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Journal of Alzheimers Disease 30 (2012) 15DOI 10.3233/JAD-2012-129011IOS Press

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Review1

The Metal Theory of Alzheimers Disease2

Ashley I. Bush3Oxidation Biology Laboratory, Mental Health Research Institute, The University of Melbourne, Parkville, VIC,

Australia

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Abstract. Brain homeostasis of transition metals is severely perturbed in Alzheimers disease (AD), with extracellular pooling

of zinc and copper in amyloid, and intraneuronal accumulation of iron. Rapidly accumulating evidence indicates that these

perturbances themselves may contribute significantly to the cognitive loss and neurodegeneration, even in the absence of AD

proteopathy. There is now strong evidence that each of the major protein participants in AD pathology has physiologically

important interactions with transition metals: APP is the neuronal iron export ferroxidase with a major interaction with

ferroportin, presenilins are needed for the import of50% of cellular copper and zinc, and tau promotes the export of neuronal

iron by facilitating the trafficking of APP to the surface. Therefore, amyloid and tau pathology arise in a milieu of constitutively

high metal flux, and the major components of AD pathology may contribute to the disease by failing in their metal transport

roles.

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Keywords: Alzheimers disease, amyloid, copper, iron, presenilin, tau, zinc15

The last five years have seen challenges for the16

amyloid hypothesis of Alzheimers disease (AD),17

with the failures of large-scale clinical trials that tar-18

get amyoid- (A). In contrast, my group has been19

involved in an alternative approach that postulates that20

the amyloid lesions of AD are not solely the cause of21

the disease, but rather, operate to exaggerate fatigue in22

metal homeostasis. A landmark in this regard is our23

demonstration for the first time in 2010, that the zinc24

released into the glutamatergic synapse through the25

activity of the ZnT3 transporter is needed to maintain26

memory and cognition [1]. ZnT3 is uniquely expressed27

in areas of grey matter affected by amyloid pathology,28

and zinc released into the glutamatergic synapse by29

ZnT3 has been shown to cause amyloid pathology in30

transgenic mice [2, 3], explaining why amyloid pathol-31

ogy is markedly enriched with zinc [4, 5].32

Correspondence to: Ashley I. Bush, Oxidation Biology Labora-

tory, Mental Health ResearchInstitute, TheUniversityof Melbourne,

30 Royal Parade, Parkville, Victoria 3052, Australia. E-mail:

ashleyib@unimelb.edu.au .

This pool of zinc, representing about 20% of the 33

zinc content of the brain [6], is an important neuro- 34

chemical system. Synaptic zinc sustains levels of key 35

functional synaptic proteins (e.g., SNAP25, PSD95, 36

AMPA receptor, NMDA receptors 2a and 2b), as well 37

as neurotrophic support proteins (e.g., doublecortin, 38

pro-BDNF, and TrkB), and neuronal spine density [6]. 39

The broad impact of extracellular zinc in modulat- 40

ing neuronal activity and viability has been an area 41

of increasing study in the last 5 years, with evidence 42

suggesting that extracellular zinc plays a role similar to 43

calcium as a second messenger system [6]. Since cor- 44

tical ZnT3 levels decline with age in mice and humans 45

and is exaggerated in AD, our group proposed that age- 46

dependent loss of trans-synaptic zinc movement leads 47

to cognitive loss. Since extracellular A is aggregated 48

by and traps this pool of zinc [4, 7, 8], the amyloid 49

lesion of AD may cause cognitive loss by trapping 50

extracellular zinc. 51

This novel reformulation of the position of amyloid 52

in AD pathogenesis has had several ramifications in 53

a short time. The lack of cycling of metal ions as a 54

ISSN 1387-2877/12/$27.50 2012 IOS Press and the authors. All rights reserved

mailto:ashleyib@unimelb.edu.aumailto:ashleyib@unimelb.edu.au

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2 A.I. Bush / The Metal Theory of Alzheimers Disease

