Four consistent features characterize the AD brain: a the presence of extracellular amyloid plaques com-prised mainly of aggregated, insoluble amyloid-b Ab peptide; b the presence of int
Trang 1The modulation of metal bio-availability as a therapeutic strategy for the treatment of Alzheimer’s disease
Peter J Crouch1, Anthony R White1and Ashley I Bush2,3
1 Department of Pathology and Centre for Neuroscience, The University of Melbourne, Australia
2 The Mental Health Research Institute of Victoria, Parkville, Australia
3 Department of Psychiatry, Massachusetts General Hospital, Charlestown, MA, USA
Introduction
Alzheimer’s disease (AD) destroys the mental health of
millions of people worldwide Sufferers lose their
inde-pendence and require dedicated on-going care, usually
from members of their own family and at enormous
economic and social cost There is no cure for AD,
and the development of effective therapeutic strategies
is hampered by a paucity of information on the
biolo-gical mechanisms underlying the disease The severity
of AD relates to a multitude of age-related cellular
processes culminating in neuronal and synaptic
dys-function Successful therapeutic strategies that target
the causative neuropathological events will have an enormous impact on treatment of AD patients Four consistent features characterize the AD brain: (a) the presence of extracellular amyloid plaques com-prised mainly of aggregated, insoluble amyloid-b (Ab) peptide; (b) the presence of intracellular neurofibrillary tangles (NFTs) containing hyperphosphorylated tau; (c) increased oxidative damage to lipids, proteins and nucleic acids; and (d) a loss of biometal homeostasis The deposition of aggregated Ab and the hyperphos-porylation of tau have been shown to cause neuronal damage and contribute substantially to the pathology
of AD [1] Therefore, both have received considerable
Keywords
Alzheimer’s disease; amyloid-b; biometals;
copper; iron; oxidative stress; tau; zinc
Correspondence
A I Bush, The Mental Health Research
Institute of Victoria, 155 Oak Street,
Parkville, Victoria 3052, Australia
Fax: +61 39387 5061
Tel: +61 39389 2962
E-mail: abush@mhri.edu.au
(Received 9 March 2007, revised 17 May
2007, accepted 18 May 2007)
doi:10.1111/j.1742-4658.2007.05918.x
The postmortem Alzheimer’s disease brain is characterized histochemically
by the presence of extracellular amyloid plaques and neurofibrillary tangles Also consistent with the disease is evidence for chronic oxidative damage within the brain Considerable research data indicates that these three crit-ical aspects of Alzheimer’s disease are interdependent, raising the possibility that they share some commonality with respect to the ever elusive initial factor(s) that triggers the development of Alzheimer’s disease Here, we discuss reports that show a loss of metal homeostasis is also an important event in Alzheimer’s disease, and we identify how metal dyshomeostasis may contribute to development of the amyloid-b, tau and oxidative stress biology
of Alzheimer’s disease We propose that therapeutic agents designed to modulate metal bio-availability have the potential to ameliorate several of the dysfunctional events characteristic of Alzheimer’s disease Metal-based therapeutics have already provided promising results for the treatment of Alzheimer’s disease, and new generations of pharmaceuticals are being developed In this review, we focus on copper dyshomeostasis in Alzheimer’s disease, but we also discuss zinc and iron
Abbreviations
AD, Alzheimer’s disease; Ab, amyloid-b; APP, amyloid-b A4 precursor protein; CHO, Chinese Hamster Ovary; Cp, ceruloplasmin; CQ, clioquinol; CNS, central nervous system; COX, cytochrome c oxidase; CSF, cerebrospinal fluid; Cu⁄ Zn-SOD, copper ⁄ zinc superoxide dismutase; GSK3, glycogen synthase kinase-3; MMP, matrix metalloproteinase; NFT, neurofibrillary tangles; NMDA, N-methyl- D -aspartate; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; SOD, superoxide dismutase; Tg, transgenic; ZnT3, Zn transporter-3.
