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Metal complexing agents as therapies for Alzheimer’s disease
Neurobiology of Aging 23 (2002) 1031–1038
Metal complexing agents as therapies for Alzheimer’s disease Ashley I. Bush∗ Oxidation Disorders Research Unit, Mental Health Research Institute of Victoria, University of Melbourne, 155 Oak Street, Parkville, VIC 3052, Australia Received 7 March 2002; received in revised form 25 July 2002; accepted 6 August 2002
Abstract Modern research approaches into drug development for Alzheimer’s disease (AD) target -amyloid (A) accumulation in the brain. The main approaches attempt to prevent A production (secretase inhibitors) or to clear A (vaccine). However, there is now compelling evidence that A does not spontaneously aggregate, but that there is an age-dependent reaction with excess brain metal (copper, iron and zinc), which induces the protein to precipitate into metal-enriched masses (plaques). The abnormal combination of A with Cu or Fe induces the production of hydrogen peroxide, which may mediate the conspicuous oxidative damage to the brain in AD. We have developed metal-binding compounds that inhibit the in vitro generation of hydrogen peroxide by A, as well as reverse the aggregation of the peptide in vitro and from human brain post-mortem specimens. Most recently, one of the compounds, clioquinol (CQ; a USP antibiotic) was given orally for 9 weeks to amyloid-bearing transgenic mice, and succeeded in markedly inhibiting A accumulation. On the basis of these results, CQ is being tested in clinical trials. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Amyloid; Alzheimer’s disease; Copper; Zinc; Oxidation; Hydrogen peroxide; Superoxide dismutase
1. Introduction Current drug therapies for Alzheimer’s disease (AD) target symptom relief and do not interdict the underlying causal pathobiology. Modern research approaches into drug development for AD propose to target the underlying disease. In this realm, the greatest emphasis is on the abolition of -amyloid (A) accumulation in the brain, the pathological hallmark of the disease [49]. Although genetic studies implicate A in the biochemistry of the disease [22], there is no certainty as to how A accumulates or how it induces dementia. A is neurotoxic at non-physiological (micromolar) concentrations in vitro, but it is also produced in health [66], and at physiological (nanomolar) concentrations it is neurotrophic in cell culture [73, 74,76]. The length of the A species is considered to be one important factor in AD pathogenesis since A1–42, a minor free soluble species in biological fluids [71], is enriched in amyloid deposits [36,48], and its concentration is elevated ∗ Present address: Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Massachusetts General Hospital, Building 114, 16th Street, Charlestown, MA 02129, USA. Tel.: +1-617-726-8244; fax: +1-617-724-1823. E-mail address: [email protected] (A.I. Bush).
as a result of familial AD-linked mutations [12]. Synthetic A1–42 appears to be more self-aggregating than A1–40 in solution [27,33]. But the self-aggregating properties of A are insufficient to explain the association of the peptide with AD. Based upon the implication of A as the culprit protein in AD, the major approaches for developing therapeutics for AD have attempted either to prevent A production (secretase inhibitors) or to clear A (vaccine). However, A1–42 may not self-aggregate at all in the absence of metal binding. The high-affinity metal-binding properties of A (K for Cu2+ :A1–42 is attomolar) [5] mediate the apparent self-aggregation of A1–42 in neutral buffers, which is abolished if the peptide is strictly quarantined from metal ions contaminating experimental buffer (Cu2+ , Zn2+ , Fe3+ ) by using high-affinity chelators [5]. Secondly, the soluble, but not the fibrillar, forms of A correlate with both mortality, and dementia-associated neuropathological features like tangles and neuritic changes [46,52,72]. The quantity of classic amyloid plaques actually inversely correlates with oxidation damage to the neocortex in AD [16]. However, not all forms of soluble A are toxic, since healthy people without AD normally have soluble A in their brains, and A is a soluble component of all biological fluids. Therefore, there may be an abnormally modified rogue form of soluble A that is toxic in AD.
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There is strong evidence that other neurochemical reactions apart from A production must contribute to amyloid formation in AD. Were elevated cortical A concentrations to be solely responsible for the initiation of amyloid, it would be difficult to explain why the amyloid deposits are focal (related to synapses, and the cerebrovascular lamina media) and not uniform in their distribution since A is ubiquitously expressed. Further evidence for microanatomical neurochemical factors initiating A accumulation, is the neuropathology of Hereditary Cerebral Hemorrhage with Amyloidosis— Dutch disease, where a mutation at residue 22 of A (Glu → Gln) [44] causes amyloid to form only in the lamina media of the cerebrovasculature and not in the brain parenchyma, despite being expressed at both sites. Another observation to argue against elevated concentrations of A as being solely sufficient to induce cortical A precipitation, is that although a handful of transgenic mouse models with amyloid pathology related to overexpression of APP and A species have been reported, similar degrees of overexpression in transgenic mice have often failed to induce amyloid deposition in other reported attempts to induce cortical amyloid pathology [29]. To attribute amyloid initiation to the presence of A1–42 alone is problematic since the peptide is a normal component of healthy CSF [71]. Finally, amyloid deposition is an age-dependent phenomenon, and if A production does not increase with age, other age-related stochastic neurochemical changes would play an essential role in the reaction that causes A to accumulate. The age-dependent changes are closely associated with oxidative damage to neuronal cells, which precedes A deposition [55–57]. Because the production of A alone is an unsatisfactory explanation for cerebral amyloidogenesis, we investigated the vulnerability of A towards other potentially abnormal neurochemicals. Our data indicate that A is a metalloprotein, whose combination with the brain’s intrinsic supply of Cu2+ and Zn2+ (and possibly Fe3+ ) mediates the peptide’s toxicity (through hydrogen peroxide production) and aggregation. On the basis of these findings, a program to develop a new class of drug therapy was undertaken.
