PK-sensitive PrP is infectious and shares basic structural features with PK-resistant PrP.

One of the main characteristics of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP(Sc) that is sensitive to PK hydrolysis (sPrP(Sc)). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP(Sc) versus the PK-resistant (rPrP(Sc)) fractions of PrP(Sc) and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP(Sc) and rPrP(Sc) fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP(Sc) to the rPrP(Sc) forms of PrP(Sc). Although the sPrP(Sc) and rPrP(Sc) fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) has recently been identified to be a possible modulator of neurodegeneration. To investigate whether PAR2 plays a role in prion infection, we inoculated PAR2-deficient (PAR2(-/-)) and wild-type (WT) mice intracerebrally with the Rocky Mountain Laboratory strain of scrapie. PAR2(-/-) mice demonstrated a delayed onset of clinical symptoms, including weight loss, and demonstrated moderate but highly significant prolongation of survival over WT controls. Concomitantly, no apparent differences in brain pathology, infectivity or features of brain prion protein between deceased WT and PAR2(-/-) mice were found. Our study suggests that PAR2 deletion modulates dynamics of the disease without gross perturbation of its pathogenesis.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice.

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

Influence of ADAM10 on prion protein processing and scrapie infectiosity in vivo.

Both the cellular prion protein (PrP(c)) and the amyloid precursor protein (APP) are physiologically subjected to complex proteolytic processing events. While for APP the proteinases involved--alpha-, beta- and gamma-secretase--have been identified in vitro and in vivo, the cleavage of PrP(c) by now has been linked only to the shedding activity of the metalloproteinase ADAM10 and/or ADAM17 in cell culture. Here we show that neuronal overexpression of the alpha-secretase ADAM10 in mice reduces all PrP(c) species detected in the brain instead of leading to enhanced amounts of specific cleavage products of PrP(c). Additionally, the incubation time of mice after scrapie infection is significantly increased in mice moderately overexpressing ADAM10. This indicates that overexpression of ADAM10 rather influences the amount of the cellular prion protein than its processing in vivo.

Increase of monoamine oxidase-B activity in the brain of scrapie-infected hamsters.

In the present study, the purpose is to determine activities of monoamine oxidases (MAO) in the brain of 263K scrapie-infected hamsters during the development of this experimental prion disease. Indeed, MAO activity modifications which have already been related in aging and neurodegenerations is suspected to be involved in the neuron loss process by elevated hydrogen peroxide formation. Monoamine oxidase type A (MAO-A) and B (MAO-B) activities were followed in the brain at different stages of the disease. MAO-A activity did not change significantly during the evolution of the disease. However, concerning the MAO-B activity, a significant increase was observed from 50 days post-infection and through the course of the disease and reached 42.9+/-5.3% at its ultimate stage. Regarding these results, MAO-B could be a potential therapeutic target then we have performed a pre-clinical treatment with irreversible (Selegiline or L-deprenyl) or and reversible (MS-9510) MAO-B inhibitors used alone or in association with an anti-scrapie drug such as MS-8209, an amphotericin B derivative. Our results show that none of the MAO-B inhibitors used was able to delay the onset of the disease. Neither these MAO-B inhibitors nor R-NMDA inhibitors (MK-801) can enhance the effects of MS-8209. The present findings clearly indicate a significant increase of cerebral MAO-B activity in scrapie-infected hamsters. Furthermore, inhibitors of MAO-B do not have any curative or palliative effect on this experimental model indicating that the raise of this activity is probably more a consequence rather than a causal event of the neurodegenerative process.

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia.

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrPC) is misfolded into an accumulating, disease-associated isoform (PrPD). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors contributing to cellular permissiveness. We previously isolated five clones from an immortalized subline of ovine microglia, two of which had demonstrated differential permissiveness to a natural isolate of sheep scrapie and distinct transcriptomic profiles. To more robustly identify factors contributing to this activity, relative permissiveness, cell proliferation, selected gene transcript level, and matrix metalloproteinase 2 (MMP2) activity were compared amongst all five clones. Differences in cell proliferation were not detected between clones; however, significant correlations were identified between relative permissiveness and genes associated with cell growth (i.e., RARRES1 and PTN), protein degradation (i.e., CTSB and SQSTM1), and heparin binding (i.e., SEPP1). MMP2 activity varied amongst clones, but did not correlate with permissiveness. These associations support the contribution of cell division and protein degradation on the permissiveness of cultured ovine microglia to PrPD.

Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.

We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.

Creutzfeldt-Jakob infection increases adenylate cyclase activity in specific regions of guinea pig brain.

Creutzfeldt-Jakob disease is a slow, infectious, progressive neurological disorder which results in human dementia. Synaptic membranes from various brain regions of guinea pigs infected with Creutzfeldt-Jakob disease show increased guanyl nucleotide- or 5-hydroxytryptamine-mediated activation of adenylate cyclase. This increased enzyme activity appears due, primarily, to facilitated 'coupling' between the GTP-binding protein which stimulates adenylate cyclase (GNs) and the catalytic moiety of that enzyme rather than increased sensitivity to 5-hydroxytryptamine. It is possible that this phenomenon is due to direct effects of the Creutzfeldt-Jakob infectious agent, or a pathological product resulting from that agent, upon synaptic membrane adenylate cyclase.

