BSE and vCJD cause disturbance to uric acid levels

Experimental Neurology 190 (2004) 233 – 244 www.elsevier.com/locate/yexnr

BSE and vCJD cause disturbance to uric acid levels Tamuna Lekishvilia, Judyth Sassoona, Andrew R. Thompsetta, Alison Greenb, James W. Ironsideb, David R. Browna,* a

Department of Biology and Biochemistry, University of Bath, Claverton Dow, Bath, BA2 7AY, UK b National CJD Surveillance Unit, Western General Hospital, Edinburgh, EH4 2XU, Scotland Received 22 April 2004; revised 9 June 2004; accepted 9 July 2004

Abstract Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) are two new members of the family of neurodegenerative conditions termed prion diseases. Oxidative damage has been shown to occur in prion diseases and is potentially responsible for the rapid neurodegeneration that is central to the pathogenesis of these diseases. An important nonenzymatic antioxidant in the brain is uric acid. Analysis of uric acid in the brain and cerebrospinal fluid (CSF) of cases of BSE and CJD showed a specific reduction in CSF levels for both BSE and variant CJD, but not sporadic CJD. Further studies based on cell culture experiments suggested that uric acid in the brain was produced by microglia. Uric acid was also shown to inhibit neurotoxicity of a prion protein peptide, production of the abnormal prion protein isoform (PrPSc) by infected cells, and polymerization of recombinant prion protein. These findings suggest that changes in uric acid may aid differential diagnosis of vCJD. Uric acid could be used to inhibit cell death or PrPSc formation in prion disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Bovine spongiform encephalopathy; Creutzfeldt-Jakob disease; Nonenzymatic antioxidant

Introduction Neuronal death is central to the fatal nature of prion diseases (Prusiner, 1998). These diseases otherwise known as transmissible spongiform encephalopathies include Creutzfeldt-Jakob disease (CJD) (Bell and Ironside, 1993), bovine spongiform encephalopathy (BSE) (Hope et al., 1988), scrapie, and chronic wasting disease (Guiroy et al., 1993). Particular interest in these diseases has been sparked by the emergence of a variant of CJD (vCJD), which is unlike the common form of this disease (sCJD) in that it has been causally linked to BSE (Hill et al., 1997). Whether this is true or not, there is quite a number of similarities between these diseases. All prion diseases are characterized by the deposition of an abnormal, protease-resistant isoform of the prion protein (PrPc) in the central nervous tissue and some

* Corresponding author. Fax: +44 1225 826 779. E-mail address: [email protected] (D.R. Brown). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.07.002

peripheral tissues (Head et al., 2004). This abnormal isoform (PrPSc) is thought to be both the infectious agent and the cause of neurodegeneration in the disease. Its detection is central to diagnosis of prion disease. However, this currently means that diagnosis can only be conclusive following death (Kubler et al., 2003). As a result, there is great interest in finding a marker for these diseases that would serve diagnostic purposes before death and would allow diagnosis before symptoms had progressed to the point where the patient could not possibly recover. The deposition in the brain of PrPSc does not cause neuronal death without the expression by neurons of the normal isoform, PrPc. This was first shown using a cell culture model (Brown et al., 1994) and later confirmed using tissue transplantation studies in mice (Brandner et al., 1996). Recently, a conditional PrP-knockout model has shown that, during the course of scrapie infection, switching off PrPc expression halts the toxic effect of PrPSc and the infected animal recovers (Mallucci et al., 2003). These findings also imply an indirect element in the mechanism of

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prion-induced neuronal death. This confirms previous findings that showed oxygen radicals produced by PrPScactivated microglia trigger apoptosis in models of prion disease (Brown et al., 1996a; Giese et al., 1998). The current view of the mechanism of cell death in prion disease is a complex one (Brown, 2002). There have been many new theories in recent years, and there clearly is much that needs to be resolved. However, there is now overwhelming evidence that oxidative stress is a critical trigger involved (Brown et al., 1996a; Guentchev et al., 2000; Milhavet et al., 2000; Wong et al., 2001). It has been shown previously that there is increased lipid oxidation in both the CSF and brains of CJD patients (Arlt et al., 2002; Wong et al., 2001). This suggests that there is either an increase in oxidative substances in the brain or a reduction in the ability of the brain’s antioxidant defense to deal with oxidative stress. The implication is that regulating defense against oxidative stress could be a viable method of treatment for prion disease. It is therefore critical to begin to understand more about changes in the inherent antioxidants in the brain during prion disease. There has been some investigation of cellular antioxidants in prion disease, but currently, little is known about nonenzymatic antioxidants. In the brain, the two main nonenzymatic antioxidants are ascorbate and uric acid. Uric acid is a product of purine metabolism and is also known to have antioxidant properties (Lopez-Torres et al., 1993). In nonprimates, the end point of purine metabolism is allantoin, but in man, the gene for urate oxidase is silent implying that the end point is uric acid. Uric acid levels in man are much higher than in other mammals, possibly as a result of this. In addition, ascorbate in man can only be gained through the diet. In comparison to ascorbate, uric acid is a more effective antioxidant at lower concentrations (Spitsin et al., 2002). Therefore, it is possible that uric acid has replaced ascorbate as the main nonenzymatic antioxidant in the human brain. It has been shown that ascorbate is reduced in the CSF of CJD patients (Arlt et al., 2002). In parallel with this, there was an increase in lipid peroxidation in the CSF of CJD patients, but Vitamin E was unaffected. Although uric acid has not been measured in prion diseases, it has been suggested to be elevated in the CSF of patients with other dementias (Degrell and Nicklasson, 1988) but is probably decreased in Alzheimer disease (Tahgi et al., 1993). In general, neurological conditions ranging from stroke to meningitis are associated with increased CSF uric acid levels (Stover et al., 1997). Whether this is beneficial is unclear. In some studies of stroke patients, there is a correlation between serum uric acid levels and the chance of a good clinical outcome. Those with higher serum uric acid levels showed lower levels of neurological impairment (Chamorro et al., 2002). However, another study has suggested the opposite (Weir et al., 2003). Despite this, the majority of evidence suggests that having higher uric acid levels might diminish the effects of neurodegenerative disease.

