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The essential elements of Alzheimer’s disease

  • Peng Lei
    Correspondence
    For correspondence: Peng Lei; Ashley I. Bush
    Affiliations
    Department of Neurology and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, P.R. China

    Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia
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  • Scott Ayton
    Affiliations
    Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia
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  • Ashley I. Bush
    Correspondence
    For correspondence: Peng Lei; Ashley I. Bush
    Affiliations
    Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia
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Open AccessPublished:November 26, 2020DOI:https://doi.org/10.1074/jbc.REV120.008207
      Treatments for Alzheimer’s disease (AD) directed against the prominent amyloid plaque neuropathology are yet to be proved effective despite many phase 3 clinical trials. There are several other neurochemical abnormalities that occur in the AD brain that warrant renewed emphasis as potential therapeutic targets for this disease. Among those are the elementomic signatures of iron, copper, zinc, and selenium. Here, we review these essential elements of AD for their broad potential to contribute to Alzheimer’s pathophysiology, and we also highlight more recent attempts to translate these findings into therapeutics. A reinspection of large bodies of discovery in the AD field, such as this, may inspire new thinking about pathogenesis and therapeutic targets.

      Keywords:

      Abbreviations:

      (amyloid-β peptide), AD (Alzheimer’s disease), ApoE (apolipoprotein E), APP (amyloid protein precursor), CSF (cerebrospinal fluid), Cp (ceruloplasmin), DFO (deferoxamine), FAD (familial AD), GPx4 (glutathione peroxidase 4), HO-1 (heme oxygenase 1), LTP (long-term potentiation), MRI (magnetic resonance imaging), NAC (N-acetylcysteine), NMDA (N-methyl-D-aspartate), PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol), PUFA (polyunsaturated fatty acids), QSM (quantitative susceptibility mapping), RTA (radical trapping agent), SOD1 (superoxide dismutase 1), ZIP (Zinc regulated transporter-like Iron regulated transporter-like Protein), ZnT (Zinc transporter)
      Alzheimer’s disease (AD), the most common form of dementia, is increasingly prevalent and a worsening healthcare burden. The cognitive deterioration of AD has remained frustratingly recalcitrant to candidate disease-modifying therapeutics despite massive efforts over the last 35 years. Most research into therapeutics has been philosophically guided by the connection of the hallmark histopathology of AD, cortical amyloid plaques, and neurofibrillary tangles, with familial dementia-causing mutations associated with their most insoluble component proteins, the amyloid-β peptide (Aβ) (
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      Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein.
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      ), and the microtubule-associated protein tau (
      • Wood J.G.
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      Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau).
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      Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.
      ). Both proteins are normal and soluble components of tissue that become denatured by events that are not simply related to overproduction.
      Alois Alzheimer himself came to the conclusion 5 years after his description of plaques and tangles that despite their dramatic appearance, these histopathologies were not the cause of neurodegeneration in AD but, rather, a signature epiphenomenon (reviewed [
      • Ayton S.
      • Bush A.I.
      β-amyloid: the known unknowns.
      ]). Yet, dogged efforts have been made in the modern era to causatively link the aggregation of these proteins to the brain atrophy, synaptic disintegration, and neuronal loss that characterize AD, through death mechanisms that remain unproven after decades of research (e.g., the Amyloid Cascade Hypothesis [
      • Hardy J.
      • Allsop D.
      Amyloid deposition as the central event in the aetiology of Alzheimer's disease.
      ]). The discovery of familial AD (FAD) causative mutations of the amyloid protein precursor (APP) and the presenilins (that cleave the carboxyl terminus of Aβ from APP) as well as mutations of tau that cause fronto-temporal dementia have been interpreted simplistically through the prism of the toxic proteinopathy theory. Efforts to investigate the neurodegeneration mechanisms of the genetic lesions of AD outside of the formation of putatively toxic aggregates have received, in our opinion, insufficient attention, for example, that pathogenic presenilin mutations cause neurodegeneration without proteinopathy through a loss of trophic function (
      • Xia D.