2. Metallochemistry mediates the aggregation and toxicity of A The original basis for our interest in brain metallochemistry, was our observation that the amyloid protein precursor (APP) possesses selective Zn2+ and Cu2+ binding sites that mediate its physicochemical behavior [7,26], and that A is rapidly precipitated by Zn2+ at low-physiological (submicromolar) concentrations [9]. Cu2+ and Fe3+ also induce marked A aggregation but only under mildly acidic conditions (e.g. pH 6.8–7.0) [4], such as those believed to occur in AD brain [78]. Cu2+ induces greater precipitation of A than Fe3+ , and even the trace (nanomolar) concentrations of Zn2+ , Cu2+ or Fe3+ in common laboratory buffers is sufficient to induce nucleation of A, which can then
lead to fibrillization of the peptide solution [4,5,54]. Significantly, rat/mouse A has amino acid substitutions that decrease metal interactions [4], perhaps explaining why these animals are exceptional among mammals for not forming cerebral A amyloid with age [70]. A possesses selective high- and low-affinity metal-binding sites, which are histidine mediated [4,8,9,53]. The Kd of high-affinity Zn2+ binding is ≈100 nM, and for low-affinity binding is ≈5 M [8]. Low-affinity Zn2+ binding mediates the precipitation of the peptide, as well as its resistance to tryptic (␣ secretase-like) cleavage [8,9]. A also possesses high- and low-affinity Cu2+ binding sites. Although the affinity of the low-affinity Cu2+ binding site is similar between A1–40 and A1–42 (5.0 × 10−9 M), the affinity of the high-affinity site on A1–42 is 7.0 × 10−18 M, which is among the highest known affinities for any Cu2+ -binding protein, and similar to that of superoxide dismutase 1 (SOD1) [4]. This is much greater that the highest observed affinity of A1–40 for Cu2+ (5.0 × 10−11 M) [4]. The higher affinity of A1–42 than A1–40 for Cu2+ correlates with enhanced precipitation of A1–42 by Cu2+ [4,5], increased SDS-resistant dimerization of A1–42 by Cu2+ [5], and with increased redox activity of the Cu2+ :A1–42 complex. A binds equimolar amounts of Cu2+ and Zn2+ at pH 7.4, but under conditions representing acidosis (pH 6.6) Cu2+ completely displaces Zn2+ from A [5]. A binds up to 2.5 equivalents of either Cu2+ or Zn2+ , suggesting that metal binding is coordinated by dimeric or oligomeric peptide assemblies [5]. The positive cooperativity in Cu2+ binding observed for A may be greater for A1–42 than for A1–40 because of the enhanced ability of the longer peptide to form a Cu2+ coordinating oligomer [17]. Zn2+ and Cu2+ -induced precipitation of A can be completely reversed by chelation, and is mediated by the ␣-helical conformation of the peptide [4,31]. Zn/Cu-selective chelators were found to markedly enhance the resolubilization of A deposits from post-mortem AD brain samples [11]. The observed increase in extractable A from post-mortem human brain specimens correlated with significant depletion in zinc (30%) and to a lesser extent, copper [11]. The ability of a chelator to extract A depended upon the presence of Mg2+ and Ca2+ , hence the chelating compound needed to be far more selective for Zn2+ and Cu2+ , than Ca2+ and Mg2+ . Higher concentrations of Cu/Zn chelator caused a paradoxical decrease in the amount of A released because the sequestration of Ca2+ and Mg2+ from the sample became substantial [11]. Copper, iron and zinc play more of a role than assembling A alone. We also found that when binding Cu2+ or Fe3+ , A reduces the metal ions and produces H2 O2 by double electron transfer to O2 (there is no evidence of O2 − formation as an intermediate) [30,32]. The origin of the electron for this reduction may be from the Met35 residue [17], which mediates A-induced oxidative stress and toxicity in cell culture [37]. However, it is also possible for H2 O2 to
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be formed catalytically by the cycling of Cu or Fe bound to A using biological reducing agents as electron donors without any net oxidation of the A peptide [58]. The generation of H2 O2 is important because there is overwhelming evidence in the literature for oxidation injury in AD that is mediated by H2 O2 [64]. H2 O2 is a pro-oxidant molecule that is the substrate for the Fenton reaction that generates the highly reactive hydroxyl radical (OH• ). H2 O2 is freely permeable across all tissue boundaries and will react with reduced metal ions (Fe2+ , Cu+ ) to generate OH• , which in turn, generates lipid peroxidation adducts, protein carbonyl modifications, and nucleic acid adducts such as 8-OH guanosine, in all cellular compartments, which typifies AD neuropathology [57,65,67]. In AD, the H2 O2 scavenging defences, e.g. catalase and glutathione peroxidase, may be overwhelmed by the catalytic generation of H2 O2 from the A metalloprotein mass. The redox activity (metal reduction, OH• and H2 O2 formation) of A variants is greatest for A42human > A40human A40mouse ≈ 0, which is a strikingly relevant relationship since A1–42 is most involved in AD amyloid and is overproduced as a consequence of the familial mutations that cause inherited forms of AD. In contrast, the redox inertness of Amouse is also important because transgenic mice expressing presenilin mutations do not form cerebral amyloid despite overproducing A42 [19]. This redox relationship also corresponds to the neurotoxicity of the respective peptide in neuronal culture, which is largely mediated by the Cu2+ :A interaction causing H2 O2 generation [32]. Therefore, we believe that this adverse redox activity is most relevant to the pathogenesis of AD, and we have focussed on blocking it as a pharmacological manoeuvre. The activity can be blocked by chelators [32] and by Zn2+ [16]. The role of Zn2+ in the pathophysiology of A appears complex. Zn2+ precipitates A to form amyloid plaques [9,68], and since Zn2+ suppresses H2 O2 production by A [16], we proposed that plaque formation might represent a defence where Zn2+ is mobilized to entomb the abnormal activity of A [16]. This would explain why H2 O2 -mediated oxidative damage in the neuropil is inversely correlated to plaque load [16]. Recent data indicate that the A from plaque is insufficiently loaded with Zn2+ to completely abolish this adverse catalytic activity [58], and therefore, Zn2+ -induced plaque formation might not be a wholly effective means of preventing abnormal H2 O2 production by A. Indeed, despite the inverse correlation between oxidative damage and plaque burden in AD, even in the cases where plaque burden is heaviest, oxidative adducts are still abnormally elevated [16]. Precipitation of A by Zn2+ may also inhibit clearance and catabolism of A [8]. Therefore, beyond a certain threshold, Zn2+ quenching of H2 O2 production may be an incomplete biochemical strategy to defend against abnormal H2 O2 production. For these reasons, interdiction of both the Zn2+ as well as the Cu2+ or Fe3+ interactions with A may be beneficial in a therapeutic compound. Therefore, the dual Zn2+ /Cu2+ -binding properties
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of clioquinol (CQ) could explain the drug’s effectiveness in inhibiting A accumulation in vivo [10]; Zn2+ binding by the chelator principally facilitating the disaggregation and clearance of A [31], while Cu2+ or Fe3+ binding by the chelator inhibits neurotoxic H2 O2 production [32]. The protective effects of Zn2+ resembles a recent proposition by the Beckman lab that altered Cu2+ coordination converts Zn-deficient SOD (which normally binds copper and zinc simultaneously) from an antioxidant to a neurotoxic pro-oxidant [20]. Intriguingly, we have found that the coordination of Cu2+ by A resembles the SOD1 active site, and that Cu binding generates an allosterically ordered membrane-penetrating A oligomer linked by SOD-like bridging histidine residues [17].