We examine levels of uric acid in the brain and CSF of BSE-infected cattle and humans with vCJD and sCJD. We found reduction in uric acid levels specific for both vCJD and BSE. In culture experiments, we found that the main brain cell that produces uric acid was microglia. Application of uric acid to in vitro test systems showed that uric acid inhibited toxicity of a prion peptide, reduced the production of PrPSc by infected cells, and inhibited prion protein aggregation. These findings suggest that uric acid might have a protective role in prion disease and that reduction of uric acid might expose neurons to increased risk of cell death. As uric acid levels can be elevated by dietary factors, then altering uric acid might be beneficial to patients with prion diseases.

Methods Tissue samples Human brain autopsy tissue samples from the National CJD Surveillance Unit Brain Bank were obtained from cases of sporadic and variant CJD in whom consent for use of autopsy tissues for research had been obtained. Use of this material has been approved by the Lothian Region Ethics Committee. Some human CSF samples were obtained from the University of Bern, Switzerland. Brain and CSF samples from BSE-positive and BSE-negative animals were obtained from the Veterinary Laboratories Agency. Cows were orally challenged with 100 g BSEinfected brain tissue. Samples were cut from the frontal lobe of brains, snap-frozen, and shipped on dry ice and were stored at 808C or prepared for assay in the following way. Brain tissue samples of 0.5 g were homogenized in 5 ml of PBS buffer (pH 7). The homogenized samples were then disrupted 2  60 s using a sonicator. Aliquots, 1 ml, were centrifuged in Eppendorf tubes at maximum speed in a MSE Micro Centaur microfuge. Supernatants were then filtered through a 0.22-Am syringe-driven MF membrane filter (Millipore) and stored at 808C until required. The majority of human CSF samples were obtained from patients studied by the National CJD Surveillance Unit (NCJDSU). CSF was available from 17 patients with variant CJD [9 males, 8 females; aged 15 to 53 (mean F SD: 30.5 F 11.3) years at notification], 10 patients with sporadic CJD [7 males, 3 females; aged 50 to 84 (mean F SD: 64.7 F 10.5 years) at notification], and 17 control patients [9 males, 8 females; aged 18 to 85 (mean F SD: 45.6 F 19.5) years at notification]. Of the 17 patients with vCJD, 14 met published neuropathological criteria (Ironside, 1998), and the remaining 3 were diagnosed with probable vCJD according to the clinical criteria previously described (Will et al., 2000). All 10 patients with sporadic CJD were diagnosed according to established neuropathological criteria (Kretzschmar et al., 1996). The control group consisted of patients with suspected CJD referred to the NCJDSU in

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whom the final diagnosis was not CJD. Of these 17 patients, 5 had neuropathological examination, 2 were found to have no evidence of CJD but no alternative diagnosis was identified, 1 patient had evidence of paraneoplastic encephalitis, 1 patient had Alzheimer disease, and the remaining patient had multiple sclerosis. Of the remaining 12 patients, 4 had dementia of unknown cause, 1 patient had Alzheimer disease, 1 patient had cerebrovasculitis, 1 had Huntington disease, 1 had an adverse drug reaction, and the diagnosis was excluded in the remaining 4 patients. Reasons for excluding the diagnosis of CJD included complete or partial recovery or a prolonged or atypical clinical course with either an alternative diagnosis probable on clinical grounds or investigations suggestive of an alternative diagnosis. All CSF samples were stored at 808C before analysis. All samples were only identified by the CJD notification number, and all analyses were undertaken blind to the patients’ diagnostic category. Determination of uric acid Uric acid was measured using a Shimadzu LC-6A highperformance liquid chromatographic (HPLC) system in conjunction with a 20-Al sample loop, C-18 guard column, and a C-18 analytical column (4.6  250 mm, ISCO). Peak detection was carried out using a Shimadzu SPD-6AV spectrophotometric detector configured in series with a Bioanalytical systems CC-5 cross-flow thin-layer cell (glassy carbon working electrode) coupled to a Petit Ampere Potentiostat. Uric acid was detected with the working electrode held at a potential of +800 mV versus Ag/AgCl. The degassed mobile phase consisted of 50 mM ammonium formate:methanol 95:5 at pH 4.0. Uric acid peaks were also detected spectrophotometrically at 232 nm. Quantification of uric acid was accomplished using standard solutions, which were run before, during, and after the analysis to verify the consistency of the chromatographic method. Stock solutions of standards were prepared daily. Enzymatic assays Uric acid and urate oxidase (uricase) were measured using Amplex Red-based assay kits (Molecular Probes). Each of these assays depended on the generation of H2O2 in the reaction which, in the presence of horseradish peroxidase, reacts stoichiometrically with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to generate the redfluorescent oxidation product, resorufin, having an absorption maximum at 563 nm. The intensity of the colored product was measured using a plate reader (Techan, Spectra). Primary cell cultures Cerebellar neuronal cultures were prepared from 6-dayold 129SV mice (Harlen). Cultures were prepared as

