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.

Abortive cell cycle events in the brains of scrapie-infected hamsters with remarkable decreases of PLK3/Cdc25C and increases of PLK1/cyclin B1.

Polo-like kinases (PLKs) consist of a family of kinases which play critical roles during multiple stages of cell cycle progression. Increase of PLK1 and decrease of PLK3 are associated with the developments and metastases of many types of human malignant tumors; however, the situations of PLKs in prion diseases are less understood. Using Western blots and immunohistochemical and immunofluorescent assays, marked increase of PLK1 and decrease of PLK3 were observed in the brains of scrapie strain 263K-infected hamsters, presenting obviously a time-dependent phenomenon along with disease progression. Similar alterations of PLKs were also detected in a scrapie infectious cell line SMB-S15. Both PLK1 and PLK3 were observed in neurons by confocal microscopy. Accompanying with the changes of PLKs in the brains of 263K-infected hamsters, Cdc25C and its phosphorylated forms (p-Cdc25C-Ser198 and p-Cdc25C-Ser216) were significantly down-regulated, whereas Cyclin B1 and PCNA were obviously up-regulated, while phospho-histone H3 remained almost unchanged. Moreover, exposure of the cytotoxic peptide PrP106-126 on the primary cultured cortical neuron cells induced similar changes of cellular PLKs and some cell cycle-related proteins, such as Cdc25C and its phosphorylated forms, phospho-histone H3. Those results illustrate obviously aberrant expressions of cell cycle regulatory proteins in the prion-infected neurons, which may lead to the cell cycle arrest at M phase. Possibly due to the ill-regulation of some key cell cycle events during prion infection, together with the fact that neurons are unable to complete mitosis, the cell cycle reentry in prion-infected neurons is definitely abortive, which may lead to neuron apoptosis and neuron degeneration.

Protein disulfide isomerase regulates endoplasmic reticulum stress and the apoptotic process during prion infection and PrP mutant-induced cytotoxicity.

BACKGROUND: Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active S-nitrosylated modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins. CONCLUSION/SIGNIFICANCE: Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO-PDI may feed as an accomplice for PDI apoptosis.

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.

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.

Na+/K+-ATPase is present in scrapie-associated fibrils, modulates PrP misfolding in vitro and links PrP function and dysfunction.

Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C), to the disease-associated form, PrP(Sc), through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C) and PrP(Sc) isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C) or to provide a scaffold ensuring correct alignment of PrP(C) and PrP(Sc) during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF) from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+)/K(+)-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+)/K(+)-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C) and Na(+)/K(+)-ATPase have previously been reported in astrocytes, our data highlight this molecule as a key link between PrP function, dysfunction and misfolding.

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.

Glutathione peroxidase (GPX) activity in blood of ewes on farms in different scrapie categories in Iceland.

BACKGROUND: Preliminary studies indicated decreased glutathione peroxidase (GPX) activity in blood of ewes on scrapie-afflicted farms. Other studies have shown decreased GPX activity in brain of prion-infected mice and in prion-infected cells in vitro. The aim of this study was to examine the GPX activity in blood as well as the distribution of GPX-activity levels from ewes on farms in scrapie-afflicted areas in Iceland. METHODS: Blood samples were collected from 635 ewes (non-pregnant [n = 297] and pregnant [n = 338]) on 40 farms in scrapie-afflicted areas during the years 2001-2005, for analysis of GPX activity. The farms were divided into three categories: 1. Scrapie-free farms (n = 14); 2. Scrapie-prone farms (earlier scrapie-afflicted, restocked farms) (n = 12); 3. Scrapie-afflicted farms (n = 14). For comparison, 121 blood samples were also collected from non-pregnant ewes on one farm (farm A) in a scrapie-free area (scrapie never registered). Chi-square test was used to test for normal distribution of GPX-results, and Kruskal-Wallis test to compare GPX-results between categories. RESULTS: The GPX-results appeared to be biphasically distributed in ewes in all three scrapie categories and on farm A. The presumptive breaking point was about 300 units g Hb-1. About 30-50% of the GPX-results from ewes in all three scrapie categories were below 300 units g Hb-1 but only about 13% of the GPX-results from ewes on farm A. The mean GPX activity was highest on farm A, and was significantly lower on scrapie-prone farms than on scrapie-free or scrapie-afflicted farms (non-pregnant and pregnant ewes: P < 0.005, respectively; non-pregnant and pregnant ewes combined: P < 0.0005). CONCLUSIONS: 1) the distribution of GPX-results in blood of Icelandic ewes apparently has a biphasic character; 2) the GPX-results were higher in ewes on one farm in a scrapie-free area than in ewes on farms in the scrapie-afflicted areas; 3) GPX-activity levels were significantly lowest on earlier scrapie-afflicted, restocked farms, which might have a bearing on the recurrence of sporadic scrapie on these farms; 4) further study on the possible role of GPX activity in the occurrence of scrapie in Iceland is warranted.