3. Brain metallobiology in Alzheimer’s disease The brain is a specialized organ that concentrates metals (Cu2+ , Zn2+ , Fe3+ ) in the neocortex. Zn2+ is released during neurotransmission from a subset of corticofugal glutatmatergic fibers, and extracellular concentrations in the vicinity of the synapse rise transiently to ≈300 M [1,28]. Less is known about the release of Cu2+ during neurotransmission in the neocortex, but the exchangeable Cu2+ concentration is observed to rise to ≈15 M [24,25]. In agreement with our observations of metal interactions with A in vitro, several reports indicate that the concentrations of zinc, copper and iron are markedly elevated in A deposits in AD [45,68]. Zn2+ in A amyloid deposits can be visualized by histological fluorescent techniques in human brain [68], and also in the deposits of APP2576 transgenic mice [42]. Zn2+ released from the glutamatergic fibers of the corticofugal system, which is the largest labile pool of brain zinc [21], may be the dominant neurochemical factor in the formation of plaque pathology [41]. The distribution of this zinc strikingly parallels the regions of the neocortex that are most prone to amyloid deposition. Zn2+ that is released during neurotransmission is packaged into synaptic vesicles by ZnT3, a synaptic vesicle membrane protein of zinc-containing neurons. In ZnT3 knockout animals, total zinc levels in the hippocampus and cortex of these mice are reduced by ≈25% [13], reflecting the pool of brain Zn2+ that is released during glutamatergic neurotransmission [21]. Interestingly, the phenotype of the ZnT3 mouse is otherwise not abnormal, and reproduces normally. The function of this labile pool of brain Zn2+ is still indeterminate, but may play a role in regulating the response of the NMDA receptor. Recently, the relationship of this pool of synaptic Zn to the formation of plaque pathology was validated by the observation that in ZnT3 knockout mice crossed with transgenic AD mice, cerebral A deposition was markedly diminished as a result of the ablation of the labile pool of neuronal Zn2+ [41]. Zn2+ release during neurotransmission may also explain the gender effect of AD because female mice exhibited
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Fig. 1. Proposed model for the role of biometals in AD. A is normally inserted into membranes after binding Cu2+ or Zn2+ [17]. Stochastic neurochemical events cause a small population of A (soluble, or non-membrane-inserted) to convert to rogue form with adverse redox activity (“rogue soluble A”). These biochemical events include oxidation of the peptide (by Cu2+ or H2 O2 [2]), an age-dependent rise in copper or iron (Cu2+ is more likely to be relevant since A has a much greater affinity for Cu2+ than for Fe2/3+ [4]), or stochastic epochs of acidosis that enhance Cu2+ binding [4,5]. The abnormally Cu-decorated “rogue soluble A” is recognized as a damaged protein, and normally cleared by scavenging machinery such as LDL receptor-related protein, LRP [39], that is modulated by apolipoprotein E (ApoE) and ␣-2-macroglobulin, whose isoforms are risk factors for AD. Alternatively, the soluble peptide might be catabolized by enzymes such as neprolysin. If this machinery cannot adequately clear the soluble A, the H2 O2 it generates may be neurotoxic. H2 O2 is a highly cell-permeable metabolic neurotoxin and pro-oxidant. The levels of soluble A in the brain, but not aggregated A or plaque load, correlate with dementia-related features of the neuropathology such as neurofibrillary tangles, synaptic loss, as well as inversely correlate with age at death [46,52,72]. The soluble A is condensed into diffuse and plaque amyloid deposits by reacting with Cu2+ and Zn2+ released from the neurons during synaptic transmission, or pooling in the extracellular milieu due to impaired cellular reuptake [3]. CQ and related compounds have twin actions. CQ removes low-affinity Cu2+ and Zn2+ (possibly also Fe3+ ) from the plaque A deposits, and from diffuse deposits of A. This alters the equilibrium of A deposition and dissolution, facilitating the dissolution reaction, and liberating soluble A, which can then be cleared by LRP or proteolysis. The second action of CQ is to inhibit catalytic H2 O2 production by soluble (or insoluble) A. This ensures that even if soluble levels of A are elevated transiently as the A deposits dissolve (as observed in the treatment of APP2576 transgenic mice) [10], that the soluble A will not be neurotoxic.
age-dependent hyperactivity of the ZnT3 transporter associated with increased amyloid deposition, which was abolished in the ZnT3 knockout cross [41]. The sequence of neurochemical events leading to metal-mediated plaque formation is still uncertain. One model we contemplate is that the affinity of A for Cu2+ and Zn2+ could be increased by the generation of a rogue, oxidized, A form (Fig. 1). The oxidized form of A could be generated by the peptide’s vulnerability to Cu-mediated oxidative damage [2]. Abnormalities of brain metal homeostasis in AD, or as a consequence of aging, could also contribute to A deposition. Recently, we have found that Cu2+ and Fe3+ levels rise markedly and invariably with aging in the Tg2576 model for AD, and its background strain [51]. The age-dependent rise in these metals may be set up chemical conditions where the A toxicity is promoted.
Like Zn2+ , both Cu2+ and Fe3+ play important normal roles in cortical physiology, however only Cu2+ and Zn2+ is released during neurotransmission. The iron in plaque is found in predominantly in neuritic processes, and probably complexed with ferritin [23], and therefore may not be directly interacting with A. Whereas genetic ablation of synaptic Zn2+ markedly diminished plaque formation in Tg2576 mice, ≈20% of A accumulation in the brains of the Tg2576 crossed mice remained. This residual may be contributed to by Cu2+ and Fe3+ from these alternative origins. However, studies of the effects of various metal chelators on post-mortem AD-affected brain tissue correlated the solubilization of precipitated A with the release of Cu and Zn, but not with Fe [11]. These findings together with the observation that Cu2+ is more effective than Fe3+ in precipitating synthetic A in vitro [4], suggest that cortical Cu2+ may play more of a direct role in precipitating the amyloid mass than Fe3+ . Both Cu2+ and Fe3+ need only be combined with A in relatively small stoichiometric ratios in order to initiate catalytic H2 O2 production [30,32], hence both metals may be of pathophysiological relevance as pharmacotherapeutic targets. Since both Cu2+ and Zn2+ are potentially neurotoxic [43], the brain has efficient homeostatic mechanisms and buffers to prevent the abnormal discompartmentalization of metal ions. Among these is a sophisticated system of metal buffering involving three isoforms of metallothionein, MT-1, MT-2 and MT-3. The brain specific isoform of metallothionein, MT-3, facilitates loading of Zn2+ by ZnT3 into synaptic vesicles [47], and is deficient in AD affected cortex [69]. It is possible that failure of incorporation of Zn2+ or other metals into the metallothionein system could lead to pooling of metals in the extracellular space and contribute to A precipitation. A further means of controlling metal levels in the brain is the blood brain barrier (BBB), which resists the transduction of fluctuating levels of plasma metal ions [59,60]. Numerous abnormalities of the BBB have been reported in AD [34,35], which could lead to a fatigue of this regulatory function, leading to an abnormal rise in extracellular metal levels, and compounding amyloid deposition.
4. Development of therapeutic drugs on the basis of metal–A interactions The principle of a pharmacotherapeutic molecule complexing a metal-binding site on a protein target is actually well developed in pharmacology. This approach is quite different pharmacologically to chelation therapy. Several well-known antibiotic, anticonvulsive, antitumor, and antiinflammatory drugs [15] exert their pharmacological effects by interacting with the Cu-, Zn- or Fe-active site of their target protein. Disulfiram, for example, blocks the enzyme activity by chelating the zinc-catalytic site of alcohol dehydrogenase [40]. Non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin, diflunisal, ibuprofen, naproxen