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described previously (Brown, 1999). Briefly, the cerebella were dissociated in Hanks media (Sigma) containing 0.5% trypsin (Sigma) and plated at 1–2  106 cells/cm2 in 24-well trays (Falcon) coated with poly-d-lysine (50 Ag/ml, Sigma). Cultures were maintained in Dulbecco minimal essential media (Sigma) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin, streptomycin) (Sigma). Cultures were maintained at 378C with 5% CO2. Mixed glial cultures were prepared from dissociating cerebral cortices of newborn mice. Four to five cortices were trypsinized in 0.05% trypsin (Sigma) and plated in a 75 cm2 culture flask (Falcon) in Dulbecco minimal essential medium (Sigma) supplemented with 10% fetal calf serum (Sigma) and 1% of antibiotic solution (penicillin, streptomycin; Sigma). Cultures were maintained at 378C with 5% CO2 for 14 days until glial cultures were confluent. Microglia were dislodged into the medium and replated in 24-well trays. Nonadhesive cells were removed after 15 min. This procedure produced pure microglial cultures on the basis of morphology. Microglia were maintained under the same conditions as for mixed glial cultures. Astrocytes were purified by taking the remaining adhesive cells from mixed cultures and trypsinizing. The cells were preplated for 30 min to remove contaminating microglia. Then astrocytes remaining in the medium were collected and plated for 2 h at 104 cells per well into 24-well trays, after which time the medium was replaced to remove less-adhesive contaminating cells. Purified astrocytes were maintained under the conditions described for mixed glial cultures. Purity of cultures was determined as previously described (Brown, 1999; Brown et al., 1996b; Daniels and Brown, 2001). Parallel wells to experimental wells contained circular coverslips onto which the cells were plated. Cells were fixed with 4% paraformaldehyde and stained with each of the following primary antibodies: NeuN (mouse; Chemicon), anti-glial fibrillary acid protein (antiGFAP, rabbit; Daco), or ferratin (Sigma). NeuN labels neuronal cells, anti-GFAP labels astrocytes, and antiferratin labels microglia. Secondary antibodies conjugated to FITC (mouse; Daco) were used to detect labeled cells. Cells were examined using fluorescence microscopy (Leitz). Only cultures with greater than 90% purity were used. For the assessment of uric acid levels, the enzymatic assay was used. Cultured cells were maintained in culture for up to 4 days following medium change. Cell culture medium was collected from the cells at either 1, 2, 3, or 4 days after this. The medium was cleared by centrifugation (14,000 rpm). At the same time, cells were also collected to determine cellular uric acid levels. The cells were washed with three changes of medium and then lysed by addition of 100 Al of double distilled water and physical disruption with a cell scraped. The collected material was centrifuged to remove cellular debris. Protein levels in the cellular supernatant was determined using a BCA assay (sigma) according to the manufacturer’s instructions.

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Cell lines F14 and F21 fusion cell lines were produced by fusing a neuroblastoma cell line with cerebellar neurons from PrPknockout mice and were as previously described (Holme et al., 2003). These cells were maintained in the same medium as for neurons and astrocytes. For experiments, the cells were plated into 24-well trays at 30% confluency. SMB and SMB-PS cells were derived from the brain of a scrapie-infected mouse. The mouse had been infected with the Chandler strain of scrapie (Clarke and Haig, 1970). The resulting cell line was recently cured of the infection by the use of pentosan sulfate (Birkett et al., 2001) to generate an uninfected cell line that served as an appropriate control. The cells were maintained at 378C and 5% CO2 in DMEM medium contain 10% fetal calf serum and 5% newborn calf serum with 1% antibiotics (penicillin/streptomycin). Levels of uric acid in these cell lines was determined in the same way as for primary cell cultures. Toxicity assay Cerebellar neurons were prepared as described above and plated in 24-well trays. The peptide PrP106-126 was applied at 80 AM with or without cotreatment with uric acid. PrP106-126 was prepared as previously described (Brown et al., 1996a). PrP106-126 had the amino acid sequence KTNMKHMAGAAAAGAVVGGLG. The peptide was solubilized in dimethyl sulfoxide (DMSO) at 50 mM and applied to the culture wells directly. An equivalent amount of vehicle was added to the control wells. The peptide was applied as a single dose at the same time as uric acid. Uric acid was also applied to similar cultures in the absence of the peptide. Uric acid on its own had no effect on cell survival (data not shown). After 4-day treatment, the survival of the cerebellar cells was determined. MTT (3, [4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide; Sigma) was diluted to 200 AM in Hanks solution and added to cultures for 30 min at 378C. The MTT formazan product was released from cells by addition of dimethyl sulfoxide (Sigma) and measured at 570 nm in a spectrophotometer (Bio50, Cary). Relative survival in comparison to control treated with the DMSO vehicle could then be determined. Assay of protease resistant PrP SMB cells were plated at 50% confluency per well in 6well plates. Cells were left for 24 h to allow for attachment. The medium was then replaced with fresh medium containing the appropriate dilution of uric acid. Uric acid was reapplied 2 days after this treatment. Four days after the initial treatment, the medium was removed and the protein extracted. Cells were lysed in PBS containing 1% Triton-X 100, 1% Igepal CA-630 for 20 min at 378C. Cell lysates were either placed on ice or treated with 80 Ag/ml proteinase