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      Presenilin-1 knockin mice reveal loss-of-function mechanism for familial Alzheimer's disease.
      ). Indeed, the dogma that all FAD causative mutations of the presenilins generate longer proaggregate forms or more Aβ has been persuasively challenged as data to the contrary accumulate (
      • Ayton S.
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      β-amyloid: the known unknowns.
      ,
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      Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase.
      ).
      Billions of dollars are being spent by big pharmaceutical companies on lowering Aβ or tau as therapeutic strategies. This approach was justified on the premise of the earliest data from murine knockouts of APP and tau, which have minimal phenotypes in youth, leading to the conclusion that these proteins therefore must be functionless rogues that humans can live without. But, the safe redundancy of tau and APP is unlikely because the adverse phenotypes relevant for neurodegeneration, particularly those related to brain metal dyshomeostasis, are, like AD itself, a product of aging and do not emerge until the postreproductive epoch (
      • Lei P.
      • Ayton S.
      • Finkelstein D.I.
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      • Ciccotosto G.D.
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      • Lam L.Q.
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      Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export.
      ,
      • Lei P.
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      • Moon S.
      • Zhang Q.
      • Volitakis I.
      • Finkelstein D.I.
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      Motor and cognitive deficits in aged tau knockout mice in two background strains.
      ,
      • Belaidi A.A.
      • Gunn A.P.
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      • Ayton S.
      • Appukuttan A.T.
      • Roberts B.R.
      • Duce J.A.
      • Bush A.I.
      Marked age-related changes in brain iron homeostasis in amyloid protein precursor knockout mice.
      ,
      • Adlard P.A.
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      • Finkelstein D.I.
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      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ). Nonetheless, clinical trials of Aβ-lowering agents proceed despite more than 30 phase 3 trials failing to demonstrate conspicuous cognitive benefit to AD patients or sometimes being harmful, even upon successful clearance of amyloid plaques (
      • Cummings J.
      • Lee G.
      • Ritter A.
      • Sabbagh M.
      • Zhong K.
      Alzheimer's disease drug development pipeline: 2019.
      ,
      • Egan M.F.
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      • Voss T.
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      Randomized trial of verubecestat for prodromal Alzheimer's disease.
      ,
      • Honig L.S.
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      • Woodward M.
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      • Bullock R.
      • Borrie M.
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      • Case M.
      • Dean R.A.
      • Hake A.
      • Sundell K.
      • Poole Hoffmann V.
      • et al.
      Trial of solanezumab for mild dementia due to Alzheimer's disease.
      ,
      • Doody R.S.
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      Phase 3 trials of solanezumab for mild-to-moderate Alzheimer's disease.
      ,
      • Salloway S.
      • Sperling R.
      • Fox N.C.
      • Blennow K.
      • Klunk W.
      • Raskind M.
      • Sabbagh M.
      • Honig L.S.
      • Porsteinsson A.P.
      • Ferris S.
      • Reichert M.
      • Ketter N.
      • Nejadnik B.
      • Guenzler V.
      • Miloslavsky M.
      • et al.
      Two phase 3 trials of bapineuzumab in mild-to-moderate Alzheimer's disease.
      ,
      • Murphy M.P.
      Amyloid-beta solubility in the treatment of Alzheimer's disease.
      ). One of these, aducanumab, has been presented for registration to the Food and Drug Administration on the basis of debatable benefits that were seen in one but not both of its two phase 3 trials and could be explained as a placebo effect caused by the unblinding when treatment is temporarily suspended upon activation of the amyloid related imaging artefact protocols, which is much more common in the active arm (
      • Gleason A.
      • Ayton S.
      • Bush A.I.
      Unblinded by the light: ARIA in Alzheimer's clinical trials.
      ). In no instance has an amyloid-lowering treatment shown a reliable and indisputable benefit.