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.

Oxidative stress in the brain at early preclinical stages of mouse scrapie.

Oxidative stress has been shown to be involved in the pathogenesis of neurodegenerative diseases including prion diseases. Although a growing body of evidence suggests direct involvement of oxidative stress in the pathogenesis of prion diseases, it is still not clear whether oxidative stress is a causative early event in these conditions or a secondary phenomenon commonly found in the progression of neurodegenerative diseases. Using a mouse scrapie model, we assessed oxidative stress in the brain at various stages of the disease progression and observed significantly increased concentration of lipid peroxidation markers, malondialdehyde and 4-hydroxyalkenals, and mRNA level of an oxidative stress response enzyme, heme oxygenase-1, at early preclinical stages of scrapie. The changes preceded dramatic synaptic loss demonstrated by immunohistochemical staining of a synaptic protein, synaptophysin. These findings imply that the brain undergoes oxidative stress even from an early stage of prion invasion into the brain. Given the well-known deleterious effects of reactive-oxygen-species-mediated damage in the brain, it is considered that the oxidative stress at the preclinical stage of prion diseases may predispose the brain to neurodegenerative mechanisms that characterize the diseases.

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.

Activation of mitogen-activated protein kinases in hamster brains infected with 263K scrapie agent.

We investigated the expression, activation and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38 MAPKs) and extracellular signal-regulated kinases (ERKs), using western blotting and immunohistochemistry, in the brains of hamsters infected with 263K scrapie agent, to clarify the role of these kinases in the pathogenesis of prion disease. The immunoblot analysis demonstrated that activation of JNK, p38 MAPK and ERK in whole brain homogenates was increased in infected animals. Phosphorylation of cAMP/calcium responsive element binding protein (CREB), a downstream transcription factor of active ERK, was significantly increased in scrapie-infected hamsters. The immunohistochemical study showed that active ERK was enhanced in infected hamsters compared with controls. Active ERK immunoreactivity was observed within neurons in the dentate gyrus and in glial fibrillary acidic protein (GFAP)-positive reactive astrocytes of infected animals. The expression level of c-Jun mRNA as well as protein, a substrate of active JNK, was increased in infected animals. A significant increase in JNK activity upon glutathione S-transferase (GST)-c-Jun was observed in infected compared with control animals. Phospho-c-Jun immunoreactivity was observed only in neurons of the thalamus in infected groups. These findings indicated that the JNK pathway was activated in the scrapie-infected group. The chronological activation of MAPKs using immunoblot analysis indicates that the kinases are sequentially activated during the pathophysiology of prion disease. Taken together, these results lend credence to the notion that MAPK pathways are dysregulated in prion disease, and also indicate an active role for this pathway in disease pathogenesis.

Increased expression of phospholipase D1 in the brains of scrapie-infected mice.

Mitochondrial dysfunction and free radical-induced oxidative damage are critical factors in the pathogenesis of neurodegenerative diseases. Recently, phospholipid breakdown by phospholipase D (PLD) has been recognized as an important signalling pathway in the nervous system. Here, we examined the expression of PLD and alteration of membrane phospholipid in scrapie brain. We have found that protein expression and enzyme activity of PLD1 were increased in scrapie brains compared with controls; in particular, there was an increase in the mitochondrial fraction. PLD1 in mitochondrial membranes from scrapie brains, but not from control brains, was tyrosine phosphorylated. Furthermore, the concentration of mitochondrial phospholipids such as phosphatidylcholine and phosphatidylethanolamine was increased and the content of phosphatidic acid, a product of PLD activity, was up-regulated in the mitochondrial membrane fractions. Immunohistochemically, PLD1 immunoreactivity was significantly increased in activated astrocytes in both cerebral cortex and hippocampus of scrapie brains. Taken together, these results suggest that PLD activation might induce alterations in mitochondrial lipids and, in turn, mediate mitochondrial dysfunction in the brains of scrapie-infected mice.