K for 1 h at 378C. Proteins were concentrated from the total cell lysate by methanol precipitation and the protein pellet resuspended and denatured by boiling for 5 min in 8 M urea. Samples were electrophoresed on a 12% acrylamide gel and transferred electrophoretically to PVDF membrane (Immobilon-P, Millipore). PrP was detected using the primary antibody DR1, as previously described (Brown, 2000), and an HRP conjugated secondary antibody (Daco). Specific protein bands were visualized using ECL Plus chemiluminescent reagent (Amersham Pharmacia Biotech) followed by autoradiography. Autoradiographs were analyzed using Scion Image densitometric software (Scion Corporation). Polymerization assay All measurements were performed on a Cary 100Bio UV-Visible spectrophotometer at 325 nm using a quartz cuvette of 5-mm path length. Recombinant mouse PrP was generated as previously described (Brown et al., 2000). The assay was based on the ability of fibrillar recombinant PrP (the seed) to initiate polymerization of soluble monomeric recombinant PrP (substrate). Substrate recombinant mouse PrP (rPrP) was refolded in the absence of metal ions, and the seed for aggregation was aged manganese refolded recombinant mouse PrP (MnPrP) prepared as previously described (Brown et al., 2000). Briefly, a seed of MnPrP induces immediate aggregation of substrate PrP, observed as an increase in solution turbidity. The resultant scattering of UV light at 325 nm results in an increased absorbance measurement. The abilities of compounds to prevent this turbidity increase were measured, and the results were expressed as a percentage of the turbidity observed with a vehicle control. PrP (50 Ag) and the test compound were preincubated in 500 Al H2O pH 6.5 for 30 min to provide a zero for the measurement. MnPrP (10 Ag) seed from a 400-Ag ml 1 stock was added to the drug/rPrP mixture, and an initial reading was obtained immediately. A second reading was measured after 5 min, and the increase in absorbance over 5 min was recorded.

Results CSF uric acid is reduced in prion disease Uric acid is one of the major soluble nonenzymatic antioxidants in the brain. We examined the levels of uric acid in both the brain and CSF of patients with CJD, vCJD and cattle with BSE using an HPLC-based system linked to an electrochemical detector. Fig. 1A shows the levels of uric acid in the CSF collected from field cases of BSE as compared to controls (n = 10 for both). The range of values was 0.9–1.5 AM for controls and 0.5–1.0 AM for BSE samples. Uric acid in the CSF of BSE cases was significantly (Student t test P b 0.05) lower than in controls. Similar analysis was carried out on samples of frontal cortex

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rosis) and shown in Fig. 2B. The values are shown for both male and female because women usually have lower uric acid levels than men. The controls of Fig. 2B (n = 16 averaging both sexes) showed an average of 34.8 F 4.5 AM and were not significantly different from those of normal patients (36.0 F 2 AM, n = 6, three males and three females). Fig. 2B shows the range of values. Patients with sCJD (male = 37.9 F 4.9 AM, female = 31.0 F 3.5 AM) did not show any significant change in uric acid levels compared to controls (male = 40.8 F 5.6 AM, female = 26.4 F 4.2 AM). However, cases of vCJD showed a

Fig. 1. Uric acid in BSE field cases. Uric acid levels were assessed using an HPLC coupled to an electrochemical detector. (A) CSF, (B) frontal cortex. Shown are the mean and SE for 10 samples each in A and 4 samples each in B.

