Metal Toxicity and Therapeutic Intervention

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					Biochemical Society Transactions (2002) Volume 30, part 4

      Metal Toxicity and Therapeutic Intervention
                                               Copper and prion diseases
                                                    D. R. Brown1
                     Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.

Abstract                                                                    include BSE, transmissible mink encephalopathy
Transmissible spongiform encephalopathies are                               and chronic wasting disease (CWD) which is a
diseases of animals and humans that are also                                sporadic disease of deer and elk. Mice and ham-
termed prion diseases. These diseases are linked                            sters are commonly used as experimental models
together because a normal brain glycoprotein                                of TSEs but these animals do not have naturally
termed the prion protein is converted to a readily                          occurring prion diseases. Additionally, there are
detectable protease-resistant isoform. There is                             no known prion disease for vertebrates other than
now strong evidence to suggest that apart from                              mammals.
this difference in resistance a major difference                                   TSEs have long incubation periods in which
between the isoforms is that the normal prion                               there are few observable changes in behaviour or
protein binds copper and has an anti-oxidant                                any clear pathophysiology [2]. Symptoms include
function. Brains from Creutzfeldt–Jakob disease                             motor changes, loss of balance and eventually
patients and brains from mice with experimental                             dementia. Neuronal death and gliosis also follow
mouse scrapie have been shown to have changes in                            leading to the behavioural changes. Deposition of
the levels of both copper and manganese. There is                           PrPSc in the form of extracellular aggregates and
growing evidence that links prion diseases to                               sometimes plaques precedes these changes. Also
disturbances of metal metabolism.                                           typical of these diseases is the vaculation that
                                                                            occurs in the nervous tissue leading to the spongi-
                                                                            form changes [3].
                                                                                 Since the 1980s, research on TSEs has fo-
History of copper and transmissible                                         cused on the role of the prion protein in these
spongiform encephalopathies (TSEs)                                          diseases [4], it is now mostly accepted that the
Prion diseases include a broad range of diseases                            abnormal isoform of the protein generated in
of both animals and humans. These include                                   the disease (PrPSc) is at least a major part of the
the human diseases Creutzfeldt–Jakob disease                                causative agent if not the sole cause of the disease.
                                                                            Transmission of the disease between individuals
(CJD; a sporadic disease), Gerstmann–Straussler–
Scheinker syndrome (a group of inherited dis-                               seems to require PrPSc but what else is required
eases), fatal familiar insomnia (inherited), Kuru (a                        besides the protein is unknown. In 1982, Stanley
disease spread by the eating of human brains),                              Prusiner proposed the protein-only hypothesis
vCJD (the variant form of CJD; cause unknown)                               with which he suggested that PrPSc could act on its
and iatrogenic CJD (caused by transplanting or                              own to transmit prion diseases [5]. However, most
injecting CJD-infected material) [1]. Inherited                             prion diseases are sporadic, meaning they occur
prion diseases, which also include familal CJD, are                         spontaneously with no apparent transmission and
all associated with the point mutations of in-                              no known cause. One must ask what is the rele-
sertions into the human gene (prnp) that encodes                            vance of this hypothesis if the majority of such
the prion protein (PrP). Animal diseases include                            diseases involve no act of transmission between
scrapie, a sheep disease so named because the sick                          individuals? Quite clearly, sporadic prion diseases
sheep scrape themselves. Other animal diseases                              arise because some factor potentiates the con-
                                                                            version of the normal prion protein (PrPc) ex-
                                                                            pressed by neurones into PrPSc. Once generated
                                                                            PrPSc can cause the conversion of further PrPc
Key words : antioxidant, Creutzfeldt–Jakob disease, prion, scrapie.         into PrPSc. Therefore the molecular mechanisms
Abbreviations used : CJD, Creutzfeldt–Jakob disease ; vCJD, variant         that initially cause the generation of PrPsc lie at the
form of CJD ; PrPc, normal prion protein ; PrPSc, abnormal isoform          very heart of understanding prion diseases.
of prion protein ; TSE, transmissible spongiform encephalopathy ;
RML, Rocky Mountains Laboratory scrapie strain ; SOD, super-
                                                                                 In recent years the most important molecular
oxide dismutase.                                                            advance in understanding the prion protein and its
  E-mail : bssdrb!                                                relation to prion disease (TSEs) has been the