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sodium, indomethacin, d-penicillamine, etc. block the heme-iron catalytic site on cyclooxygenase/arachidonic acid pathway [61,62]. On the basis of this growing pool of information about Zn2+ and Cu2+ interactions with A in AD, we recently embarked on a trial of copper/zinc-chelators to attempt to inhibit A accumulation in APP2576 transgenic mice. Oral treatment with CQ, a retired USP antibiotic and orally bioavailable Cu/Zn chelator, induced a 49% decrease in brain A deposition (−375 g/g wet weight, P = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks [10]. There was no evidence of neurotoxicity or increased non-amyloid pathology. General health and body weight parameters were significantly more stable in the treated animals, which were conspicuously improved after only 16 days of treatment. The drug may work by a combined action that facilitates metal-mediated disaggregation of the A collections, while also inhibiting H2 O2 production even by soluble forms of A (Fig. 1). The drug’s rapid onset of benefit in the transgenic mice [10] may have been due to a rapid fall in cerebral H2 O2 concentrations caused by the inhibition of its catalytic production by A, since the 16 days interval might be too soon to expect any marked difference in the amount of amyloid accumulation itself between the CQ-treated and control mice. Importantly, CQ treatment did not induce a loss in metal levels systemically, probably because it is a relatively weak chelator and the metals are redistributed rather than excreted. Therefore, the benefits of the drug appear to be due to its ability to bind selectively to the A–metal complex, and are not due to metal depletion of brain tissue. The affinity of CQ for Cu2+ and Zn2+ is not as strong as standard chelators, like EDTA or TETA. Nevertheless, CQ was as effective as high-affinity chelators in blocking the production of H2 O2 by A in vitro, in preventing precipitation of synthetic A by Zn2+ and Cu2+ , and in extracting A from post-mortem AD brain specimens [10]. The decrease in A accumulation in the CQ-treated Tg2576 mice may be due to a combination of inhibition of A precipitation, as well as dissolution of established A aggregates. A collections which have been precipitated by metals are reversible with chelation [11,31]. Cu and Fe binding to A engenders H2 O2 production by A [6,30,32], which may inhibit LRP-mediated clearance mechanisms [75], leading to A accumulation. The inhibition of metal-mediated H2 O2 production from A by CQ could, therefore, facilitate A clearance mechanisms. The resolubilized A may then either be removed into the blood, as observed in A-immunized transgenic mice [18], or degraded by intracellular uptake and hydrolysis. This may explain the small increase in soluble brain A (but far greater decrease in total brain A) that is observed in Tg2576 mice treated with CQ [10]. We hypothesize that the hydrophobic nature of CQ (and possibly other stereochemical properties) facilitates the drug access to the metal-binding site on A. There is a Cu2+ binding site on A1–42 with attomolar affinity [5], which
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is unlikely to dissociate Cu2+ in the presence of CQ. However, A1–40 and A1–42 have up to 2.5 Cu2+ binding sites [5], depending upon pH, probably as an oligomeric complex [17]. These include lower (nM) affinity sites which modulate peptide precipitation, and may be redox active [5]. The affinity of CQ for Cu2+ is sufficient (nM) to dissociate low-affinity bound Cu2+ from A, and we have observed CQ inducing Cu2+ dissociation from A by NMR spectroscopy [10]. In addition, redox activity of the Cu2+ bound to A might also be blocked by the formation of ternary complexes with CQ. The affinity of CQ for Fe ions is not known, but likely to be similar to that for other transition metal ions. Since Fe ions are less implicated in the generation of plaque, CQ activity is less likely to be mediated by complexing Fe in A deposits. However, CQ may block the neurotoxic generation of H2 O2 caused by Fe interaction with A [30,63]. CQ treatment of non-transgenic mice significantly decreases brain levels of Cu, Zn and Fe [77]. However, treatment of 21-month-old APP2576 mice with CQ for 9 weeks paradoxically elevated brain Cu by 19% and Zn by 13% while markedly inhibiting A deposition. This paradoxical result is explained by our recent findings that brain Cu and Zn levels are relatively decreased by APP transgene expression in APP2576 mice, despite A levels accumulating several hundred fold from 2.8 to 18 mo. The decrease in Cu and Zn levels that were observed were, therefore, not a consequence of plaque pathology, and were relative to a marked increase (≈50%) in Cu and Fe levels that occurred after 6 months of age [51]. This relative decrease must either be due to secreted APP and/or A promoting the efflux of the metal ions, or APP/A preventing their uptake. Supporting this latter possibility is evidence that A scavenges extracellular Cu2+ , possibly to prevent oxidation [38]. In light of our recent findings, the paradoxical increase in Cu and Zn in CQ-treated APP2576 mice may be explained by CQ preventing Cu2+ and Zn2+ from complexing with extracellular A, so securing metal for uptake into metal-deficient brain tissue instead of being sequestered into amyloid. Therefore, despite being a chelator, CQ treatment may be able to restore homeostatic defects of normal brain metal metabolism which may occur in AD. The consequent lowering of extracellular metal concentrations inhibited the formation, or facilitated the dissolution, of amyloid deposits. In contrast, TETA (triene), a high-affinity copper/zinc chelator that does not penetrate the BBB, failed to inhibit amyloid deposition in the transgenic mice [10]. TETA, like penicillamine, is used for the treatment of the copper overload disorder, Wilson’s disease. Wilson’s disease commences as a hepatic disorder but advanced complications include cataracts and neurological complications. There is no known association with AD, which is not surprising because copper accumulation is intracellular in Wilson’s disease, whereas it is extracellular in AD. The ineffectiveness of a traditional copper chelator (TETA) in inhibiting amyloid depostion in transgenic mice [10], indicates that
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systemic metal depletion (e.g. “chelation therapy”), is not likely to be a useful therapeutic strategy for AD. Indeed, the transgenic mice successfully treated with CQ exhibited a small but significant increase in brain zinc and copper suggesting that these metals had been trapped by the amyloid aggregates, and prevented from constitutive entry into the tissue. CQ treatment induced a ≈50% decrease in total (mainly precipitated) A levels in transgenic mice compared to sham-treated controls (23-month-old animals) which was proportionally less than the best reported effects of the A vaccination in older mice (≈60 and 80% decreases in 15and 18-month-old PDAPP mice, respectively). However, the absolute reduction in A induced by CQ was ≈30 times greater and was achieved more rapidly with CQ (9 weeks) than with the vaccine protocol (4 and 7 months). Therefore, CQ treatment appears, like the vaccine therapy, to be a potent inhibitor of A accumulation. CQ may be the first credible drug candidate based on the amyloid hypothesis of AD, and a phase II double-blind clinical trial on the effects of oral CQ in patients with AD has recently been completed. One previous trial of a metal chelator (desferrioxamine, DFO) was reported to induce a significant slowing in the rate of progression of dementia [14]. This effect was attributed to complexing aluminum but the drug has high-affinity for zinc, copper and iron, as well. Further clinical research into the effects of DFO may have been met with diminished enthusiasm since the administration of DFO is associated with discouraging difficulties including the non-specific problems of systemic metal ion depletion (e.g. anemia), and the problem of administration of a twice daily, painful intramuscular injection. Also, DFO is a charged molecule that does not easily penetrate the blood–brain barrier and is easily degraded after it is administered [50]. Unlike DFO, the approach with CQ is selectively targeted at a metalloprotein and attempts to avoid unfavorable tissue metal depletion.
5. Conclusion The metal-binding site on A is a promising target for pharmacological interventions that may benefit AD. This approach involves medicinal chemistry approaches to identify compounds that block the adverse generation of H2 O2 that is catalyzed by the site, as well as the secondary effect of metal-induced aggregation, which may be mediated by an alternative metal-binding site. Similar compounds may have utility in other degenerative diseases where abnormal metalloprotein biochemistry is implicated such as Parkinson’s disease, cataracts, prion diseases, and ALS.