from BSE-positive cattle compared to controls. Fig. 1B shows that uric acid in frontal cortex was reduced in BSE. In humans, uric acid is the end product of purine metabolism and is excreted from the body. However, humans are unable to express uricase (urate oxidase), an enzyme that converts uric acid to allantoin. Allantoin is the normal end product of purine metabolism in most mammals, and allantoin is excreted in cattle. Uricase is poorly expressed in the brain. Nevertheless, there is a possibility that the differences measured in BSE and control bovine samples are a result of differences in uricase activity. Using an Amplex Red kit, uricase was measured in bovine liver at 47.4 F 5.0 U/mg protein (four samples). Using the same assay, uricase was undetectable in either the CSF or frontal cortex of any bovine samples (four samples each). Uric acid levels were also measured in the CSF from patients with various neurological diseases and compared to that of normal patients. As can be seen in Fig. 2A, uric acid levels were significantly (P b 0.05) decreased in some disorders such as Alzheimer disease and Parkinson disease but increased in disorders that effect the blood–brain barrier. Further analyses of samples from either sCJD or vCJD were carried out in comparison to control samples from patients with other neurological disorders (diagnoses included dementia with Lewy bodies, astrocytosis, and arterioscle-

Fig. 2. Uric acid in human samples from patients with neurological conditions. Uric acid levels were assessed using an HPLC coupled to an electrochemical detector. (A) CSF from various neurological conditions (normal, n = 6; Alzheimer, n = 4; Parkinson, n = 6; BBB = blood–brain barrier disorder, n = 5). (B) CSF from vCJD and sCJD; (C) Frontal cortex of vCJD and sCJD. Shown are the mean and SE for five patients per group.

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symptoms. For each of the four time points, four challenged and four unchallenged animals were sampled. Animals had begun to show early symptoms of BSE before the third time point and had either early or full clinical signs by the last time point. BSE was confirmed in these animals following autopsy. The results indicate a significant (P b 0.05) reduction in uric acid levels in BSE-challenged animals before the development of symptoms. This reduction continued through the rest of the course of the disease at the time points monitored. The enzymatic uric acid assay was used to confirm this finding by comparing the 3-month and 29-month time points (Fig. 3D). Uric acid levels in bovine frontal cortex were also assessed for experimental BSE over a similar time course using the enzymatic assay. The results are shown in Fig. 4. Uric acid levels in frontal cortex began to be significantly lower from 18 months onwards. This result suggests that changes in uric acid levels are an early change in the disease process.

significant (P b 0.05) reduction in the levels of uric acid (male = 22.2 F 2.2 AM, female = 17.4 F 2.5 AM). The implication is that vCJD, unlike sCJD, causes a decrease in CSF uric acid levels. Uric acid levels in samples of frontal cortex of patients with vCJD and sCJD were measured and compared to those with other dementias (as for CSF). There was no significant difference in uric levels between any of the groups (Fig. 2C). The results suggest that BSE and vCJD are similar in that both diseases show a decrease in CSF uric acid. Reduction of uric acid levels in bovine brain in any disease has not previously been reported. Therefore, this finding is worthy of further investigation. Spectrophotometric-based enzymatic assays for uric acid are commercially available. The sensitivity of such an assay was compared to that detected using HPLC and electrochemical detection using uric acid at concentrations found in CSF (Figs. 3A and B). As can be seen, both assays showed the same range of sensitivity. This implies that an enzymatic assay could be used to detect uric acid in CSF. Samples of CSF from cattle orally challenged with BSE were tested in both assays. These were compared to unchallenged age-matched controls. These experimental BSE-challenged animals were sampled at various times after the date of challenge. Analyses of these samples by HPLC analysis (Fig. 3C) gave information on changes in uric acid levels of CSF during the time course of BSE. These measurements therefore indicated the levels of uric acid in BSE-challenged cattle before and after the onset of BSE

Cellular release of uric acid Little is known about which cells produce and release uric acid in the brain. Cell culture experiments were undertaken to determine which cells in the brain produce the most uric acid and determine how PrPc expression influences uric acid expression levels. Primary cultures of cerebellar neurons, purified microglia, and purified astrocytes were produced from wild-type mice and maintained in

Fig. 3. Uric acid levels in a BSE time course. Sensitivity of the HPLC-electrochemical detector was compared to that of an enzymatic assay using a concentration curve (n = 3). The curve for the HPLC method (A) was similar to that for the enzymatic assay (B). Both assays were used to measure uric acid levels in bovine CSF from BSE orally challenge cattle (C, D). The enzymatic assay of the 3-month and 29-month time points (D) gave similar measurements to the HPLC-based assay of the whole time course (C). BSE-infected animals ( ) were compared to unchallenged, age-matched controls (o). Four animals were assessed for each time point except the final time point which was for three animals only. In D, open bars are the control animals, and black bars are the BSEchallenged animals.

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Fig. 4. Uric acid in bovine frontal cortex. The enzymatic assay was used to assess the uric acid levels in extracts of bovine frontal cortex from BSEchallenged ( ) and unchallenged animals (o). The concentrations were determined by comparison to a standard curve of known uric acid concentrations. Shown are the mean and SE for four animals per time point.