# 2002 Biochemical Society                                            742
                                                        Biometals 2002 : Third International Biometals Symposium

investigation of the protein’s ability to bind cop-               In 1992 Pan and colleagues reported a method
per. Initially the reports of Hornshaw and col-              of purification of prion protein from the hamster
leagues [6,7] from 1995 were dismissed because               brain [15]. The technique was based on immobi-
the affinity constants for the binding of copper to            lized metal affinity chromatography. In this tech-
octameric repeats or hexameric repeats of mam-               nique copper was immobilized on a column
malian and chicken prion protein were too low for            and the copper-binding proteins were trapped on
the protein to be accepted as being a genuine                the copper. Selective elution with salt led to the
copper-binding protein. However, since 1997                  isolation of the prion protein. It was also found
when the present author and colleagues in Toronto            that metabolically cleaved prion protein lacking
and Germany reported that prion protein knock-               the N-terminus did not bind to this column.
out mice have disturbances that could be related to               The work of Martin Hornshaw and colleagues
copper metabolism [8] an increase in the study of            began the new wave of interest in copper and
the interaction between copper and the prion                 the prion protein [6,7]. This was driven by the
protein has flourished. It is now possible to access          historical link between the work of Kimberlin at
75 citations of papers on PubMed when ‘ copper               Compton and the interest in this link that re-
AND prion ’ or ‘ copper AND scrapie ’ is used as a           mained at the Institute of Animal Health. These
search term. Additionally, a number of reports               two small studies using synthetic peptides based
have also shown that the true affinity for copper is           on the octameric and hexameric repeats identified
indeed that expected for a copper-binding protein            that copper, but no other metal, interacted weakly
[9]. However, copper and prion diseases have been            with the peptides. That same year (1995) the
associated, if coincidentally, since they were first          present author began experiments to examine
described.                                                   whether the octameric repeat region could inhibit
     Creutzfeldt’s original reports of patients with         the toxicity of copper to neurones. This turned out
what was later termed CJD included a case which              to be the case. In parallel David Westaway’s group
later turned out to be Wilson’s disease. Wilson’s            began to examine the affinity of copper for a larger
disease is a neurological condition linked to                fragment of the prion protein [8]. The resulting
mutations in a copper-transporting ATPase. The               collaboration mediated by Hans Kretzschmar led
pathology in the brain caused by the disease                 to the publication of the first important paper
often results in spongiform changes.                         addressing the issue of copper and its relation to
     CWD, the TSE of deer and elk, was originally            prion protein function.
believed to be caused by copper deficiency [10].
Deficiencies in trace elements such as copper and
selenium have long been known to cause neuro-                Copper and prion protein
logical or other diseases in animals. It has been            There is now considerable evidence from many
known for some time that particular regions of the           independent laboratories that the prion protein
world with low levels of copper are associated with          binds copper. There has emerged considerable
scrapie. The regions of Colorado where CWD was               evidence for two kinds of interaction with the
first identified are known to be regions where                 prion protein molecule. First is the interaction
copper supplementation was necessary.                        of between one and four atoms of copper with
     Researchers looking at both scrapie and the             the octameric repeat region [16]. Secondly is the
toxicity of a copper chelator, cuprizone, noticed a          interaction of copper with sites outside this region
similarity between the two diseases in the                   [9]. Although there is evidence that both inter-
1960s–1970s. At that time the prion protein was              actions cause conformational change there is only
unknown [11,12]. However, biochemists includ-                consistent evidence of beneficial change for inter-
ing Kimberlin identified a number of similarities,            action with the octameric repeat region. There
including the level of metabolites that were similar         is now evidence in the literature that the copper
between cuprizone toxicity and scrapie [13,14].              binds at the octameric repeat region with a low
Unfortunately at that time experiments combining             femtomolar affinity [9]. Additionally we have
cuprizone and scrapie failed to identify any sig-            shown that native PrPc can be isolated with three
nificant effect or relationship. The discovery of the          copper atoms bound [17]. Our studies with native
scrapie prion protein by Prusiner and colleagues             protein showed that when we cultured neurones
changed the face of research into these diseases             with increasing concentrations of copper we were
and such ‘ woolly science ’ became outdated with             able to raise the amount of copper bound to four
the rapid advance of molecular biology.                      atoms per molecule. The only way we could