result of aging or disease has lead to the coining of55

the term metallostasis [9], and work by our group56

has shown that this fatigue in metal homeostasis not57

only leads to A aggregation and deposition, but also,58

secondarily, to iron accumulation withinneurons, lead-59

ing to oxidative injury and neurodegeneration. Most60

importantly, this work has advanced the mechanism of61

action of a class of disease-modifying drug candidates62

(exemplified by PBT2), and propelled clinical testing.63

PBT2, an 8-OH quinolone, is the second-generation64

analogue of clioquinol, an early disease-modifying65

candidate that we identified [10]. In 2008, we reported66

that oral PBT2 therapy of APP transgenic mice67

induced marked cognitive recovery that was clear68

within 6 days of commencing treatment [11]. A similar69rapid cognitive effect was also reported in the subse-70

quent phase 2 RCT of PBT2 [12, 13]. In this trial,71

PBT2 significantly improved executive function above72

baseline levels on a Neuropsychological Test Battery73

withinonly 12 weeks of treatmentof patients with early74

(mild) AD. While this was only a small (n = 78) trial, it75

was randomized, placebo controlled and double-blind.76

To further validate the efficacy signal, a ROC statis-77

tical re-analysis of the trial data [14] was performed78

that is more appropriate for pioneering studies (a sim-79

ilar ROC analysis used to judge the efficacy signal80

on the first (6-month) donepezil Phase II trial [15]),81

where a poweranalysisis notpossible, andwhereleast-82

square means differentials are too crude a yardstick to83

measure small differences in short interval trials. The84

ROC analysis revealed that PBT2 induced highly sig-85

nificant improvement in 12 weeks on both executive86

function and composite cognitive z-scores, and even87

improved (p = 0.056) ADAS-cog scores. Since PBT2,88

unlike donepezil, is a disease-modifying approach, the89

expectation is that the effect size will be exaggerated90

with longer clinical trial periods. While the economic91

crisis has cruelled the development of non-mainstream92

approaches, PBT2 is moving ahead and another93

Phase II trial (12 months, placebo-controlled, RCT)94

using PiB and FDG PET as markers, has just com-95

menced.96

Apart from the drug development itself, another cru-97

cial aspect of the work on PBT2 is the elaboration of98

the mechanism of action. While PBT2 significantly99

lowered cerebrospinal fluid levels of A42 in the 12-100

week Phase II clinical trial [12], in concordance with101

the drug rapidly lowering interstitial A levels in trans-102

genic mouse brain [11], we built off our work on zinc103

being needed for cognition [1] to determine that the104

benefits of PBT2 are related to more than just amy-105

loid clearance. We first recognized that PBT2, and106

its precursor clioquinol, are not chelators, but rather 107

ionophores or metal uptake chaperones [11]. In other 108

words, the compounds promote the uptake of cop- 109

per and zinc (PBT2 > clioquinol, in register with their 110

efficacies). We more recently published that PBT2 has 111

neurotrophic benefits in transgenic mice that are medi- 112

ated by the restored uptake of metal ions [9]. Brains 113

of AD transgenic mice exhibit deficits in spine density 114

and functional proteins (CamKII, spinophilin, NMDA 115

receptors, pro-BDNF, and BDNF) that are significantly 116

rescued by PBT2 treatment.PBT2-treatment increased 117

neurite outgrowth in cultured cells, and in a manner 118

dependent on copper or zinc uptake. We subsequently 119

found that zinc trapped by amyloid outside of neurons 120

(in culture) was prevented from entering the neuron 121and inhibiting calcineurin [16]. This could explain 122