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culture. The purity of these cultures was determined according to the criteria in the methods. Following medium change, both the medium and the cells were collected at

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various time points. The medium was cleared by centrifugation, and the levels of uric acid were determined using the enzymatic assay (Fig. 5A and B). The level of uric acid was determined following washing of the cells with at least three changes of fresh medium and lysis with distilled water. The enzymatic uric acid assay was used to assess uric acid levels. The highest cellular levels of uric acid were found in neurons. The levels within astrocytes and microglia were at a similar levels. Accumulation of uric acid in the medium of the cells increased with time. However, microglia produced substantially more uric acid than any other cell type while neurons secreted the least. These results suggest that uric acid present in the CSF is secreted by microglia. To the assess the effect of PrPc expression on uric acid levels, we compared the uric acid produced by two cell lines. These cell lines were produced by the fusion of cerebellar granule cells and neuroblastoma cell lines (Holme et al., 2003). The cerebellar cell line was generated from PrP-knockout mice, and thus the resulting cell line, F14, is deficient in PrPc expression. A similarly generated cell line, F21, has wild-type levels of PrPc expression. Analysis of uric acid expression by these cell lines did not show a significant difference (P N 0.05) in the levels of uric acid within the cells. However, F21 cell secreted significantly

Fig. 5. Uric acid produced from cultured cells. The enzymatic assay was used to determine uric acid concentrations either secreted into culture medium or within the cells. (A) Uric acid within cerebellar neurons (open bars), astrocyte (black bars), or microglia (gray bars). (B) Uric acid levels secreted from neurons (open bars), astrocyte (black bars), or microglia (gray bars) over 4 days. (C) Uric acid within or secreted from two neuronal cell lines differing in prion protein expression levels. F14 lacks PrPc, and F21 express PrPc. Open bars = within F21, gray bars = within F14, hatched bars = secreted by F21, and black bars = secreted by F14. (D) Uric acid levels in cell lines that are constitutively infected with PrPSc (SMB) and the cured line without infection (SMB-PS). Open bars = within SMB-PS, gray bars = within SMB, hatched bars = secreted by SMB-PS, and black bars = secreted by SMB. Shown are the mean and SE for three to four experiments.

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(P b 0.5) more uric acid after 2–4 days in culture (Fig. 5C). However, the amount of uric acid produced by F21 cells was equivalent to neurons and much lower than for microglia. Nevertheless, the implication is that PrPc expression levels could influence uric acid secretion in neuronal-like cells. The SMB cell line (Birkett et al., 2001) was produced from the brains of scrapie-infected mice and maintains scrapie infection. A control cell line SMB-PS was cured of the infection by pentosan sulfate (Birkett et al., 2001). Uric acid levels in these cell lines were equivalent, but that secreted by infected SMB cells was significantly lower than for the uninfected controls (Fig. 5D). This implies that scrapie infection could reduce uric acid release by cells. Uric acid and toxicity Uric acid is an antioxidant. Cell death in prion disease has been linked to oxidative stress. A neurotoxic prion protein peptide, PrP106-126, has been shown to cause neuronal death in culture. Dispersed monolayer cultures of 6-day-old mouse cerebellar granule neurons were prepared. As previously shown, 80 AM PrP106-126 causes a reduction in cell survival of approximately 50%. Assessment of survival was determined using an MTT assay. Neuronal cells in 24-well trays were treated in triplicate. The survival after 4 days was determined by comparison to controls treated with the vehicle only (DMSO). Furthermore, PrP106-126 was applied to cultures at 80 AM and cotreated with uric acid for 4 days at various concentrations. MTT assays for survival were also carried out following these treatments. Fig. 6 shows the effect of uric acid on peptide toxicity. Although uric acid did not block the toxicity

Fig. 7. Uric acid treatment of infected cells. SMB cells were treated with increasing concentration of uric acid (UA) for 4 days. At the end of that time, protein from the cells was extracted and precipitated. Half of the extracted protein was digested with proteinase K (PK). Digested and undigested fractions were run in parallel. After electrophoresis, the protein was Western blotted and PrP detected with a specific antibody (DR1). As can be seen in the upper panel, the majority of PrP produced by SMB cells is generated in a cleaved form even before proteinase K digestion. PK digestion was only effective for sample treated with high amounts of uric acid. The level of PK-resistant protein for SMB cells treated with various concentrations of UA was determined for four separate experiments and compared as a percentage to that produced by untreated cells. Uric acid concentrations of 10 AM and above significantly reduced PK-resistant PrP levels (P b 0.05).

completely, concentrations above 10 AM showed significant protection (Student t test, P b 0.05). Uric acid and scrapie infection

Fig. 6. Uric acid inhibits PrP106-126 toxicity. Survival of cerebellar neurons in the presence of 80 AM PrP106-126 and increasing concentrations of uric acid was determined using and MTT assay after 4 days of treatment. The values were compared to the reduction in survival of cells treated with the peptide alone. Values shown are the percentage inhibition of the peptide’s toxic effect. Shown are mean and SE for four experiments.

SMB cells maintain a standing infection of mousepassaged Chandler scrapie strain. This makes this cell line interesting for testing compounds that could inhibit scrapie infection. We tested the ability of uric acid to inhibit scrapie infection in SMB cells. Uric acid was applied to SMB cells over 4 days. At the end of 4 days, total protein was extracted from the cell and precipitated with methanol, and the resulting protein was treated with proteinase K at 50 Ag/ml for 30 min. Protease-resistant protein was detected after Western blot using antiserum DR1, specific for PrP (Brown, 2000). Comparison of the resulting bands to untreated controls showed that uric acid treatment diminished the levels of PrPSc generated by SMB cells without diminishing the total level of PrP expressed by the cells (Fig. 7). This

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implies that uric acid has a strong effect decreasing the levels of protease-resistant protein generated by SMB cells.