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A.I. Bush / Neurobiology of Aging 23 (2002) 1031–1038 [21] Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 1989;31:145–328. [22] Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, et al. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature 1991;349:704–6. [23] Grundke-Iqbal I, Fleming J, Tung YC, Lassmann H, Iqbal K, Joshi JG. Ferritin is a component of the neuritic (senile) plaque in Alzheimer dementia. Acta Neuropathol (Berl) 1990;81:105–10. [24] Hartter DE, Barnea A. Brain tissue accumulates 67 copper by two ligand-dependent saturable processes. J Biol Chem 1988;263:799– 805. [25] Hartter DE, Barnea A. Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67 copper. Synapse 1988;2:412–5. [26] Hesse L, Beher D, Masters CL, Multhaup G. The betaA4 amyloid precursor protein binding to copper. FEBS Lett 1994;349:109–16. [27] Hilbich C, Kisters-Woike B, Reed J, Masters CL, Beyreuther K. Aggregation and secondary structure of synthetic amyloid  A4 peptides of Alzheimer’s disease. J Mol Biol 1994;218:149–63. [28] Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 1984;308:736–8. [29] Hsiao KK, Borchelt DR, Olson K, Johannsdottir R, Kitt C, Yunis W, et al. Age related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron 1995;15:1203–18. [30] Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, et al. The A peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 1999;38:7609–16. [31] Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel J-P, Tanzi RE, et al. Zinc-induced Alzheimer’s A1–40 aggregation is mediated by conformational factors. J Biol Chem 1997;272:26464–70. [32] Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall J, Hanson GR, et al. Cu(II) potentiation of Alzheimer A neurotoxicity: correlation with cell-free hydrogen peroxide production and metal reduction. J Biol Chem 1999;274:37111–6. [33] Jarrett JT, Berger EP, Lansbury Jr PT. The carboxy terminus of the  amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer’s disease. Biochemistry 1993;32:4693–7. [34] Kalaria RN, Harik SI. Reduced glucose transporter at the blood–brain barrier. J Neurochem 1989;53:1083–8. [35] Kalaria RN, Hedera P. Differential degeneration of the cerebral microvasculature in Alzheimer’s disease. NeuroReport 1995;6:477– 80. [36] Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, et al. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 1987;325:733–6. [37] Kanski J, Varadarajan S, Aksenova M, Butterfield DA. Role of glycine-33 and methionine-35 in Alzheimer’s amyloid beta-peptide 1–42-associated oxidative stress and neurotoxicity. Biochim Biophys Acta 2002;1586:190–8. [38] Kontush A, Berndt C, Weber W, Akopyan V, Arlt S, Schippling S, et al. Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Radic Biol Med 2001;30:119–28. [39] Kounnas MZ, Moir RD, Rebeck GW, Bush AI, Argraves WS, Tanzi RE, et al. LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted -amyloid precursor protein and mediates its degradation. Cell 1995;82:331–40. [40] Langeland BT, McKinley-McKee JS. The effects of disulfiram on equine hepatic alcohol dehydrogenase and its efficiency against alcoholism: vinegar effect. Alcohol 1996;31:75–80. [41] Lee J-Y, Cole TB, Palmiter RD, Suh SW, Koh J-Y. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci USA 2002;99:7705–10.
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Metal complexing agents as therapies for Alzheimer’s disease Ashley I. Bush∗ Oxidation Disorders Research Unit, Mental Health Research Institute of Victoria, University of Melbourne, 155 Oak Street, Parkville, VIC 3052, Australia Received 7 March 2002; received in revised form 25 July 2002; accepted 6 August 2002
Abstract Modern research approaches into drug development for Alzheimer’s disease (AD) target -amyloid (A) accumulation in the brain. The main approaches attempt to prevent A production (secretase inhibitors) or to clear A (vaccine). However, there is now compelling evidence that A does not spontaneously aggregate, but that there is an age-dependent reaction with excess brain metal (copper, iron and zinc), which induces the protein to precipitate into metal-enriched masses (plaques). The abnormal combination of A with Cu or Fe induces the production of hydrogen peroxide, which may mediate the conspicuous oxidative damage to the brain in AD. We have developed metal-binding compounds that inhibit the in vitro generation of hydrogen peroxide by A, as well as reverse the aggregation of the peptide in vitro and from human brain post-mortem specimens. Most recently, one of the compounds, clioquinol (CQ; a USP antibiotic) was given orally for 9 weeks to amyloid-bearing transgenic mice, and succeeded in markedly inhibiting A accumulation. On the basis of these results, CQ is being tested in clinical trials. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Amyloid; Alzheimer’s disease; Copper; Zinc; Oxidation; Hydrogen peroxide; Superoxide dismutase
1. Introduction Current drug therapies for Alzheimer’s disease (AD) target symptom relief and do not interdict the underlying causal pathobiology. Modern research approaches into drug development for AD propose to target the underlying disease. In this realm, the greatest emphasis is on the abolition of -amyloid (A) accumulation in the brain, the pathological hallmark of the disease [49]. Although genetic studies implicate A in the biochemistry of the disease [22], there is no certainty as to how A accumulates or how it induces dementia. A is neurotoxic at non-physiological (micromolar) concentrations in vitro, but it is also produced in health [66], and at physiological (nanomolar) concentrations it is neurotrophic in cell culture [73, 74,76]. The length of the A species is considered to be one important factor in AD pathogenesis since A1–42, a minor free soluble species in biological fluids [71], is enriched in amyloid deposits [36,48], and its concentration is elevated ∗ Present address: Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Massachusetts General Hospital, Building 114, 16th Street, Charlestown, MA 02129, USA. Tel.: +1-617-726-8244; fax: +1-617-724-1823. E-mail address: [email protected] (A.I. Bush).
as a result of familial AD-linked mutations [12]. Synthetic A1–42 appears to be more self-aggregating than A1–40 in solution [27,33]. But the self-aggregating properties of A are insufficient to explain the association of the peptide with AD. Based upon the implication of A as the culprit protein in AD, the major approaches for developing therapeutics for AD have attempted either to prevent A production (secretase inhibitors) or to clear A (vaccine). However, A1–42 may not self-aggregate at all in the absence of metal binding. The high-affinity metal-binding properties of A (K for Cu2+ :A1–42 is attomolar) [5] mediate the apparent self-aggregation of A1–42 in neutral buffers, which is abolished if the peptide is strictly quarantined from metal ions contaminating experimental buffer (Cu2+ , Zn2+ , Fe3+ ) by using high-affinity chelators [5]. Secondly, the soluble, but not the fibrillar, forms of A correlate with both mortality, and dementia-associated neuropathological features like tangles and neuritic changes [46,52,72]. The quantity of classic amyloid plaques actually inversely correlates with oxidation damage to the neocortex in AD [16]. However, not all forms of soluble A are toxic, since healthy people without AD normally have soluble A in their brains, and A is a soluble component of all biological fluids. Therefore, there may be an abnormally modified rogue form of soluble A that is toxic in AD.
0197-4580/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 5 8 0 ( 0 2 ) 0 0 1 2 0 - 3
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There is strong evidence that other neurochemical reactions apart from A production must contribute to amyloid formation in AD. Were elevated cortical A concentrations to be solely responsible for the initiation of amyloid, it would be difficult to explain why the amyloid deposits are focal (related to synapses, and the cerebrovascular lamina media) and not uniform in their distribution since A is ubiquitously expressed. Further evidence for microanatomical neurochemical factors initiating A accumulation, is the neuropathology of Hereditary Cerebral Hemorrhage with Amyloidosis— Dutch disease, where a mutation at residue 22 of A (Glu → Gln) [44] causes amyloid to form only in the lamina media of the cerebrovasculature and not in the brain parenchyma, despite being expressed at both sites. Another observation to argue against elevated concentrations of A as being solely sufficient to induce cortical A precipitation, is that although a handful of transgenic mouse models with amyloid pathology related to overexpression of APP and A species have been reported, similar degrees of overexpression in transgenic mice have often failed to induce amyloid deposition in other reported attempts to induce cortical amyloid pathology [29]. To attribute amyloid initiation to the presence of A1–42 alone is problematic since the peptide is a normal component of healthy CSF [71]. Finally, amyloid deposition is an age-dependent phenomenon, and if A production does not increase with age, other age-related stochastic neurochemical changes would play an essential role in the reaction that causes A to accumulate. The age-dependent changes are closely associated with oxidative damage to neuronal cells, which precedes A deposition [55–57]. Because the production of A alone is an unsatisfactory explanation for cerebral amyloidogenesis, we investigated the vulnerability of A towards other potentially abnormal neurochemicals. Our data indicate that A is a metalloprotein, whose combination with the brain’s intrinsic supply of Cu2+ and Zn2+ (and possibly Fe3+ ) mediates the peptide’s toxicity (through hydrogen peroxide production) and aggregation. On the basis of these findings, a program to develop a new class of drug therapy was undertaken.