Trang 2research attention as potential therapeutic targets
Pla-ques and NFTs, however, cannot be regarded as
‘up-stream’ causative factors in the development of AD
While aggregated, insoluble Ab found within plaques
can cause neurotoxicity, soluble intermediate Ab
oligo-mers are substantially more toxic [2] Similarly, NFTs
can also contribute to neurodegeneration, but their
development is the result of an aberrant shift in
activ-ity of tau kinases and phosphatases [3] Thus, although
targeting plaques and NFTs may ameliorate some of
the consequences of AD and no doubt lessen the
bur-den of the disease, the biological mechanisms that
caused them to develop will remain unchecked The
fundamental research aim in AD research is to identify
the up-stream biological mechanisms that trigger the
development of AD Identifying these mechanisms will
substantially facilitate the development of more
effect-ive therapeutic strategies
Metal dyshomeostasis and AD
Transition metals such as Cu and Fe are essential for
normal cell functionality Due to their capacity to
move between transition states, they are most
abun-dant within redox enzymes such as Cu⁄ Zn-superoxide
dismutase (Cu⁄ Zn-SOD), tyrosinase, and cytochrome c
oxidase (COX) In these enzymes, the redox potential
of the metals is harnessed to provide the enzymes with
their electron transfer capabilities Paradoxically, it is
the same redox potential of the metals that makes
them potentially toxic to the cell Under conditions
where they are allowed to accumulate freely, redox
act-ive metals contribute directly to cellular oxidatact-ive
dam-age by generating the highly reactive and toxic OH•
via Fenton and Haber–Weiss reactions [4] Cells have
therefore developed sophisticated regulation and
trans-fer systems to ensure tight control of metals within the
cell [5,6] The toxic effects of Fe and Cu and their role
in numerous neurodegenerative and age-related
dis-eases have been reviewed recently [7]
Equally detrimental to the cell is metal deficiency
Deficient metal bio-availability causes decreased
activ-ity of critical enzymes because activactiv-ity of the enzymes
is dependent on optimal metal loading Cu deficiency
is central to the recessive Menkes and Wilson diseases,
and has been implicated in AD Just as excess Cu can
contribute to oxidative damage by catalysing the
pro-duction of OH•, so too can deficient Cu by preventing
normal activity of cuproenzymes important in
main-taining cellular oxidative homeostasis
The cortical glutamatergic synapse, where amyloid
pathology first commences in AD, contains
exception-ally high concentrations of Zn and Cu, which are
released during neurotransmission Zn2+ is released together with glutamate from presynaptic terminals to achieve concentrations in the order of 300 lm [8] The
Zn2+ is concentrated into glutamate vesicles by Zn transporter-3 (ZnT3), which is only expressed in gluta-matergic neurons [9] Ionic Cu is released into the cleft following postsynaptic stimulation of the
N-methyl-d-aspartate (NMDA) receptor [10,11] and is concen-trated into postsynaptic vesicles by the Menkes Cu7aATPase [11]
Cu dyshomeostasis is evident within the brain during normal aging [12–15], but is substantially more pro-nounced within the aged AD-affected brain [16,17] Lovell et al [17] demonstrated that Cu levels in the unaffected (i.e plaque-free) neuropil of the AD brain are approximately 400% higher than in the neuropil of the healthy brain, and that within the AD brain itself
Cu levels are approximately 30% higher within the amyloid plaques compared to plaque-free regions These data indicate that an accumulation of Cu within the brain, be it a cause or consequence, is consistent with the development of AD Despite these gross increases in cerebral extracellular Cu, intracellular Cu appears to be deficient in the AD brain [18] The cupro-enzymes COX and peptidylglycine a-amidating mono-oxygenase have significantly decreased activities in AD brain and cerebrospinal fluid (CSF), respectively [19,20], and deficiencies in COX activity may be responsible for the deficit in energy metabolism charac-teristic of AD brain [21] There are conflicting reports