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zation (Fig. 8B). Uric acid showed a significant effect at 10 AM and above (P b 0.05). In comparison, folic acid at 100 AM did not have this effect (data not shown).

Uric acid and prion protein polymerization A polymerization assay was used to assess the effect of uric acid on aggregation of recombinant prion protein. Recombinant mouse prion protein was generated in two forms. The first was a soluble form and the second was an aggregated form, rich in h-sheet structure and proteaseresistant. The second was generated by refolding the protein in the presence of manganese (Brown et al., 2000). This second form of PrP was used to bseedQ the polymerization of soluble PrP. The progress of the reaction could be assessed using a spectrophotometer with and without addition of uric acid (Fig. 8A). It was found that there was a dose-dependent effect of uric acid that limited the extent of this polymeri-

Fig. 8. Uric acid and PrP polymerization. (A) Time course of a single polymerization assay from the point of addition of seeding protein. Baseline for the unpolymerized substrate protein is determined before addition of seeding protein. Addition of seed has no effect on the baseline of the spectrophotometric absorbance. Uric acid is added to the assay before addition of seed. The light line indicates the change in absorbance on addition of the seed protein without uric acid. The dark line is the same reaction in the presence of 100 AM uric acid. (B) Dose response of PrP polymerization to the presence of uric acid. Uric acid was added to the polymerization assay at the concentrations indicated and the extent of polymerization measured. This was compared to a parallel assay where uric acid was not added. The percentage inhibition of polymerization was then determined. Shown are the mean and SE of four separate assays.

Discussion The findings described here support the notion that the CSF level of uric acid is decreased in the two novel prion diseases, BSE and vCJD. Uric acid is now accepted as a major nonenzymatic antioxidant. As there have been numerous reports suggesting that oxidative damage occurs in prion disease (Guentchev et al., 2000; Milhavet et al., 2000; Wong et al., 2001), the finding that a particular antioxidant is decreased is quite significant. Other human neurodegenerative orders that are also characterized by oxidative damage have also been reported to show decreases in CSF uric acid (Stover et al., 1997). This means that decreased uric acid levels are not specific to prion diseases and could not be used to diagnose them on their own. Nevertheless, vCJD levels were the lowest observed in this study, and in comparison to vCJD levels, sCJD levels were not depressed. As demonstrated in this study, a simple commercial assay can be used for assessing uric acid levels in CSF. Therefore, it is possible, subject to further confirmation, that a uric acid assay could be used in parallel with other diagnostic tests. This does not imply that uric acid could diagnose vCJD as uric acid levels were also decreased in Parkinson and Alzheimer diseases (Fig. 2). However, as the majority of vCJD patients are not in the age group susceptible to these diseases, then this assay used in conjunction with other current diagnostic tools might be beneficial in reaching an earlier diagnosis. CSF levels of various proteins and other molecules have been assessed previously to determine if any molecule could be used as a marker for prion disease. Such molecules include ascorbic acid (Arlt et al., 2002), 14-3-3 (Zerr et al., 2000; Laplanche et al., 2000), tau (Clark et al., 2003), and S100 (Beaudry et al., 1999). Of these, only 14-3-3 has any repeatable correlation with diagnosis of CJD (Van Everbroeck et al., 2003). Uric acid levels are influenced by diet, especially levels of meat (Loenen et al., 1990). Although CSF levels are lower than in serum, both have been seen to be affected by diet (Reiber et al., 1993). This implies that the diets of patients with neurodegenerative conditions would have to be closely monitored if uric acid levels were to be studied. As this information is not available for the patients of this study, it is unknown how differences in patient diets might have affected the results in this study. Independently of the findings for human disease, analyses of CSF from BSE-challenged cows suggested that changes in uric acid levels not only occur as a result of BSE challenge but also are manifest before onset of neurological symptoms of the disease. The implication of this finding is that changes in uric acid levels are a potential surrogate marker for BSE. Little is known about uric acid levels in