      With amyloid being challenged as a therapeutic target, other neurochemical changes in AD have attracted growing interest. Hence, the subject of this monograph. Biometals such as zinc, copper, and iron, which have essential roles in normal physiology, have been implicated in AD pathogenesis for more than 25 years, while commanding a tiny fraction of the research and clinical trial resources committed to proteinopathy research. These are physiologically essential metal ions, but their nutritional (or genetic) dysregulation causes neurotoxicity and neurological damage. These metal ions are stringently regulated by multiple handling systems because excess can also be neurotoxic. These should not be called “trace” metal ions because their concentrations in the brain are within the same order of magnitude as magnesium. Also, the epithet “heavy metal” should not be applied to these versatile and essential metal ions but should be reserved for metals such as lead, mercury, and cadmium that are conspicuously neurotoxic and serve no biochemical purpose. Although aluminum has been investigated as a neurotoxicant that may influence AD, we place it outside of this review of essential elements because it is a nonessential “light” metal with no biochemical function but is very abundant in the environment (present at low micromolar concentrations in plasma as an environmental contaminant) and only potentially toxic at high concentrations (
      • Good P.F.
      • Perl D.P.
      • Bierer L.M.
      • Schmeidler J.
      Selective accumulation of aluminum and iron in the neurofibrillary tangles of Alzheimer's disease: a laser microprobe (LAMMA) study.
      ).
      Ionic zinc was first reported in 1994 to induce histological amyloid structures rapidly out of soluble Aβ (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ). Later, ionic copper and iron were found to facilitate Aβ aggregation as well as catalyze reactive oxygen species generation from the ternary complexes (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.
      ,
      • Huang X.
      • Cuajungco M.P.
      • Atwood C.S.
      • Hartshorn M.A.
      • Tyndall J.D.
      • Hanson G.R.
      • Stokes K.C.
      • Leopold M.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Saunders A.J.
      • Lim J.
      • Moir R.D.
      • Glabe C.G.
      • et al.
      Cu(II) potentiation of Alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.
      ,
      • Cherny R.A.
      • Legg J.T.
      • McLean C.A.
      • Fairlie D.P.
      • Huang X.
      • Atwood C.S.
      • Beyreuther K.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion.
      ,
      • Rottkamp C.A.
      • Raina A.K.
      • Zhu X.W.
      • Gaier E.
      • Bush A.I.
      • Atwood C.S.
      • Chevion M.
      • Perry G.
      • Smith M.A.
      Redox-active iron mediates amyloid-beta toxicity.
      ,
      • Opazo C.
      • Huang X.
      • Cherny R.A.
      • Moir R.D.
      • Roher A.E.
      • White A.R.
      • Cappai R.
      • Masters C.L.
      • Tanzi R.E.
      • Inestrosa N.C.
      • Bush A.I.
      Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2).
      ). Over this time, evidence has accumulated to indicate that these biological elements impact Aβ and tau production, posttranslational modification, aggregation, and toxicity. Sensitive multielement assay technology, e.g., inductively coupled plasma mass spectrometry, has enabled metallomics (“elementomics”, actually, because the array of elements measured simultaneously frequently includes nonmetals, such as selenium [Se]) to be adapted to examining biological samples. Furthermore, biological metal dyshomeostasis alone has been shown to cause neuronal and cognitive dysfunction. Here, we review the associations of the brain’s three most abundant physiological transition metals, iron, zinc, and copper, with the pathophysiology and neuropathology of AD. Because the iron-dependent regulated cell death pathway, ferroptosis, is so closely involved with the selenoenzyme glutathione peroxidase 4 (GPx4) (
      • Friedmann Angeli J.P.
      • Schneider M.
      • Proneth B.
      • Tyurina Y.Y.
      • Tyurin V.A.
      • Hammond V.J.
      • Herbach N.
      • Aichler M.
      • Walch A.
      • Eggenhofer E.
      • Basavarajappa D.
      • Rådmark O.
      • Kobayashi S.
      • Seibt T.
      • Beck H.
      • et al.
      Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.
      ,
      • Chen L.
      • Hambright W.S.
      • Na R.
      • Ran Q.
      Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis.
      ), we also discuss the role of the essential trace metal Se.