2. Metallochemistry mediates the aggregation and toxicity of A The original basis for our interest in brain metallochemistry, was our observation that the amyloid protein precursor (APP) possesses selective Zn2+ and Cu2+ binding sites that mediate its physicochemical behavior [7,26], and that A is rapidly precipitated by Zn2+ at low-physiological (submicromolar) concentrations [9]. Cu2+ and Fe3+ also induce marked A aggregation but only under mildly acidic conditions (e.g. pH 6.8–7.0) [4], such as those believed to occur in AD brain [78]. Cu2+ induces greater precipitation of A than Fe3+ , and even the trace (nanomolar) concentrations of Zn2+ , Cu2+ or Fe3+ in common laboratory buffers is sufficient to induce nucleation of A, which can then
lead to fibrillization of the peptide solution [4,5,54]. Significantly, rat/mouse A has amino acid substitutions that decrease metal interactions [4], perhaps explaining why these animals are exceptional among mammals for not forming cerebral A amyloid with age [70]. A possesses selective high- and low-affinity metal-binding sites, which are histidine mediated [4,8,9,53]. The Kd of high-affinity Zn2+ binding is ≈100 nM, and for low-affinity binding is ≈5 M [8]. Low-affinity Zn2+ binding mediates the precipitation of the peptide, as well as its resistance to tryptic (␣ secretase-like) cleavage [8,9]. A also possesses high- and low-affinity Cu2+ binding sites. Although the affinity of the low-affinity Cu2+ binding site is similar between A1–40 and A1–42 (5.0 × 10−9 M), the affinity of the high-affinity site on A1–42 is 7.0 × 10−18 M, which is among the highest known affinities for any Cu2+ -binding protein, and similar to that of superoxide dismutase 1 (SOD1) [4]. This is much greater that the highest observed affinity of A1–40 for Cu2+ (5.0 × 10−11 M) [4]. The higher affinity of A1–42 than A1–40 for Cu2+ correlates with enhanced precipitation of A1–42 by Cu2+ [4,5], increased SDS-resistant dimerization of A1–42 by Cu2+ [5], and with increased redox activity of the Cu2+ :A1–42 complex. A binds equimolar amounts of Cu2+ and Zn2+ at pH 7.4, but under conditions representing acidosis (pH 6.6) Cu2+ completely displaces Zn2+ from A [5]. A binds up to 2.5 equivalents of either Cu2+ or Zn2+ , suggesting that metal binding is coordinated by dimeric or oligomeric peptide assemblies [5]. The positive cooperativity in Cu2+ binding observed for A may be greater for A1–42 than for A1–40 because of the enhanced ability of the longer peptide to form a Cu2+ coordinating oligomer [17]. Zn2+ and Cu2+ -induced precipitation of A can be completely reversed by chelation, and is mediated by the ␣-helical conformation of the peptide [4,31]. Zn/Cu-selective chelators were found to markedly enhance the resolubilization of A deposits from post-mortem AD brain samples [11]. The observed increase in extractable A from post-mortem human brain specimens correlated with significant depletion in zinc (30%) and to a lesser extent, copper [11]. The ability of a chelator to extract A depended upon the presence of Mg2+ and Ca2+ , hence the chelating compound needed to be far more selective for Zn2+ and Cu2+ , than Ca2+ and Mg2+ . Higher concentrations of Cu/Zn chelator caused a paradoxical decrease in the amount of A released because the sequestration of Ca2+ and Mg2+ from the sample became substantial [11]. Copper, iron and zinc play more of a role than assembling A alone. We also found that when binding Cu2+ or Fe3+ , A reduces the metal ions and produces H2 O2 by double electron transfer to O2 (there is no evidence of O2 − formation as an intermediate) [30,32]. The origin of the electron for this reduction may be from the Met35 residue [17], which mediates A-induced oxidative stress and toxicity in cell culture [37]. However, it is also possible for H2 O2 to
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be formed catalytically by the cycling of Cu or Fe bound to A using biological reducing agents as electron donors without any net oxidation of the A peptide [58]. The generation of H2 O2 is important because there is overwhelming evidence in the literature for oxidation injury in AD that is mediated by H2 O2 [64]. H2 O2 is a pro-oxidant molecule that is the substrate for the Fenton reaction that generates the highly reactive hydroxyl radical (OH• ). H2 O2 is freely permeable across all tissue boundaries and will react with reduced metal ions (Fe2+ , Cu+ ) to generate OH• , which in turn, generates lipid peroxidation adducts, protein carbonyl modifications, and nucleic acid adducts such as 8-OH guanosine, in all cellular compartments, which typifies AD neuropathology [57,65,67]. In AD, the H2 O2 scavenging defences, e.g. catalase and glutathione peroxidase, may be overwhelmed by the catalytic generation of H2 O2 from the A metalloprotein mass. The redox activity (metal reduction, OH• and H2 O2 formation) of A variants is greatest for A42human > A40human A40mouse ≈ 0, which is a strikingly relevant relationship since A1–42 is most involved in AD amyloid and is overproduced as a consequence of the familial mutations that cause inherited forms of AD. In contrast, the redox inertness of Amouse is also important because transgenic mice expressing presenilin mutations do not form cerebral amyloid despite overproducing A42 [19]. This redox relationship also corresponds to the neurotoxicity of the respective peptide in neuronal culture, which is largely mediated by the Cu2+ :A interaction causing H2 O2 generation [32]. Therefore, we believe that this adverse redox activity is most relevant to the pathogenesis of AD, and we have focussed on blocking it as a pharmacological manoeuvre. The activity can be blocked by chelators [32] and by Zn2+ [16]. The role of Zn2+ in the pathophysiology of A appears complex. Zn2+ precipitates A to form amyloid plaques [9,68], and since Zn2+ suppresses H2 O2 production by A [16], we proposed that plaque formation might represent a defence where Zn2+ is mobilized to entomb the abnormal activity of A [16]. This would explain why H2 O2 -mediated oxidative damage in the neuropil is inversely correlated to plaque load [16]. Recent data indicate that the A from plaque is insufficiently loaded with Zn2+ to completely abolish this adverse catalytic activity [58], and therefore, Zn2+ -induced plaque formation might not be a wholly effective means of preventing abnormal H2 O2 production by A. Indeed, despite the inverse correlation between oxidative damage and plaque burden in AD, even in the cases where plaque burden is heaviest, oxidative adducts are still abnormally elevated [16]. Precipitation of A by Zn2+ may also inhibit clearance and catabolism of A [8]. Therefore, beyond a certain threshold, Zn2+ quenching of H2 O2 production may be an incomplete biochemical strategy to defend against abnormal H2 O2 production. For these reasons, interdiction of both the Zn2+ as well as the Cu2+ or Fe3+ interactions with A may be beneficial in a therapeutic compound. Therefore, the dual Zn2+ /Cu2+ -binding properties
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of clioquinol (CQ) could explain the drug’s effectiveness in inhibiting A accumulation in vivo [10]; Zn2+ binding by the chelator principally facilitating the disaggregation and clearance of A [31], while Cu2+ or Fe3+ binding by the chelator inhibits neurotoxic H2 O2 production [32]. The protective effects of Zn2+ resembles a recent proposition by the Beckman lab that altered Cu2+ coordination converts Zn-deficient SOD (which normally binds copper and zinc simultaneously) from an antioxidant to a neurotoxic pro-oxidant [20]. Intriguingly, we have found that the coordination of Cu2+ by A resembles the SOD1 active site, and that Cu binding generates an allosterically ordered membrane-penetrating A oligomer linked by SOD-like bridging histidine residues [17].