Unchanged scrapie pathology in brain tissue of tyrosine kinase Fyn-deficient mice.

Fyn is a 59-kDa member of the Src family of tyrosine kinases synthesized on cytosolic polysomes and then targeted to the plasma membrane where it clusters in caveolae-like membrane microdomains. The cellular isoform of the prion protein (PrP) has also been identified to be a caveolar constituent and to participate in signal transduction events concerning cell survival and differentiation via recruitment of Fyn. We studied the scrapie infection of mice deficient for Fyn (Fyn(-/-)) to clarify the role of Fyn in an in vivo model of transmissible spongiforme encephalopathies. Fyn(-/-) mice died on average 9 days earlier than wild-type control mice, but no differences were seen regarding activation of astrocytes, vacuolization of the neuropil, and accumulation of misfolded prion protein. The experimental model suggests that a deficiency for Fyn is detrimental in prion diseases, although it has no major effect on the clinical course of an experimental prion infection of the CNS.

Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein.

Prion diseases are characterized by accumulation of misfolded prion protein (PrP(Sc)), and neuronal death by apoptosis. Here we show that nanomolar concentrations of purified PrP(Sc) from mouse scrapie brain induce apoptosis of N2A neuroblastoma cells. PrP(Sc) toxicity was associated with an increase of intracellular calcium released from endoplasmic reticulum (ER) and up-regulation of several ER chaperones. Caspase-12 activation was detected in cells treated with PrP(Sc), and cellular death was inhibited by overexpression of a catalytic mutant of caspase-12 or an ER-targeted Bcl-2 chimeric protein. Scrapie-infected N2A cells were more susceptible to ER-stress and to PrP(Sc) toxicity than non-infected cells. In scrapie-infected mice a correlation between caspase-12 activation and neuronal loss was observed in histological and biochemical analyses of different brain areas. The extent of prion replication was closely correlated with the up-regulation of ER-stress chaperone proteins. Similar results were observed in humans affected with sporadic and variant Creutzfeldt-Jakob disease, implicating for the first time the caspase-12 dependent pathway in a neurodegenerative disease in vivo, and thus offering novel potential targets for the treatment of prion disorders.

Prion protein is secreted in soluble forms in the epididymal fluid and proteolytically processed and transported in seminal plasma.

The presence of prion protein in sperm and fluids collected from different parts of the ram genital tract was investigated by immunoblotting with monoclonal antibodies. A slightly immunoreactive 25- to 30-kDa protein was recognized on Western blots of testicular and epididymal sperm extracts. Immunoreactivity increased on ejaculated sperm extracts and 2 other bands at 35 and 43 kDa also reacted. Seminal plasma showed several immunoreactive bands, the main bands being detected at 43 and 35 kDa, whereas less reactive bands were observed at 30, 25, 20, and <14 kDa. All these bands strongly decreased in the seminal plasma after vasectomy, indicating a testicular or an epididymal origin. Testicular fluid showed almost no reactivity, whereas caudal epididymal fluid contained the 2 strong immunoreactive bands at 43 and 35 kDa and in some cases a faint 30-kDa band. The 43-kDa band was also found in the fluid from the proximal caput, whereas the 35-kDa band appeared in the distal caput. Immunoprecipitation of (35)S-labeled proteins secreted in the epididymal fluid indicated that the 43-kDa form was synthesized in caput and caudal regions and the 35-kDa form in the distal caput to the distal corpus. Treatment of caudal fluid and seminal plasma by N-glycosidase resulted in the formation of 3 bands: 1 highly reactive at about 25 kDa, a second less reactive at about 28 kDa, and a third at approximately 20 kDa. The pattern of prion protein distribution in epididymal fluids was found to be similar in scrapie-infected rams to that of healthy rams. Cauda epididymal fluid and seminal plasma from infected animals could not be treated directly with proteinase K, because of the presence of protease inhibitors. However, the prion protein immunoprecipitated from these fluids was completely cleaved by proteinase K, whereas in the same conditions this from an infected sheep brain gave the usual resistant band pattern.

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.

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.

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.