      Zinc

      Zinc is essential for brain function, and it participates in protein structure stabilizing and catalytic reactions in living organisms. It is concentrated in the gray matter of the brain, where 20 to 30% of brain zinc is located in glutamatergic vesicles (
      • Danscher G.
      • Stoltenberg M.
      Zinc-specific autometallographic in vivo selenium methods: tracing of zinc-enriched (ZEN) terminals, ZEN pathways, and pools of zinc ions in a multitude of other ZEN cells.
      ), which results in an extraordinary level of Zn2+ in the synaptic cleft during neurotransmission (100–300 μM) (
      • Howell G.A.
      • Welch M.G.
      • Frederickson C.J.
      Stimulation-induced uptake and release of zinc in hippocampal slices.
      ,
      • Vogt K.
      • Mellor J.
      • Tong G.
      • Nicoll R.
      The actions of synaptically released zinc at hippocampal mossy fiber synapses.
      ). The high flux of zinc in the synapse contributes to synaptic plasticity, and long-term potentiation (LTP) in the hippocampal CA3 region is modulated by zinc at presynaptic and postsynaptic targets (
      • Pan E.
      • Zhang X.A.
      • Huang Z.
      • Krezel A.
      • Zhao M.
      • Tinberg C.E.
      • Lippard S.J.
      • McNamara J.O.
      Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse.
      ). Synaptic zinc turnover is therefore highly energetic but fatigues with age (
      • Datki Z.
      • Galik-Olah Z.
      • Janosi-Mozes E.
      • Szegedi V.
      • Kalman J.
      • Hunya A.G.
      • Fulop L.
      • Tamano H.
      • Takeda A.
      • Adlard P.A.
      • Bush A.I.
      Alzheimer risk factors age and female sex induce cortical Aβ aggregation by raising extracellular zinc.
      ), highlighting the potential for zinc dysregulation to contribute to cognitive impairment in AD. Zinc homeostasis is mostly regulated by the SLC39 family (zinc regulated transporter-like iron regulated transporter-like proteins, ZIPs), which has 14 members that transport Zn2+ into the cytoplasm (from organelles and cellular uptake), and the SLC30 family (zinc transporters, ZnTs), which has 10 members that transport Zn2+ out of the cytoplasm (extracellularly and into organelles) (
      • Xu Y.
      • Xiao G.
      • Liu L.
      • Lang M.
      Zinc transporters in Alzheimer's disease.
      ). These two families of transporters are believed to be relative selective for Zn2+, but a few ZIPs and ZnTs transport other metals, such and Fe, Mn, and Cd. The expression of various members of these families is tissue-specific. ZnT3 expression is selectively expressed in cortical tissue, accounting for 20% of total brain zinc content, and, by loading Zn2+ into glutamatergic synaptic vesicles, is responsible for the high concentrations of extracellular Zn2+ released during neurotransmission (
      • Palmiter R.D.
      • Cole T.B.
      • Quaife C.J.
      • Findley S.D.
      ZnT-3, a putative transporter of zinc into synaptic vesicles.
      ). ZnT3 and its associated synaptic Zn2+ release is strongly implicated in deteriorating cognitive function in AD and in the pathogenesis of the hallmark amyloid pathology.
      After the discovery that Aβ is normally secreted by neurons as a soluble peptide (
      • Haass C.
      • Schlossmacher M.G.
      • Hung A.Y.
      • Vigo-Pelfrey C.
      • Mellon A.
      • Ostaszewski B.L.
      • Lieberburg I.
      • Koo E.H.
      • Schenk D.
      • Teplow D.B.
      • Selkoe D.J.
      Amyloid beta-peptide is produced by cultured cells during normal metabolism.
      ), factors inducing Aβ aggregation became of interest. Zn2+ was found to bind to Aβ with affinity in the high nM to low μM range and to induce its rapid aggregation and precipitation (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ,
      • Bush A.I.
      • Pettingell W.H.
      • Paradis M.D.
      • Tanzi R.E.
      Modulation of Aβ adhesiveness and secretase site cleavage by zinc.
      ), with up to 3 eq. of Zn2+ per mole of Aβ in the precipitate (
      • Atwood C.S.