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Comparison of NADPH diaphorase activity in the brains of hamsters infected with scrapie strains 139H or 263K or with normal hamster brain homogenate.

Previous studies showed that the histopathological changes found in the brains of scrapie-infected animals included amyloid plaque formation, vacuolation, gliosis and neuronal and neurite degeneration. There were differences in the histopathological findings as a function of the scrapie strain-host combination. NADPH-diaphorase (NADPH-d) has been shown to be a selective histochemical marker for neurons containing nitric oxide (NO) synthase. Neuronal cell damage caused by NOS in brain has been reported to be associated with many neurodegenerative diseases. In this study, we used NADPH-d histostaining to investigate changes in the NOS system in brains of 139H- and 263K-infected hamsters and compared the results to normal hamster brain (NHB) injected animals. We observed that some of the NADPH-d histostaining neurons in the cortex of scrapie-infected hamsters appeared to be atrophic: the neurons were smaller and had fewer neurites. The NADPH-d histostaining intensity of neurons or astrocytes in septum, thalamus, hypothalamus and amygdala of 139H- and 263K-infected hamsters was greater than in control hamsters. Astrocytes in the thalamus, hypothalamus and lower part of the cortex (layers 4 to 6) in 263K-infected hamsters were more intensely stained for NADPH-d than in either 139H-infected hamsters or controls. Our results suggest that changes in NADPH-d system might play a role in the diversity of scrapie induced neurodegenerative changes.

Activation of Fas and caspase 3 precedes PrP accumulation in 87V scrapie.

The sequence of events involved in the neurodegeneration caused by transmissible spongiform encephalopathies is not yet known. Using a murine scrapie model in which neurodegeneration in the hippocampus is restricted to the CA2, we show an up-regulation of the proapoptotic markers Fas and caspase 3 early in the incubation period prior to disease-specific prion protein (PrP) deposition and clinical signs. These results suggest that activation of Fas and caspase 3 are involved in the early pathological sequence of events during murine scrapie, and that these proapoptotic markers may be a specific method for early detection of neurodegeneration.

Early changes in tyrosine hydroxylase and glutamate decarboxylase activity in the golden hamster striatum after intracerebral inoculation of the nigrostriatal system with scrapie agent (strain 263 K).

Unilateral inoculation of hamster substantia nigra (SN) with scrapie agent led to an early decrease in tyrosine hydroxylase (TH) activity in the corresponding striatum, which was detectable by the 5th day. This decrease was accompanied by an increase in glutamate decarboxylase (GAD) observed on the 20th day. Local phenomena related to administration of the agent were investigated by intrastriatal inoculation followed by local measurement of TH, GAD and choline acetyltransferase (ChAT) activities. A rise in GAD activity was observed 20 days later. The decrease in TH activity which occurred 5 days after inoculation of the substantia nigra with scrapie agent constitutes an extremely early indication in hamsters of the slow pathological processes at work: at clinical and behavioural levels, these can be detected at best only 80 days after the intracerebral inoculation.

Increased expression of CaM kinase II alpha in the brains of scrapie-infected mice.

We investigated the distribution of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the brains of mice infected with ME7 scrapie strain. CaM kinase II is an enzyme that plays a major role in the regulation of long-term potentiation, a form of synaptic plasticity associated with learning and memory. Immunoreactivity of CaM kinase II alpha, measured by Western blot, increased markedly in scrapie-infected brains compared with control brains. Immunohistochemically, CaM kinase II alpha immunoreactivity was upregulated in the cerebral cortex and hippocampal CA1 area of scrapie-positive mice infected with ME7 scrapie strain. This result implies that this enzyme is associated with aberrant function of synaptic transmission and LTP of the pyramidal neurons in the hippocampal CA1 area of mice infected with ME7 scrapie strain.

Choline acetyltransferase activity and [3H]quinuclidinylbenzilate binding in brains of scrapie-infected hamsters.

Choline acetyltransferase (ChAT) activity and [3H]quinuclidinylbenzilate binding were studied in the brain of scrapie-infected hamsters and sham inoculated controls. Although scrapie-infected hamsters showed no reduction of ChAT activity compared to the controls, they showed a decrease in the affinity and maximum number of post-synaptic muscarinic receptors. Scrapie virus thus alters the cholinergic system at the post-synaptic rather than at the pre-synaptic level.

Induction of heme oxygenase-1 in the brains of scrapie-infected mice.