why calcineurin is overactive in AD and AD mod- 123

els. Importantly, PBT2 facilitated the migration of zinc 124

from being trapped in amyloid, to entering the neuron 125

and inhibiting calcineurin, with an array of favorable 126

consequences including decreased tau hyperphospho- 127

rylation [16], as seen in PBT2-treated AD transgenic 128

mice [11]. Taken together, this work has evolved a 129

novel therapeutic concept: that in AD, an ionophore 130

drug (e.g., PBT2) may restore the uptake of physi- 131

ological metal ions trapped within extracellular A 132

aggregates and so induce biochemical and anatomical 133

changes to rescue cognitive function. 134

Our work on the mechanism of action of PBT2 135

and related drug candidates revealed that these bio- 136

logically active drugs were correcting an underlying 137

problem of metal distribution in AD, where essential 138

metal ions are depleted in some brain compartments 139

whilezinc and copper accumulate in extracellular amy- 140

loid, and iron in dystrophic neurites. This leads to 141

another hypothesis that is currently being tested: that 142

the reason that metals are maldistributed in the brain 143

in AD, and are enriched in AD pathology, is because 144

the major proteins implicated in AD physiologically 145

interact with these metals, e.g., for homeostatic metal 146

regulation. 147

Moving forward, we are exploring the functional 148

interaction of metal transport systems and proteins 149

involved in neurodegeneration. This tests the hypoth- 150

esis that the reason that certain proteins involved in 151

neurodegeneration are denatured by metals is because 152

these proteins fail in a specific functional role in 153

metal regulation, and then become overwhelmed by 154

the metals that they handle. Evidence has ramified to 155

encompass all of the major protein targets implicated 156

in the disease (i.e., not just A, but also APP, tau, 157

presenilin, and BACE1). 158

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A.I. Bush / The Metal Theory of Alzheimers Disease 3

We have identified that APP has a function in regu-159

lating iron levels within the brain and other organs [17,160

18]. We identified functional homology between the161

active site of ferritin heavy chain [17], and a motif on162

APP close to Tsunao Saitohs RERMS neurotrophic163

motif [19]. Like H-ferritin, APP is ferroxidase that164

resembles ceruloplasmin in that it interacts with ferro-165

portin to remove iron from cells [17]. Since neurons166

lack ceruloplasmin, APP appears to be the dedicated167

export ferroxidase of neurons. APP ferroxidase spe-168

cific activity is 75% decreased in AD cortical (but not169

cerebellar) tissue, and contributes to the elevation of170

pro-oxidant iron in neurons in AD, first described by171

the late Mark Smith and colleagues [20]. We found172

that APP levels were not decreased in AD corti-173cal tissue, but rather that APP ferroxidase activity174

is decreased due to inhibition by zinc that transfers175

from the extracellular amyloid mass [20]. Remarkably,176

APP ferroxidase activity therefore inversely corre-177

lated with cortical A burden, and we found that it178

was the zinc content of the amyloid that was respon-179

sible for inhibiting the activity. This paper introduces180

a completely new mechanism of toxicity for A and181

defines a pathological relationship between extracel-182

lular zinc accumulation in amyloid, and intraneuronal183

iron accumulation in AD.184

This relationship resembles the tandem association185

of amyloid with intracellular tau in neurofibrillary tan-186

gles. Since iron binds to tangle pathology and induces187

oxidative stress (again, work of Mark Smith and col-188

leagues [21]), our theory surmised that tau might have189

a physiological role in neuronal iron homeostasis.190

Indeed, we found that tau directs the trafficking of191

APP to the neuronal surface [18], where it inter-192

acts with ferroportin (most of the APP in the brain193

co-IPs with ferroportin [17]). In tau knockout mice,194

iron accumulates in neurons, and the mice develop195

a dementia with parkinsonism phenotype with age196

(12+ months) [18]. Previous reports of phenotyping197

tau knockout mice had not persevered with aging the198

animals beyond 7 months. Like the cognitive pheno-199

type of ZnT3 knockout mice, the neurodegenerative200

phenotype did not emerge until the animals were aged201

a little more. So we propose that tau knockout mice202

are a useful model of AD/PD neurodegeneration, and203

that relative iron accumulation may explain the MAPT204

risk alleles in common for both diseases. We also pro-205

pose that it is the loss of soluble, functional, tau that206

contributes to neuronal iron accumulation in AD, PD,207

and tauopathies. Soluble tau loss, of course, can come208

about due to the precipitation of tau, so the observa-209

tions are not incompatible with current thinking about210

the adverse consequences of tau hyperphosphoryla- 211

tion. Importantly, the iron accumulation, the cognitive 212

losses, and the parkinsonism in the tau knockout mice 213

is completely rescued by treatment with clioquinol, the 214

prototype to PBT2 [18]. 215

The value of 8-OH quinoline class of drug candi- 216

dates, and its members clioquinol and PBT2, recently 217

was given strong endorsement by Susan Lindquist, 218

Massachusetts Institute of Technology, in her recent 219

publication [22]. In this paper, Lindquist and col- 220

leagues describe the identification of selected 8-OH 221

quinoline compounds as protective against neurode- 222

generative models due to their influence on metal 223

homeostasis, as metal chaperones and ionophores. 224

They corroborate the possibility that tailoring 8-OH 225quinoline activity to a particular neurodegenerative 226