in the literature about levels of the cuproprotein cerulo-plasmin (Cp) in AD brain [22,23], but Cp activity has been reported to be decreased in plasma [24], despite being elevated in CSF [25] and in plasma [26] Further-more, the antioxidant Cu⁄ Zn-SOD shows decreased activity in both AD brain and transgenic (Tg) animal models of AD despite increased protein expression [27] The loss of Cu⁄ Zn-SOD activity likely results from a deficiency in active site Cu because activity can be restored by dietary Cu supplementation [27]
Homeostasis of the transition metal Fe is also altered
in the AD brain It accumulates within extracellular amyloid plaques [17,28] and localizes within NFTs [29] Like Cu, Fe is essential within the cell because of its redox potential, and Fe dyshomeostasis can similarly contribute to cellular dysfunction when in excess (by catalysing OH• production) and when deficient (decreased enzyme activity) Of particular relevance to
Fe dyshomeostasis in AD is Cp As described above, Cp
is a cuproprotein, but its functional role within the cell
is to detoxify and remove excess Fe Decreased Cp levels
in the brain [22], possibly due to decreased Cu homeo-stasis, may contribute to Fe accumulation in AD [30]
Trang 3because Jeong and David [31] have recently shown that
Cp deficiency leads to increased Fe levels in the CNS
By contrast to Cu and Fe, Zn is redox-silent, and
therefore does not contribute directly to redox
reac-tions Its role within the brain, however, is essential
nonetheless It is required for the activity of enzymes
such as Cu⁄ Zn-SOD [32] and matrix
metalloproteinas-es [33] where it is required in a structural role rather
than a redox-active role Perhaps the most critical role
for Zn within the brain is in neurotransmission across
the glutamatergic synapse [34] Within the synaptic
cleft, Zn concentrations can reach approximately
300 lm [35] where it is believed to function as a
coun-ter ion for the high concentrations of glutamate
present and quenches the response of the NMDA
receptor [36] Like Cu and Fe, considerable data
indi-cates a loss of Zn homeostasis in AD Abnormally
high concentrations of Zn are associated with amyloid
plaques in AD brain [17,37,38] and AD Tg mice [39]
In a pertinent study performed in vivo, Lee et al [40]
crossed AD Tg mice (Tg2576) with mice deficient in
ZnT3, the protein responsible for loading Zn into
syn-aptic vesicles for release into the synsyn-aptic cleft These
mice exhibited a 50% decrease in amyloid plaque
bur-den compared to Tg2576 littermates, indicating that
the pool of Zn essential for glutamatergic
neurotrans-mission may contribute to plaque formation The
syn-aptic Zn has recently been demonstrated to be in
communication with the plasma, and contributes to
amyloid congophilic angiopathy, which is abolished in
Tg2576 mice where the gene for ZnT3 is ablated [41]
Amyloid-b
After the 4.5 kDa Ab peptide was identified as a
major component of the amyloid plaques in AD brain
[42,43], global AD research focused on this peptide as
a causative agent in the disease The 39–43 amino acid
cleavage product of the Ab A4 precursor protein
(APP) is initially present as a soluble, unaggregated
species, and it is only through the processes of
olig-omerization, aggregation and fibrilization that Ab
forms amyloid plaques As amyloid plaques are
prom-inent in the postmortem AD brain, early research
the-ories placed the accumulation of extracellular,
insoluble forms of Ab as central to the disease process
[44] However, as studies emerged reporting that
sol-uble, intermediate Ab oligomers were more toxic than
fibrillar Ab [2,45–47], it became evident that, although
amyloid plaques no doubt contribute to neuronal
dys-function, the occurrence of plaques may be several
steps downstream from the more critical causes of
AD
Ab readily binds Cu and Zn via its three N-terminal histidine residues [48–51], and several compelling stud-ies have shown that the interaction between Ab and these metals can promote the formation of Ab oligo-mers, aggregates and fibrils [48,52–55] Metal mediated oligomerization of Ab may therefore contribute to the potent inhibition of synaptic transmission mediated by
Ab Several studies have now shown that Ab mediated inhibition of synaptic transmission is dependent on the presence of Ab oligomers, and that Ab monomers are relatively nontoxic in these assays [56,57] As described above, synaptically released Zn is required for the for-mation of amyloid plaques in Tg mice [41] Although amyloid plaques contain predominantly higher order
