BSE and vCJD cause disturbance to uric acid levels

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Experimental Neurology

BSE and vCJD cause disturbance to uric acid levels

Tamuna Lekishvilia, Judyth Sassoona, Andrew R. Thompsetta, Alison Greenb,

James W. Ironsideb, David R. Browna,*

aDepartment of Biology and Biochemistry, University of Bath, Claverton Dow, Bath, BA2 7AY, UKbNational 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

0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2004.07.002

* Corresponding author. Fax: +44 1225 826 779.

E-mail address: [email protected] (D.R. Brown).

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

190 (2004) 233–244

T. Lekishvili et al. / Experimental Neurology 190 (2004) 233–244234

prion-induced neuronal death. This confirms previous

findings that showed oxygen radicals produced by PrPSc-

activated 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 antioxi-

dants in the brain during prion disease. There has been

some investigation of cellular antioxidants in prion disease,

but currently, little is known about nonenzymatic antioxi-

dants. 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 proper-

ties (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 BSE-

infected 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 (meanF SD: 30.5F11.3) 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 cri-

teria (Kretzschmar et al., 1996). The control group consisted

of patients with suspected CJD referred to the NCJDSU in

T. Lekishvili et al. / Experimental Neurology 190 (2004) 233–244 235

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 ence-

phalitis, 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 high-

performance 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 peroxi-

dase, reacts stoichiometrically with the Amplex Red reagent

(10-acetyl-3,7-dihydroxyphenoxazine) to generate the red-

fluorescent oxidation product, resorufin, having an absorp-

tion 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-day-

old 129SV mice (Harlen). Cultures were prepared as

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, strepto-

mycin; 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 con-

tained 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 (anti-

GFAP, 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 super-

natant was determined using a BCA assay (sigma) accord-

ing to the manufacturer’s instructions.


Cell lines

F14 and F21 fusion cell lines were produced by fusing a

neuroblastoma cell line with cerebellar neurons from PrP-

knockout 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 AMin 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 6-

well 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 lysateswere 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 (Immo-

bilon-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 chemilumi-

nescent 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 recombi-

nant 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 measure-

ment. 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

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.

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-

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 AMand 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


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 enzy-

matic 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

Fig. 3. Uric acid levels in a BSE time course. Sensitivity of the HPLC-electr

concentration curve (n = 3). The curve for the HPLC method (A) was similar to th

levels in bovine CSF from BSE orally challenge cattle (C, D). The enzymatic assay

the HPLC-based assay of the whole time course (C). BSE-infected animals (.) we

assessed for each time point except the final time point which was for three anima

challenged animals.

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.

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 astro-

cytes were produced from wild-type mice and maintained in

ochemical detector was compared to that of an enzymatic assay using a

at for the enzymatic assay (B). Both assays were used to measure uric acid

of the 3-month and 29-month time points (D) gave similar measurements to

re compared to unchallenged, age-matched controls (o). Four animals were

ls only. In D, open bars are the control animals, and black bars are the BSE-

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 BSE-

challenged (.) 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.


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

Fig. 5. Uric acid produced from cultured cells. The enzymatic assay was used to

within the cells. (A) Uric acid within cerebellar neurons (open bars), astrocyte (blac

(open bars), astrocyte (black bars), or microglia (gray bars) over 4 days. (C) Uric a

expression levels. F14 lacks PrPc, and F21 express PrPc. Open bars = within F21

secreted by F14. (D) Uric acid levels in cell lines that are constitutively infected wi

within SMB-PS, gray bars = within SMB, hatched bars = secreted by SMB-PS, and

experiments.

various time points. The medium was cleared by centrifu-

gation, 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

determine uric acid concentrations either secreted into culture medium or

k bars), or microglia (gray bars). (B) Uric acid levels secreted from neurons

cid within or secreted from two neuronal cell lines differing in prion protein

, gray bars = within F14, hatched bars = secreted by F21, and black bars =

th PrPSc (SMB) and the cured line without infection (SMB-PS). Open bars =

black bars = secreted by SMB. Shown are the mean and SE for three to four

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


(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. 6. Uric acid inhibits PrP106-126 toxicity. Survival of cerebellar

neurons in the presence of 80 AM PrP106-126 and increasing concen-

trations 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.

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

SMB cells maintain a standing infection of mouse-

passaged 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


implies that uric acid has a strong effect decreasing the

levels of protease-resistant protein generated by SMB cells.

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 protease-

resistant. 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.

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).

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


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), support-

ing 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. Coproduc-

tion of uric acid might protect them from their own toxic by-

products. 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 concen-

tration detected in humans (17.4 AM), uric acid was able to

block formation of PrPSc by 40% and inhibit PrP polymeri-

zation 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 macro-

phages (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 consid-

erably 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 cardiac-

related 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


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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BSE and vCJD cause disturbance to uric acid levels

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