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cattle and the factors that affect its secretion into the CSF. Therefore, there is no information about other diseases of cattle that might decrease uric acid levels. Little is known about the role of uric acid in the brain, apart from its potential as an antioxidant. Indeed, it is unclear which cells secrete uric acid, the mechanism of secretion, and its regulation. Our findings suggest that the most uric acid is secreted by glial cells. This is logical given that glia are known to have a protective role in many experimental systems. High uric acid levels have been associated with gliosis (O’Neill and Lowry, 1995), supporting our suggestion that uric acid in the CSF is produced by glia. In particular, we have shown that microglia produce the most uric acid. During respiratory burst activity, microglia can produce large amounts of oxygen radicals. Coproduction of uric acid might protect them from their own toxic byproducts. In prion disease, microglia proliferate (Giese et al., 1998). Therefore, it would seem logical to assume that uric acid levels would increase in the brain in proportion to the increase in microglia. As this was not observed, then it is likely that uric acid production by microglia is inhibited in both vCJD and BSE. The second part of this study was to assess the potential of uric acid to inhibit or reduce characteristics of prion diseases, namely, cell death, generation of PrPSc, and protein aggregation. Our findings indicate that uric acid inhibits the toxicity of a prion protein peptide, PrP106-126. This is not surprising as it has been shown before that antioxidants inhibit the toxicity of this peptide (Brown et al., 1996a). Uric acid has been shown to protect neurons in culture from glutamate toxicity and reduce the neuronal damage resulting from ischemic insult (Yu et al., 1998). Uric acid also inhibited both the formation of PrPSc and the aggregation of recombinant protein. Interestingly, the level of uric acid that has these protective effects is equivalent to that found in humans without neurological disease. The most interesting of these findings is the potential of uric acid to decrease the formation of PrPSc by permanently infected cells. Currently, a number of compounds have been shown to inhibit production in infected cells (Birkett et al., 2001; Peretz et al., 2001; Rudyk et al., 2000; Supattapone et al., 2001). However, this is the first time that a compound actually present in the brain has been shown to decrease the levels of PrPSc. The implication of these findings is that drugs that increase uric acid production in the brain could potentially be a valuable resource for treatment of these diseases. Furthermore, diet has been shown to increase uric acid levels (Brule et al., 1992). Therefore, altering patients’ diet by, for example, increasing their meat intake could help the treatment of prion diseases. Despite this, uric acid was unable to completely block polymerization or PrPSc formation. At the lowest concentration detected in humans (17.4 AM), uric acid was able to block formation of PrPSc by 40% and inhibit PrP polymerization by 30% (see Figs. 7 and 8). Therefore, uric acid at this concentration would not inhibit prion disease in the

brain. However, this effect of uric acid was from exogenous application, applied above that produced by the cells (see Fig. 5). The levels of uric acid in the human brain might have an inhibitory effect, and if there was no uric acid present, the progression of vCJD might be more rapid. Although there is currently no evidence that the normal human levels of uric acid diminish the severity of prion disease, a small increase in uric acid levels could be beneficial. In our assay, 10 AM uric acid does have an inhibitory effect. Therefore, if uric acid levels in vCJD patients were raised from 17 to 27 AM or higher then, as suggested by our in vitro assays, the patients might have an improved prognosis. Although we have shown a potential for uric acid to be protective against characteristics of prion disease, our results do not indicate the possible mechanism by which uric acid levels are decreased in prion disease. Both PrPSc-infected cell and cells not expressing PrP have reduced levels of uric acid production. Potentially, the production of PrPSc could inhibit or reduce purine metabolism or inhibit one of the component enzymes such as xanthine oxidase. However, the reduction of uric acid levels in vCJD, but not sCJD, suggests that there are specific effects. Whatever the cause, the mechanism of uric acid production in the brain and its regulation in both health and disease requires further investigation. In addition, although uric acid has been assessed to be a major brain antioxidant, it is also known that high uric acid levels have other effects that might complicate any treatment based on elevating uric acid levels. These other effects include inhibition of Na/K ATPase activity (Bavaresco et al., 2004), induction of the release of proinflammitory proteins (Ryckman et al., 2004), and stimulation of the release of nitric oxide from macrophages (Jaramillo et al., 2004). Many studies have looked at the correlation between uric acid levels and neurological disease. Most of these studies have looked at serum levels. As serum levels are considerably different to those of the CSF, it is unclear as to weather such studies indicate what uric acid is doing to the brain. Some reports concerning serum uric acid levels in stoke patients have suggested that elevated levels may not be protective (Weir et al., 2003) or deleterious (Kanellis and Johnson, 2003) or are associated with higher risk of cardiacrelated death following stroke (Wong et al., 2002). There has been a reported correlation between serum urate levels and stroke in diabetics (Lehto et al., 1998). In contrast, other studies suggest that stroke victims with high serum urate are more likely to make a good recovery from stroke (Chamorro et al., 2002). It is possible that increased uric acid levels in stroke patients are a result of a protective response in those likely to have a stroke rather than something causative. The true value of elevated uric acid levels in neurological conditions needs further investigation. In summary, we have shown a reduction in uric acid produced by the brain and particularly the CSF in BSE and vCJD, but not sCJD. This finding further supports the

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notion of a similarity between vCJD and BSE. In addition, the finding that an antioxidant is reduced in these diseases further supports the notion that deregulation of defense against oxidative stress is important in prion diseases. Potentially, increasing production of uric acid by microglia in the brain could diminish some of the resulting neuronal damage that occurs in prion disease, either by a direct antioxidant effect or by the inhibition of PrPSc formation. The link between uric acid levels and diet should be considered as a possible means to increase uric acid levels in patients with prion diseases.

Acknowledgments The authors thank Dr. J.-M. Burgunder (University of Bern) for CSF from normal patients and some of the CSF samples from patients with neurological conditions other than CJD. Also thanks to the Veterinary Laboratories Agency at Weybridge for bovine samples. DRB is supported by a senior fellowship from the BBSRC of the UK. The research was supported by a grant from DEFRA of the UK (SE1774). Thanks to Kay Sinclair for proofreading the manuscript.

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