Ab aggregates, an initial formation of toxic Ab oligo-mers within the synaptic cleft may be determined by
Zn released from the presynaptic terminus Similarly,
Cu released into the synaptic cleft following activation
of postsynaptic NMDA receptors [10,11] may also facilitate extracellular Ab oligomerization Shankar
et al [58] have reported that the loss of hippocampal synapses in rat organotypic slices is mediated by Ab oligomers and is dependent on the activity of NMDA-type glutamate receptors
The concentrations of metal required to induce Ab oligomerization and aggregation are relatively low, and well within the physiological ranges that could be expected within the brain The capacity for metals to facilitate this process may therefore be a critical factor
in the Ab mediated pathology of the AD brain Subse-quent to an early report demonstrating that the Cu- and Zn-induced aggregation of Ab could be pre-vented by EDTA [53], disrupting Ab–metal interac-tions has been an attractive therapeutic target In this regard, a salient study demonstrated that the amyloid plaque burden in brains decreased by 49% when Tg2576 mice were treated orally with the 8-hydroxy-quinoline derivative clioquinol (CQ) [59] CQ is a moderate metal chelator capable of crossing the blood–brain barrier, and it was believed that CQ solu-bilized the Ab plaques by stripping them of their metal content This supported the in vitro work previously reported [53], and was consistent with the notion that excess extracellular metals contribute to the amyloid pathology of AD
Relative to the soluble Ab burden of the AD brain,
a recent study described a mechanism by which decreased intracellular metal bioavailability may con-tribute to the accumulation of soluble Ab outside the cell White et al [60] treated Chinese Hamster Ovary cells over-expressing human APP (CHO-APP) with
CQ complexed to Cu or Zn, and found that the metal-CQ treatment substantially decreased the levels
Trang 4of soluble Ab present in the cell culture medium.
Metal-CQ treatment decreased the levels of
extracellu-lar Ab not by preventing an Ab–metal interaction
outside the cell, but by facilitating the delivery of
metals into the cell White et al [60] demonstrated
that CQ facilitated the delivery of Cu and Zn across
the plasma membrane, as determined by inductively
coupled plasma mass spectrometry Once inside the
cell, Cu and Zn, but not Fe, activated
phosphoinosi-tol 3-kinase mediated protein kinase pathways, which
ultimately led to an increase in the secretion of matrix
metalloproteinases (MMPs) The capacity for MMPs
to degrade Ab has been reported by several groups
[61–64] Treatment with CQ may therefore have a
two-fold effect on Ab; by binding extracellular metals,
it prevents metal mediated Ab aggregation and toxicity
and, by then delivering the bound metals into the cell,
it activates specific protein kinases that induce an
increase in the production of Ab-degrading MMPs
Neurotoxicity generated by the interaction between
Ab and metals may be more complex than the
cata-lysis of Ab aggregation Numerous studies have now
shown that several potential mechanisms of
neurotox-icity for soluble Ab are exacerbated by, if not
depend-ent on, the presence of metals This indicates that
Ab–metal interactions, possibly occurring within the
cell, may induce mechanisms of neurotoxicity that
involve soluble Ab oligomers, and that the mechanisms
of toxicity precede Ab aggregation and accumulation
Curtain et al [49,65] demonstrated that the capacity
for Ab to bind Zn and Cu determined its ability to
penetrate and disrupt membranes; Crouch et al
demonstrated that Ab-mediated inhibition of
cyto-chrome c oxidase requires the presence of at least
equimolar concentrations of Cu [66], and that the
inhibition was not supported by Zn or Fe [67]; and
Huang et al [68] demonstrated that the potential for
Ab to generate neurotoxic H2O2 is dependent on the
presence of Cu These metal-mediated toxic effects of
Ab were abolished by preventing the Ab–metal
interac-tion with chelators such as EDTA, raising the
possibil-ity that therapeutics designed to disrupt Ab–metal