3. Brain metallobiology in Alzheimer’s disease The brain is a specialized organ that concentrates metals (Cu2+ , Zn2+ , Fe3+ ) in the neocortex. Zn2+ is released during neurotransmission from a subset of corticofugal glutatmatergic fibers, and extracellular concentrations in the vicinity of the synapse rise transiently to ≈300 M [1,28]. Less is known about the release of Cu2+ during neurotransmission in the neocortex, but the exchangeable Cu2+ concentration is observed to rise to ≈15 M [24,25]. In agreement with our observations of metal interactions with A in vitro, several reports indicate that the concentrations of zinc, copper and iron are markedly elevated in A deposits in AD [45,68]. Zn2+ in A amyloid deposits can be visualized by histological fluorescent techniques in human brain [68], and also in the deposits of APP2576 transgenic mice [42]. Zn2+ released from the glutamatergic fibers of the corticofugal system, which is the largest labile pool of brain zinc [21], may be the dominant neurochemical factor in the formation of plaque pathology [41]. The distribution of this zinc strikingly parallels the regions of the neocortex that are most prone to amyloid deposition. Zn2+ that is released during neurotransmission is packaged into synaptic vesicles by ZnT3, a synaptic vesicle membrane protein of zinc-containing neurons. In ZnT3 knockout animals, total zinc levels in the hippocampus and cortex of these mice are reduced by ≈25% [13], reflecting the pool of brain Zn2+ that is released during glutamatergic neurotransmission [21]. Interestingly, the phenotype of the ZnT3 mouse is otherwise not abnormal, and reproduces normally. The function of this labile pool of brain Zn2+ is still indeterminate, but may play a role in regulating the response of the NMDA receptor. Recently, the relationship of this pool of synaptic Zn to the formation of plaque pathology was validated by the observation that in ZnT3 knockout mice crossed with transgenic AD mice, cerebral A deposition was markedly diminished as a result of the ablation of the labile pool of neuronal Zn2+ [41]. Zn2+ release during neurotransmission may also explain the gender effect of AD because female mice exhibited
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Fig. 1. Proposed model for the role of biometals in AD. A is normally inserted into membranes after binding Cu2+ or Zn2+ [17]. Stochastic neurochemical events cause a small population of A (soluble, or non-membrane-inserted) to convert to rogue form with adverse redox activity (“rogue soluble A”). These biochemical events include oxidation of the peptide (by Cu2+ or H2 O2 [2]), an age-dependent rise in copper or iron (Cu2+ is more likely to be relevant since A has a much greater affinity for Cu2+ than for Fe2/3+ [4]), or stochastic epochs of acidosis that enhance Cu2+ binding [4,5]. The abnormally Cu-decorated “rogue soluble A” is recognized as a damaged protein, and normally cleared by scavenging machinery such as LDL receptor-related protein, LRP [39], that is modulated by apolipoprotein E (ApoE) and ␣-2-macroglobulin, whose isoforms are risk factors for AD. Alternatively, the soluble peptide might be catabolized by enzymes such as neprolysin. If this machinery cannot adequately clear the soluble A, the H2 O2 it generates may be neurotoxic. H2 O2 is a highly cell-permeable metabolic neurotoxin and pro-oxidant. The levels of soluble A in the brain, but not aggregated A or plaque load, correlate with dementia-related features of the neuropathology such as neurofibrillary tangles, synaptic loss, as well as inversely correlate with age at death [46,52,72]. The soluble A is condensed into diffuse and plaque amyloid deposits by reacting with Cu2+ and Zn2+ released from the neurons during synaptic transmission, or pooling in the extracellular milieu due to impaired cellular reuptake [3]. CQ and related compounds have twin actions. CQ removes low-affinity Cu2+ and Zn2+ (possibly also Fe3+ ) from the plaque A deposits, and from diffuse deposits of A. This alters the equilibrium of A deposition and dissolution, facilitating the dissolution reaction, and liberating soluble A, which can then be cleared by LRP or proteolysis. The second action of CQ is to inhibit catalytic H2 O2 production by soluble (or insoluble) A. This ensures that even if soluble levels of A are elevated transiently as the A deposits dissolve (as observed in the treatment of APP2576 transgenic mice) [10], that the soluble A will not be neurotoxic.
age-dependent hyperactivity of the ZnT3 transporter associated with increased amyloid deposition, which was abolished in the ZnT3 knockout cross [41]. The sequence of neurochemical events leading to metal-mediated plaque formation is still uncertain. One model we contemplate is that the affinity of A for Cu2+ and Zn2+ could be increased by the generation of a rogue, oxidized, A form (Fig. 1). The oxidized form of A could be generated by the peptide’s vulnerability to Cu-mediated oxidative damage [2]. Abnormalities of brain metal homeostasis in AD, or as a consequence of aging, could also contribute to A deposition. Recently, we have found that Cu2+ and Fe3+ levels rise markedly and invariably with aging in the Tg2576 model for AD, and its background strain [51]. The age-dependent rise in these metals may be set up chemical conditions where the A toxicity is promoted.
Like Zn2+ , both Cu2+ and Fe3+ play important normal roles in cortical physiology, however only Cu2+ and Zn2+ is released during neurotransmission. The iron in plaque is found in predominantly in neuritic processes, and probably complexed with ferritin [23], and therefore may not be directly interacting with A. Whereas genetic ablation of synaptic Zn2+ markedly diminished plaque formation in Tg2576 mice, ≈20% of A accumulation in the brains of the Tg2576 crossed mice remained. This residual may be contributed to by Cu2+ and Fe3+ from these alternative origins. However, studies of the effects of various metal chelators on post-mortem AD-affected brain tissue correlated the solubilization of precipitated A with the release of Cu and Zn, but not with Fe [11]. These findings together with the observation that Cu2+ is more effective than Fe3+ in precipitating synthetic A in vitro [4], suggest that cortical Cu2+ may play more of a direct role in precipitating the amyloid mass than Fe3+ . Both Cu2+ and Fe3+ need only be combined with A in relatively small stoichiometric ratios in order to initiate catalytic H2 O2 production [30,32], hence both metals may be of pathophysiological relevance as pharmacotherapeutic targets. Since both Cu2+ and Zn2+ are potentially neurotoxic [43], the brain has efficient homeostatic mechanisms and buffers to prevent the abnormal discompartmentalization of metal ions. Among these is a sophisticated system of metal buffering involving three isoforms of metallothionein, MT-1, MT-2 and MT-3. The brain specific isoform of metallothionein, MT-3, facilitates loading of Zn2+ by ZnT3 into synaptic vesicles [47], and is deficient in AD affected cortex [69]. It is possible that failure of incorporation of Zn2+ or other metals into the metallothionein system could lead to pooling of metals in the extracellular space and contribute to A precipitation. A further means of controlling metal levels in the brain is the blood brain barrier (BBB), which resists the transduction of fluctuating levels of plasma metal ions [59,60]. Numerous abnormalities of the BBB have been reported in AD [34,35], which could lead to a fatigue of this regulatory function, leading to an abnormal rise in extracellular metal levels, and compounding amyloid deposition.