      • Scarpa R.C.
      • Huang X.
      • Moir R.D.
      • Jones W.D.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      Characterization of copper interactions with Alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42.
      ). Histidine–Zn2+ bridges mediate the reversible assembly of these precipitates (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ,
      • Bush A.I.
      • Pettingell W.H.
      • Paradis M.D.
      • Tanzi R.E.
      Modulation of Aβ adhesiveness and secretase site cleavage by zinc.
      ), and Asp7 is also essential for the interaction (
      • Polshakov V.I.
      • Mantsyzov A.B.
      • Kozin S.A.
      • Adzhubei A.A.
      • Zhokhov S.S.
      • van Beek W.
      • Kulikova A.A.
      • Indeykina M.I.
      • Mitkevich V.A.
      • Makarov A.A.
      A binuclear zinc interaction fold discovered in the homodimer of Alzheimer's amyloid-beta fragment with Taiwanese mutation D7H.
      ), which can be abolished by phosphorylation (
      • Barykin E.P.
      • Petrushanko I.Y.
      • Kozin S.A.
      • Telegin G.B.
      • Chernov A.S.
      • Lopina O.D.
      • Radko S.P.
      • Mitkevich V.A.
      • Makarov A.A.
      Phosphorylation of the amyloid-beta peptide inhibits zinc-dependent aggregation, prevents Na,K-ATPase inhibition, and reduces cerebral plaque deposition.
      ). The metal binding site on Aβ is not specific for Zn2+ and overlaps with residues that coordinate (and reduce) Cu2+ and Fe2/3+ (vide infra). The complex of Aβ–zinc is resistant to proteolysis (
      • Bush A.I.
      • Pettingell W.H.
      • Paradis M.D.
      • Tanzi R.E.
      Modulation of Aβ adhesiveness and secretase site cleavage by zinc.
      ), promoting the stability of Aβ aggregates (Fig. 1). Importantly, the rat/mouse homolog of Aβ is exceptional among mammalian sequences for having a His13Arg substitution that attenuates Zn2+ binding and Zn2+-induced precipitation (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ), which may help explain why these rodents do not develop amyloid plaques (
      • Vaughan D.W.
      • Peters A.
      The structure of neuritic plaque in the cerebral cortex of aged rats.
      ) unless made transgenic to overexpress the human Aβ sequence. These substitutions also impair the binding of Cu2+ and Fe3+ at an overlapping binding site (vide infra). Curiously, APP possesses an ectodomain high-affinity zinc-binding site remote from the Aβ sequence that promotes the affinity of APP for heparin (
      • Bush A.I.
      • Multhaup G.
      • Moir R.D.
      • Williamson T.G.
      • Small D.H.
      • Rumble B.
      • Pollwein P.
      • Beyreuther K.
      • Masters C.L.
      A novel zinc(II) binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer's disease.
      ,
      • Bush A.I.
      • Pettingell W.H.
      • de Paradis M.
      • Tanzi R.E.
      • Wasco W.
      The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily.
      ). Little is known of the physiological purpose of this site, although it overlaps with a palmitoylation site that modulates APP binding and hence cleavage to generate the N-terminus of Aβ (
      • Bhattacharyya R.
      • Barren C.
      • Kovacs D.M.
      Palmitoylation of amyloid precursor protein regulates amyloidogenic processing in lipid rafts.
      ,
      • Bhattacharyya R.
      • Fenn R.H.
      • Barren C.
      • Tanzi R.E.
      • Kovacs D.M.
      Palmitoylated APP forms dimers, cleaved by BACE1.
      ).