interactions may prevent more than Ab aggregation
and plaque formation
Tau
The native function of the microtubule-associated
pro-tein tau is to maintain integrity of the cytoskeleton by
promoting assembly and stability of microtubules Tau
isolated from a healthy brain is partially
phosphorylat-ed [69,70], indicating that the normal function of tau
requires some phosphorylation However, in AD, tau
is hyperphosphorylated, and hyperphosphorylated tau
is the form that aggregates in NFTs [71] The loss of functional tau from the microtubule network can be compensated for by the other microtubule-associated proteins, MAP1A⁄ MAP1B and MAP2 It is the toxic gain of function exhibited by hyperphosphorylated tau that renders it most harmful towards the cell Hyper-phosphorylated tau is capable of sequestering normal tau as well as MAP1A⁄ MAP1B and MAP2 [72,73], and this compounding loss of essential proteins desta-bilizes the microtubule network, contributing to neuro-fibrillary degeneration
Tau hyperphosphorylation occurs because of an imbalance in the activity of tau kinases and phospha-tases [3] One particular tau kinase pertinent to metal dyshomeostasis in AD is glycogen synthase kinase-3 (GSK3) GSK3 has recently been implicated as a crit-ical kinase involved in the hyperphosphorylation of tau [74] Only active (nonphosphorylated) GSK3 con-tributes to tau hyperphosphorylation, and Plattner
et al [74] demonstrated that the negative regulation of GSK3 (i.e its phosphorylation) is lost in aged, but not young, Tg p25 mice This loss of regulation resulted in
an increase in GSK3 activity and tau hyperphosphory-lation Whether the change in GSK3 regulation in these mice occurred in response to an age-related decline in intracellular metals was not examined, but the study by White et al [60], described above, provi-ded evidence for a possible connection When the bio-availability of intracellular Cu and Zn was increased in CHO-APP cells by treating with CuCQ or ZnCQ complexes, a downstream target of the activated protein kinase pathways was GSK3 By contributing
to an increase in GSK3 phosphorylation, this metal mediated effect therefore decreased potential phos-phorylation of tau by GSK3 The study of White et al [60] did not present data on tau phosphorylation, but the possibility that metal mediated modulation of GSK3 represents a strong candidate therapeutic target for preventing tau hyperhosphorylation has been strengthened by a recent study Malm et al [75] treated AD Tg mice with the Cu ligand pyrrolidine dithiocarbamate (PDTC) and reported that the treat-ment increased brain Cu levels and activated the same protein kinases previously reported by White et al [60] Treatment with PDTC led to an increase in GSK3 phosphorylation and a substantial decrease in tau phosphorylation [75] Therapeutic modulation of metal bio-availability, such as that described by White
et al [60] and Malm et al [75], may therefore repre-sent a potential therapeutic strategy for preventing the tau hyperphosphorylation and NFT formation characteristic of AD
Trang 5Oxidative stress in AD
A significant, consistent feature of AD is that the
affected brain is under chronic oxidative stress An
early report in 1986 [76] described an increase in
activ-ity of enzymes from the hexose monophosphate
path-ways in postmortem AD brain samples compared to
age-matched controls, and proposed that this reflected
increased oxidative stress in the AD brain Numerous
reports have since provided direct data to show
exten-sive oxidative damage in the AD brain [77]
Oxidative-ly damaged lipids, proteins and nucleic acids have all
been reported [78–80]
A critical factor in oxidative stress within the AD
brain is intracellular Cu Insufficient intracellular Cu
can contribute to an increase in oxidative stress
(des-cribed above), and several lines of evidence indicate
that intracellular Cu deficiency in AD may involve Ab
and APP Ab and its precursor APP both bind Cu,
and over-expression of a C-terminal fragment of APP
or full length APP, both containing the Ab domain,
results in an overall decrease in Cu within the brain of
Tg mice [81] Conversely, APP knockout mice show a
40% increase in Cu levels within the cerebral cortex
[82] Furthermore, APP gene expression is
down-regu-lated by decreased availability of intracellular Cu [83]