                                                       743                           # 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 4

increase copper binding to the native protein was             neuroblastoma cells transfected to express high
by denaturation in the presence of urea and                   levels of the prion protein are more resistant to
refolding the protein with high concentrations of             both copper toxicity and superoxide toxicity.
copper. Using this method we could fill a fifth site.           When those same cells are infected with mouse
In the culture model it is possible that the fifth             scrapie this increased resistance is lost. This
atom was lost during the purification process.                 suggests that conversion of PrPc to PrPSc causes a
However, given the high affinity for this site                  loss of antioxidant protection for the infected cells.
proposed by Jackson et al. [9] this seems unlikely.                A recently completed study examined the
Studies with recombinant protein also tentatively             metal content of mice infected with scrapie [21].
suggest a fifth site outside the octameric repeat re-          The Rocky Mountains Laboratory scrapie strain
gion. However, deletion of the octameric repeat               (RML)-induced prion disease has been studied by
region abolishes the majority of copper-binding               many groups and the time course of the disease is
sites and the fifth site is again only occupied when           well characterized. Brain, liver, muscle and blood
the protein is refolded in the presence of milli-             from RML-infected and control mice of similar
molar concentrations of copper. Recent reports                age were taken and mass spectroscopy techniques
about the location of the fifth site currently suggest         were used to determine the metals in those tissues.
two possibilities. One is that reported in the                We found a decrease in copper in the brains of
publications by Robert Cappai and colleagues                  mice that reached a maximum at the onset of
[17a] and Jackson et al. [9]. The other is suggested          clinical symptoms. At the same time there was an
to be somewhere in the C-terminal domain of the               increase in the level of manganese but other metals
protein as suggested by Cereghetti et al. [18].               did not change. In the liver there was only an
     The binding of copper to the prion protein               increase in copper but no change in any other
may have a number of consequences biologically.               metal examined. There was a small increase in
The first is that the prion protein can aid copper             blood copper after onset of clinical symptoms but
uptake into cells [19]. Synaptosomal fractions                there was an elevation of manganese that occurred
from the brains of mice have increased levels of              within the first 30–60 days after the intra-
copper compared with those of prion protein-                  cerebrallar injection with the RML inoculum.
knockout mice [8]. Wild-type synaptosomal frac-               Muscle showed some elevation in manganese but
tions of cells rapidly lose copper within minutes of          no other change. We also examined the SOD-like
isolation, unlike those from prion protein-knock-             activity of prion protein purified from the brains of
out mice. Additionally the presence of copper                 the infected mice and compared it with controls.
bound to prion protein as expressed by cells in               There was a considerable loss of SOD activity
culture protects them against oxidative damage.               from the protein after 60 days’ post-infection.
Purified protein has been shown by us to have an               This loss of activity was accompanied by a change
anti-oxidant activity similar to that of a superoxide         in the metal occupancy of the purified protein with
dismutase (SOD) [20]. The importance of this in               copper being lost from the protein and a sub-
vivo, depends on the amount of copper bound                   stitution with manganese occurring Other anti-
to the protein. Further, we now have evidence that            oxidant proteins such as Cu\Zn SOD also showed
the binding of copper to the prion protein might              decreased activity during the incubation period.
inhibit abnormal interactions of the prion protein                 A study on metals and prion protein from the
with other proteins such as plasminogen and                   brains of CJD patients also showed similar
plasminogen-activating factors. The single under-             changes [22]. These changes included reduced
lying theme of all this evidence is that the binding          levels of copper and increased levels of manganese.
of copper to the prion protein has a protective               Additionally prion protein purified from the same
effect. Given the high localization of the prion               CJD patient brains also lacked the SOD activity
protein with the synapse, it is now reasonable to             that could be detected in purified PrPc from the
conclude that the function of the prion protein is            brains of control patients [22]. Therefore metal
to protect the synapse.                                       changes occur in both experimental and human
                                                              prion diseases. The reason for these changes and
                                                              their relationship to both the pathology in the
Copper and disease                                            diseases and conversion of PrPc to PrPSc have yet
Cultured cells from mice lacking the expression of            to be determined. However, it has been shown that
the prion protein show an increased sensitivity to            the substitution of manganese for copper causes
the toxicity of both copper and superoxide. N2A               conversion to a protease-resistant isoform [16].