4. Development of therapeutic drugs on the basis of metal–A interactions The principle of a pharmacotherapeutic molecule complexing a metal-binding site on a protein target is actually well developed in pharmacology. This approach is quite different pharmacologically to chelation therapy. Several well-known antibiotic, anticonvulsive, antitumor, and antiinflammatory drugs [15] exert their pharmacological effects by interacting with the Cu-, Zn- or Fe-active site of their target protein. Disulfiram, for example, blocks the enzyme activity by chelating the zinc-catalytic site of alcohol dehydrogenase [40]. Non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin, diflunisal, ibuprofen, naproxen
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sodium, indomethacin, d-penicillamine, etc. block the heme-iron catalytic site on cyclooxygenase/arachidonic acid pathway [61,62]. On the basis of this growing pool of information about Zn2+ and Cu2+ interactions with A in AD, we recently embarked on a trial of copper/zinc-chelators to attempt to inhibit A accumulation in APP2576 transgenic mice. Oral treatment with CQ, a retired USP antibiotic and orally bioavailable Cu/Zn chelator, induced a 49% decrease in brain A deposition (−375 g/g wet weight, P = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks [10]. There was no evidence of neurotoxicity or increased non-amyloid pathology. General health and body weight parameters were significantly more stable in the treated animals, which were conspicuously improved after only 16 days of treatment. The drug may work by a combined action that facilitates metal-mediated disaggregation of the A collections, while also inhibiting H2 O2 production even by soluble forms of A (Fig. 1). The drug’s rapid onset of benefit in the transgenic mice [10] may have been due to a rapid fall in cerebral H2 O2 concentrations caused by the inhibition of its catalytic production by A, since the 16 days interval might be too soon to expect any marked difference in the amount of amyloid accumulation itself between the CQ-treated and control mice. Importantly, CQ treatment did not induce a loss in metal levels systemically, probably because it is a relatively weak chelator and the metals are redistributed rather than excreted. Therefore, the benefits of the drug appear to be due to its ability to bind selectively to the A–metal complex, and are not due to metal depletion of brain tissue. The affinity of CQ for Cu2+ and Zn2+ is not as strong as standard chelators, like EDTA or TETA. Nevertheless, CQ was as effective as high-affinity chelators in blocking the production of H2 O2 by A in vitro, in preventing precipitation of synthetic A by Zn2+ and Cu2+ , and in extracting A from post-mortem AD brain specimens [10]. The decrease in A accumulation in the CQ-treated Tg2576 mice may be due to a combination of inhibition of A precipitation, as well as dissolution of established A aggregates. A collections which have been precipitated by metals are reversible with chelation [11,31]. Cu and Fe binding to A engenders H2 O2 production by A [6,30,32], which may inhibit LRP-mediated clearance mechanisms [75], leading to A accumulation. The inhibition of metal-mediated H2 O2 production from A by CQ could, therefore, facilitate A clearance mechanisms. The resolubilized A may then either be removed into the blood, as observed in A-immunized transgenic mice [18], or degraded by intracellular uptake and hydrolysis. This may explain the small increase in soluble brain A (but far greater decrease in total brain A) that is observed in Tg2576 mice treated with CQ [10]. We hypothesize that the hydrophobic nature of CQ (and possibly other stereochemical properties) facilitates the drug access to the metal-binding site on A. There is a Cu2+ binding site on A1–42 with attomolar affinity [5], which
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is unlikely to dissociate Cu2+ in the presence of CQ. However, A1–40 and A1–42 have up to 2.5 Cu2+ binding sites [5], depending upon pH, probably as an oligomeric complex [17]. These include lower (nM) affinity sites which modulate peptide precipitation, and may be redox active [5]. The affinity of CQ for Cu2+ is sufficient (nM) to dissociate low-affinity bound Cu2+ from A, and we have observed CQ inducing Cu2+ dissociation from A by NMR spectroscopy [10]. In addition, redox activity of the Cu2+ bound to A might also be blocked by the formation of ternary complexes with CQ. The affinity of CQ for Fe ions is not known, but likely to be similar to that for other transition metal ions. Since Fe ions are less implicated in the generation of plaque, CQ activity is less likely to be mediated by complexing Fe in A deposits. However, CQ may block the neurotoxic generation of H2 O2 caused by Fe interaction with A [30,63]. CQ treatment of non-transgenic mice significantly decreases brain levels of Cu, Zn and Fe [77]. However, treatment of 21-month-old APP2576 mice with CQ for 9 weeks paradoxically elevated brain Cu by 19% and Zn by 13% while markedly inhibiting A deposition. This paradoxical result is explained by our recent findings that brain Cu and Zn levels are relatively decreased by APP transgene expression in APP2576 mice, despite A levels accumulating several hundred fold from 2.8 to 18 mo. The decrease in Cu and Zn levels that were observed were, therefore, not a consequence of plaque pathology, and were relative to a marked increase (≈50%) in Cu and Fe levels that occurred after 6 months of age [51]. This relative decrease must either be due to secreted APP and/or A promoting the efflux of the metal ions, or APP/A preventing their uptake. Supporting this latter possibility is evidence that A scavenges extracellular Cu2+ , possibly to prevent oxidation [38]. In light of our recent findings, the paradoxical increase in Cu and Zn in CQ-treated APP2576 mice may be explained by CQ preventing Cu2+ and Zn2+ from complexing with extracellular A, so securing metal for uptake into metal-deficient brain tissue instead of being sequestered into amyloid. Therefore, despite being a chelator, CQ treatment may be able to restore homeostatic defects of normal brain metal metabolism which may occur in AD. The consequent lowering of extracellular metal concentrations inhibited the formation, or facilitated the dissolution, of amyloid deposits. In contrast, TETA (triene), a high-affinity copper/zinc chelator that does not penetrate the BBB, failed to inhibit amyloid deposition in the transgenic mice [10]. TETA, like penicillamine, is used for the treatment of the copper overload disorder, Wilson’s disease. Wilson’s disease commences as a hepatic disorder but advanced complications include cataracts and neurological complications. There is no known association with AD, which is not surprising because copper accumulation is intracellular in Wilson’s disease, whereas it is extracellular in AD. The ineffectiveness of a traditional copper chelator (TETA) in inhibiting amyloid depostion in transgenic mice [10], indicates that
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systemic metal depletion (e.g. “chelation therapy”), is not likely to be a useful therapeutic strategy for AD. Indeed, the transgenic mice successfully treated with CQ exhibited a small but significant increase in brain zinc and copper suggesting that these metals had been trapped by the amyloid aggregates, and prevented from constitutive entry into the tissue. CQ treatment induced a ≈50% decrease in total (mainly precipitated) A levels in transgenic mice compared to sham-treated controls (23-month-old animals) which was proportionally less than the best reported effects of the A vaccination in older mice (≈60 and 80% decreases in 15and 18-month-old PDAPP mice, respectively). However, the absolute reduction in A induced by CQ was ≈30 times greater and was achieved more rapidly with CQ (9 weeks) than with the vaccine protocol (4 and 7 months). Therefore, CQ treatment appears, like the vaccine therapy, to be a potent inhibitor of A accumulation. CQ may be the first credible drug candidate based on the amyloid hypothesis of AD, and a phase II double-blind clinical trial on the effects of oral CQ in patients with AD has recently been completed. One previous trial of a metal chelator (desferrioxamine, DFO) was reported to induce a significant slowing in the rate of progression of dementia [14]. This effect was attributed to complexing aluminum but the drug has high-affinity for zinc, copper and iron, as well. Further clinical research into the effects of DFO may have been met with diminished enthusiasm since the administration of DFO is associated with discouraging difficulties including the non-specific problems of systemic metal ion depletion (e.g. anemia), and the problem of administration of a twice daily, painful intramuscular injection. Also, DFO is a charged molecule that does not easily penetrate the blood–brain barrier and is easily degraded after it is administered [50]. Unlike DFO, the approach with CQ is selectively targeted at a metalloprotein and attempts to avoid unfavorable tissue metal depletion.
5. Conclusion The metal-binding site on A is a promising target for pharmacological interventions that may benefit AD. This approach involves medicinal chemistry approaches to identify compounds that block the adverse generation of H2 O2 that is catalyzed by the site, as well as the secondary effect of metal-induced aggregation, which may be mediated by an alternative metal-binding site. Similar compounds may have utility in other degenerative diseases where abnormal metalloprotein biochemistry is implicated such as Parkinson’s disease, cataracts, prion diseases, and ALS.
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