis of some transmissible spongiform encephalopathies such as sheep scrapie, we investigated whether CWD-affected elk and deer differ in distribution or quantity of disease-associated prion protein (PrPres) in lymphoid tissues. Immunoblot quantification of PrPres from tonsil and retropharyngeal lymph nodes showed much higher levels of PrPres in deer than in elk. This difference correlated with the natural prevalence of CWD in these species and suggested that CWD-infected deer may be more likely than elk to transmit the disease to other cervids and have a greater potential to transmit CWD to noncervids.

Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains.

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

Enhanced antiscrapie effect using combination drug treatment.

Combination treatment with pentosan polysulfate and Fe(III)meso-tetra(4-sulfonatophenyl)porphine in mice beginning 14 or 28 days after scrapie inoculation significantly increased survival times. This increase may be synergistic, implying that the compounds act cooperatively in vivo. Combination therapy may therefore be more effective for treatment of transmissible spongiform encephalopathies and other protein-misfolding diseases.

Prion-induced amyloid heart disease with high blood infectivity in transgenic mice.

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 10(7) 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Gene expression alterations in brains of mice infected with three strains of scrapie.

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML. RESULTS: In symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function. CONCLUSION: These studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.

Potent antiscrapie activities of degenerate phosphorothioate oligonucleotides.

Although transmissible spongiform encephalopathies (TSEs) are incurable, a key therapeutic approach is prevention of conversion of the normal, protease-sensitive form of prion protein (PrP-sen) to the disease-specific protease-resistant form of prion protein (PrP-res). Here degenerate phosphorothioate oligonucleotides (PS-ONs) are introduced as low-nM PrP-res conversion inhibitors with strong antiscrapie activities in vivo. Comparisons of various PS-ON analogs indicated that hydrophobicity and size were important, while base composition was only minimally influential. PS-ONs bound avidly to PrP-sen but could be displaced by sulfated glycan PrP-res inhibitors, indicating the presence of overlapping binding sites. Labeled PS-ONs also bound to PrP-sen on live cells and were internalized. This binding likely accounts for the antiscrapie activity. Prophylactic PS-ON treatments more than tripled scrapie survival periods in mice. Survival times also increased when PS-ONs were mixed with scrapie brain inoculum. With these antiscrapie activities and their much lower anticoagulant activities than that of pentosan polysulfate, degenerate PS-ONs are attractive new compounds for the treatment of TSEs.

A porphyrin increases survival time of mice after intracerebral prion infection.

Prion diseases, including scrapie, are incurable neurodegenerative disorders. Some compounds can delay disease after a peripheral scrapie inoculation, but few are effective against advanced disease. Here, we tested multiple related porphyrins, but only Fe(III)meso-tetra(4-sulfonatophenyl)porphine injected into mouse brains after intracerebral scrapie inoculation substantially increased survival times.

The most infectious prion protein particles.

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

Evaluation of new cell culture inhibitors of protease-resistant prion protein against scrapie infection in mice.

In vitro inhibitors of the accumulation of abnormal (protease-resistant) prion protein (PrP-res) can sometimes prolong the lives of scrapie-infected rodents. Here, transgenic mice were used to test the in vivo anti-scrapie activities of new PrP-res inhibitors, which, because they are approved drugs or edible natural products, might be considered for clinical trials in humans or livestock with transmissible spongiform encephalopathies (TSEs). These inhibitors were amodiaquine, thioridazine, thiothixene, trifluoperazine, tetrandrine, tannic acid and polyphenolic extracts of tea, grape seed and pine bark. Test compounds were administered for several weeks beginning 1-2 weeks prior to, or 2 weeks after, intracerebral or intraperitoneal 263K scrapie challenge. Tannic acid was also tested by direct preincubation with inoculum. None of the compounds significantly prolonged the scrapie incubation periods. These results highlight the need to assess TSE inhibitors active in cell culture against TSE infections in vivo prior to testing these compounds in humans and livestock.

Scrapie-specific neuronal lesions are independent of neuronal PrP expression.

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.

Inactivation of transmissible spongiform encephalopathy (prion) agents by environ LpH.

Agents causing transmissible spongiform encephalopathy (TSE) diseases are resistant to inactivation by several conventional decontamination methods. Using an animal bioassay, we compared the TSE agent disinfectant efficacy of a commercially available product referred to alternatively as LpH-SE, LpH-AG, or LpH-st to that of a similarly named but differently formulated product, Environ LpH, which was found to be an effective TSE agent disinfectant in a previous study. Here, we found LpH-SE to be at least 10(4)-fold to 10(5)-fold less effective than Environ LpH.

Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer, elk, sheep, and cattle.

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.