disease may be a viable therapeutic strategy. In par- 227

ticular, they corroborate that some 8-OH quinolones 228

act more as zinc/copper ionophores (like PBT2), and 229

that clioquinol is relatively more targeted to iron with 230

a chelation action. In other words, 8-OH quinoline 231

molecules can, by side group adjustment, be tailored 232

to have either ionophore (transporting or increasing 233

metals) or chelation (withdrawing or complexing met- 234

als) properties. This versatile property allows these 235

drug candidates to be relatively targeted to specific 236

metal-related signature lesions associated with differ- 237

ent aspects of neurodegeneration: in AD, the collection 238

of zinc in extracellular amyloid, and the accumulation 239

of iron intraneuronally. This important paper supports 240

this therapeutic strategy for treating AD. 241

Another new line of research within the theme 242

of the functions of the AD-related proteins, capital- 243

ized on the functional homology between APP and 244

ceruloplasmin [17]. While APP and ceruloplasmin 245

are not structurally homologous, they are both fer- 246

roxidases with strikingly similar enzymatic activity. 247

We noted that in addition to its ferroxidase activity, 248

ceruloplasmin is also an amine oxidase, and the new 249

reportdescribes that APP alsopossesses robust amine 250

oxidase activity [23]. APP oxidizes catecholamines 251

catalytically (e.g., Km norepinephrine = 0.27 mM), 252

through a site encompassing its ferroxidase motif 253

and selectively inhibited by zinc. Accordingly, APP 254

knockout mice have significantly higher levels of 255

dopamine, norepinephrine, and epinephrine in brain, 256

plasma, and select tissues, and concomitant physiolog- 257

ical changes. These findings support a role for APPin 258

extracellular catecholaminergic clearance. Since zinc 259

is already known to inhibit APP ferroxidase activity 260

in AD cortex [17], the interaction of zinc within amy- 261

loid has the potential to interfere with catecholamine 262

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4 A.I. Bush / The Metal Theory of Alzheimers Disease

neurochemistry in AD (a subject of rapidly growing263

interest in AD research) as well as in Down syndrome,264

and is a promising line of exploration.265

The metal theory has also advanced to incorporate266

presenilins. The presenilins were found to play a major267

role in normal cellular zinc and copper uptake [24].268

The presenilins mediated this role in several cell types269

and tissues, including brain. The effect of presenilin270

knockout (even of one presenilin allele) was to drive271

down the specific activity of CuZn superoxide dismu-272

tase (SOD1), by lowering the expression of its metal273

ion chaperone, CCS1. The mechanism of presenilin-274

associated metal ion uptake is still to be determined,275

and could be mediated by endosome formation or276

acidification. However, the implication is that prese-277nilin failure (e.g., as postulated by Jie Shen and others278

[25]) could be an upstream event that, in turn, would279

foster extracellular metal pooling (i.e., metallostasis),280

and so promote A aggregation in AD. The impact281

of pathogenic presenilin mutations awaits to be deter-282

mined, but identifying a major biochemical interaction283

between endogenous presenilin and brain metal chem-284

istry paves the way for some intriguing research.285

BACE1 has also been shown to have metal-based286

interactions, with the copper chaperone for SOD1287

(CCS1) and also binds copper directly [26]. The inter-288

action of copper with A

biogenesis may actually be289

caused by neuronal copper deficiency, as seen in AD,290

which promotes A production [27] and paradoxically291

enriches the remaining copper together with A into292

cholesterol-rich lipid rafts [28]. Copper is essential for293

many metabolic activities, but in AD-affected cortical294

tissue becomes biologically unavailable, and increases295

in its pathological, freely ionic, form that correlates296

with increased oxidative tissue damage [29].297

The vast majority of effort in Alzheimer science and298

drug making has been focused on the perils of amy-299

loid. Clearly, this focus is struggling, possibly because300

failed drug trials have targeted the disease too late in301

its natural history, but also possibly because the toxic302

amyloid hypothesis is too simplistic. The considerable303

evidence that has developed to support the role of met-304

als as being a denominator in the big picture of AD305

means that with patience and perseverance this work306

is poised to make important contributions in the near307

future.308

DISCLOSURE STATEMENT309

The authors disclosure is available online.

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