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin, carbon monoxide and iron, and its expression can be used as a marker for oxidative stress. Oxidative stress has been reported to be associated with neurodegenerative diseases including Alzheimer's disease. It is possible that oxidative stress is also involved in the disease process seen in scrapie, the archetype transmissible spongiform encephalopathy. In this study, we report that HO-1 is significantly increased in the scrapie-infected group compared to an age-matched control group. Immunohistochemistry showed a pronounced increase of immunostaining of this protein in the infected group compared to the minimal amount of staining in the control group. These results support that oxidative stress is closely associated with the pathogenesis of scrapie and that it might contribute to neurodegeneration in this disease.

Brain glutamate decarboxylase and cholinergic enzyme activities in scrapie.

C57BL/6J mice, age 6-8 weeks were inoculated intracerebrally with brain homogenate from mice previously infected with the 139A strain of scrapie; control mice were identically treated with brain homogenate from non-infected normal mice. The activities of choline acetyltransferase (CAT), acetyl cholinesterase (AChE), and glutamic acid decarboxylase (GAD) were determined in the forebrain and hindbrain of these animals after 67, 126 and 151 days post-inoculation. There were no significant differences in the activities of CAT and GAD between scrapie and control mice at early, middle or late stages of the disease in the scrapie-infected animals; there was an about 20% decline in AChE activity in the scrapie brain.

Plasmalogenase is elevated in early demyelinating lesions.

Plasmalogenase catalyzes the hydrolysis of ethanolamine plasmalogens to long-chain aldehydes and 2-acyl-sn-glycero-3-phosphoethanolamines. During development, plasmalogenase activity parallels myelination. The enzyme is most concentrated within oligodendroglial cells and is absent from myelin. The normal function of plasmalogenase in white matter may be related to its specificity for plasmalogens that contain most of the thromboxane and prostaglandin precursors. Plasmalogenase activities are elevated in demyelinating CNS tissues including canine white matter with lesions due to distemper virus. Elevated plasmalogenase activity precedes cellular invasion and lysosomal activation as indicated by beta-glucuronidase, acid proteinase and neutral proteinase activities. The elevation of plasmalogenase activity was 4.9-fold greater than normal in an early demyelinating lesion caused by the Snyder-Hill strain of distemper virus. Phospholipases acting on phosphatidyl ethanolamine were not activated in this tissue and have activities much lower than plasmalogenase in control tissues. Plasmalogenase activities are also elevated after intracerebral injections of complement-dependent anti-myelin antibody and after ischemia. Plasmalogenase acting on the oligodendrocyte plasma membrane may be responsible for necrosis of the oligodendrocyte that results in demyelination.

Intraneuronal enzymic inclusions in the histological diagnosis of scrapie.

Cumulative results are presented of histopathological and enzyme histochemical findings in sheep naturally or experimentally infected with scrapie compared with healthy controls or animals with other diseases. Two hundred and sixty-eight sheep were examined, including 210 cases of clinical or suspected scrapie. According to the nature and distribution of histopathological changes, especially neuropil vacuolation and neuronal vacuoles, scrapie-affected sheep were classified into groups. In particular, examination of medulla oblongata revealed a consistently different pattern of lesions between natural scrapie (type A) and experimental scrapie (type B). Cytoplasmic enzymic inclusions demonstrated by methods for beta-glucuronidase and for acid phosphatase were regularly found in neurones from scrapie sheep but not from control animals. They appeared to be more prevalent in type B than in type A scrapie and were demonstrable even in tissue affected by post-mortem autolysis. The distribution of enzymic inclusions ranged from Betz cells of cerebral cortex to grey matter of the lumbar region of spinal cord. The detection of enzymic inclusions is a useful diagnostic criterion and has been incorporated into histological diagnostic procedures for scrapie.

Total CK and CK-BB activity in serum from sheep with scrapie.

Total CK and iso-enzyme CK-BB activity was measured in serum from four sheep with scrapie and in serum from four healthy control sheep. Blood samples were taken weekly for about six months. There was a clear overlap between the total CK and CK-BB activity in serum from sheep with scrapie and that in serum from control sheep. Thus measurement of these enzymes does not aid the clinical diagnosis of scrapie.

Alterations of arginase activity in scrapie-infected mice and in amyotrophic lateral sclerosis.

We followed the dynamics of arginase activity, the ultrastructural changes, and accumulation of the scrapie agent in the CNS of scrapie-infected mice. The arginase activity has been shown to increase 5-fold within the first 3-4 months of the incubation period followed by subsequent fall at its end. The peak of increased arginase activity coincided with appearance of multilayer membranes, whereas the decrease of this activity was associated with the greatest development of status spongiosus, synaptic degeneration, accumulation of five-layer membranes as well as with maximal accumulation of the scrapie agent in the CNS. Examination of 100 patients with different forms of amyotrophic lateral sclerosis (ALS) revealed a 2-8-fold increase of arginase levels in the cerebrospinal fluid (CSF) as compared with the background levels in the comparative groups of control patients. Similar enzyme alterations in scrapie--and in ALS--are discussed in terms of possible resemblance of their pathogenesis.