and up-regulated by increased availability of Cu [84]
Collectively, these data present a strong case for
the native role of APP⁄ Ab in regulating intracellular
Cu: Cu alters APP gene expression [83,84], and the
APP⁄ Ab produced binds then transports Cu out of the
cell However, once intracellular Cu levels become too
low, possibly because of the aberrant increase in Ab
production consistent with AD, the antioxidant
capa-city of the cell may be compromised, leading to an
increase in oxidative stress The study by Busciglio
et al [85] in this regard is of particular relevance
These authors demonstrated that an increase in
oxida-tive stress alters APP processing and generates an
increase in Ab production If an oxidative
stress-induced increase in Ab production promotes excess Cu
transport out of the cell, further oxidative stress due to
deficient cellular Cu may be created, therefore creating
a vicious cycle Support for this possibility was
presen-ted in a recent review [86]
In addition to promoting oligomerization and
aggre-gation, interactions between Cu and Ab result in free
radical generation in vitro Synthetic Ab reduces Cu(II)
to Cu(I) with subsequent reduction of O2giving rise to
H2O2[14] H2O2 is itself toxic and can diffuse through
the cell membrane to oxidize lipids and intracellular
pro-teins However, greater oxidative damage is induced
when H2O2 interacts with Ab-bound Cu(I) resulting in
OH• [15] OH• reacts with lipids, proteins and nucleic acids, resulting in extensive modifications that are often irreversible and impede normal cellular turnover of these components Furthermore, OH• interaction with
Ab itself can increase Ab aggregation through the di-tyrosine-mediated cross-linking of Ab peptides [16–18] This is consistent with the high di-tyrosine con-tent observed in AD brain tissue [17]
Oxidative stress within the AD brain is also closely related to tau hyperphosphorylation For example, Gomez-Ramos et al [87] demonstrated that the pres-ence of acrolein, a peroxidation product from arachi-donic acid, induces considerable tau phosphorylation and, in a subsequent review article, this group proposed that tau hyperphosphorylation and the formation of NFTs may even represent a normal, protective cellular response to increased oxidative stress [88] Further-more, protein kinase signalling pathways sensitive to oxidative stress and known to be altered in AD have been implicated in the phosphorylation of tau [89] Such data indicate that an increase in cellular oxidative stress, be it through the generation of products of oxi-dative damage or the activation of specific cell signal-ling pathways, leads to tau hyperphosphorylation and NFT formation
Summary
Oxidative stress, tau hyperphosphorylation and the Ab biology of AD are all intricately linked, and consider-able research data now exist to indicate that they inter-act in a series of dysfunctional mechanisms that can ultimately lead to cognitive decline The early event(s) that initiates this neurodegenerative cycle has not been
Fig 1 Potential relationship between decreased intracellular metal bio-availability and the oxidative stress, tau hyperphosphorylation and extracellular Ab accumulation characteristic of AD.
Trang 6established, but the role for metal dyshomeostasis in
all aspects is clear In the AD affected brain, metal
dyshomeostasis is evident in the form of a substantial
increase in the levels of extracellular metals and a
decrease in the levels of intracellular metals Here, we
have presented evidence to show that decreased metal
bio-availability within the cell is consistent with
increased oxidative stress, a loss of regulation of Ab
production, and an increase in tau
hyperphosphoryla-tion Furthermore, an increase in extracellular metals
can catalyse Ab oligomerization and aggregation, and
the amyloid plaques that subsequently form may then
exacerbate intracellular metal deficiency by
sequester-ing metals outside the cell Figure 1 summarizes these
interdependent dysfunctional events With the loss of
biometal homeostasis placed central to all of these
AD-related neurodegenerative mechanisms, the
modu-lation of metal bio-availability has strong potential in
the therapeutic treatment of AD
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