# 2002 Biochemical Society                              744
                                                                       Biometals 2002 : Third International Biometals Symposium

     These results show that changes in anti-                               11  Pattison, I. H. and Jebbett, J. N. (1971) Nature (London)
oxidant defence may occur during prion disease.                                 230, 115–117
                                                                            12 Pattison, I. H. and Jebbett, J. N. (1971) Res. Vet. Sci. 12,
However, in particular, changes in the metabolism
of the metals copper and manganese may lie at the                           13 Kimberlin, R. H., Collis, S. C. and Walker, C. A. (1976)
heart of these diseases.                                                        J. Comp. Pathol. 86, 135–142
                                                                            14 Kimberlin, R. H. and Millson, G. C. (1976) J. Comp. Pathol.
                                                                                86, 489–495
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     J. Vet. Med. Sci. 58, 849–854                                          Received 25 January 2002

       Toxicity of myoglobin and haemoglobin : oxidative stress in patients with
                    rhabdomyolysis and subarachnoid haemorrhage
             B. J. Reeder*1, M. A. Sharpe†, A. D. Kay‡, M. Kerr§, K. Moore¶ and M. T. Wilson*
*Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K., †Department
     of Neurochemistry, Institute of Neurology, Queen Square, London WC1N 3BG, U.K., ‡Department of
Neurosurgery, Institute of Neurological Science, Southern General Hospital, Glasgow G53 4TF, U.K., §Department
 of Neurosurgery, University of Pittsburgh, Pittsburgh, PA, U.S.A., and ¶Department of Medicine, Royal Free and
                           University College Medical School, London NW3 2QG, U.K.

Abstract                                                                    haem–protein cross-linked myoglobin in the urine
Haemolytic events, such as those following                                  of patients suffering from rhabdomyolysis and
rhabdomyolysis and subarachnoid haemorrhage,                                haem–protein cross-linked haemoglobin in the
often result in pathological complications such                             cerebrospinal fluid of patients following sub-
as vasoconstriction. Haem–protein cross-linked                              arachnoid haemorrhage. These findings provide
myoglobin and haemoglobin are generated by                                  strong evidence that these respiratory haem pro-
ferric-ferryl redox cycling, and thus can be used                           teins can be involved in powerful oxidation
as markers of oxidative stress. We have found                               processes in vivo. We have previously proposed
                                                                            that these oxidation processes in rhabdomyolysis
                                                                            include the formation of potent vasoconstrictor
Key words : cerebrospinal fluid, haem proteins, haem–protein                molecules, generated by the myoglobin-catalysed
cross-linking.                                                              oxidation of membranes, inducing nephrotoxicity
Abbreviations used : CSF, cerebrospinal fluid ; Hb, haemoglobin ;           and renal failure. Haem–protein cross-linked hae-
Mb, myoglobin ; Hb-H and Mb-H, haem–protein cross-linked form
of Hb and Mb respectively ; SAH, subarachnoid haemorrhage.
                                                                            moglobin in cerebrospinal fluid suggests that a
  To whom correspondence should be addressed (e-mail                        similar mechanism of lipid oxidation is present
reedb!                                                         and that this may provide a mechanistic basis for

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