      Figure thumbnail gr1
      Figure 1Neuronal zinc homeostasis is dysregulated in Alzheimer's disease. Zn2+ enters neuronal cytoplasm through ZIPs, whereas efflux from the cytoplasm is regulated by ZnTs. There are many types of ZIPs and ZnTs expressed in neurons, but ZnT3 is implicated in cognitive loss with aging and amyloid formation in AD. ZnT3 concentrates Zn2+ in glutamatergic synaptic vesicles that is released upon synaptic activity and then is normally rapidly taken up by unidentified energy-dependent mechanisms. During aging, mitochondrial energy is decreased, leading to more sluggish reuptake of extracellular Zn2+. Loss of estrogen, as occurs during menopause, increases ZnT3 protein levels, potentially increasing Zn2+ release. Extracellular Zn2+ binds to Aβ and induces its aggregation, becoming trapped in the amyloid. Intracellularly, metallothioneins, as the major Zn2+-buffering peptides, maintain free Zn2+ at appropriate levels, but neuronal Metallothionein III levels are depleted in AD. Increased cytoplasmic free Zn2+ enhances tau phosphorylation by activating CDK5, GSK3β, ERK1/2, or JNK kinases and by inhibiting PP2A activity. Aβ, amyloid β; APP, amyloid precursor protein; CDK5, cyclin-dependent kinase 5; ERK1/2, extracellular signal-regulated protein kinase 1/2; GSK3β, glycogen synthase kinase 3β; JNK, c-Jun N-terminal kinase; MTs, metallothioneins; NFTs, neurofibrillary tangles; PP2A, protein phosphatase 2A; ZIPs, zinc regulated transporter-like iron regulated transporter-like proteins; ZnTs, zinc transporter proteins.
      Zn2+ can induce different forms of Aβ aggregates depending on the ratio between Aβ and zinc: stoichiometric concentrations of zinc induce nonfibrillary aggregates enriched in the reversible α-helical structure, whereas fibrillar, β-sheet–enriched aggregates are formed with substoichiometric concentrations of zinc as a consequence of seeding (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.P.
      • Tanzi R.E.
      • Bush A.I.
      Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors.
      ,
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer's Abeta peptides.
      ). This is a major differentiation between the fibrillar pathway of amyloid formation that occurs with a micromolar concentration of peptide in vitro at a slow rate through hydrophobic β-sheet forces, taking days, compared with the millisecond reversible precipitation of Aβ by Zn2+, mediated by an ionic histidine bridge (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ,
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.
      ,
      • Shi H.
      • Kang B.
      • Lee J.Y.
      Zn(2+) effect on structure and residual hydrophobicity of amyloid beta-peptide monomers.
      ,
      • Chen Y.R.
      • Glabe C.G.
      Distinct early folding and aggregation properties of Alzheimer amyloid-beta peptides Abeta40 and Abeta42: stable trimer or tetramer formation by Abeta42.
      ,
      • Ha C.
      • Ryu J.
      • Park C.B.
      Metal ions differentially influence the aggregation and deposition of Alzheimer's beta-amyloid on a solid template.
      ,
      • Tõugu V.
      • Karafin A.
      • Zovo K.
      • Chung R.S.
      • Howells C.
      • West A.K.
      • Palumaa P.
      Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.
      ,
      • Miller Y.
      • Ma B.
      • Nussinov R.
      Zinc ions promote Alzheimer Abeta aggregation via population shift of polymorphic states.
      ,
      • Chen W.T.
      • Hong C.J.
      • Lin Y.T.
      • Chang W.H.
      • Huang H.T.
      • Liao J.Y.
      • Chang Y.J.
      • Hsieh Y.F.
      • Cheng C.Y.
      • Liu H.C.
      • Chen Y.R.
      • Cheng I.H.
      Amyloid-beta (Abeta) D7H mutation increases oligomeric Abeta42 and alters properties of Abeta-zinc/copper assemblies.
      ,
      • Lee M.C.
      • Yu W.C.
      • Shih Y.H.
      • Chen C.Y.
      • Guo Z.H.
      • Huang S.J.
      • Chan J.C.C.
      • Chen Y.R.
      Zinc ion rapidly induces toxic, off-pathway amyloid-beta oligomers distinct from amyloid-beta derived diffusible ligands in Alzheimer's disease.
      ). Zn2+ can compete with Cu2+ for Aβ, silencing its redox activity and peroxide formation and suppressing oxidation in the vicinity of plaques (
      • Cuajungco M.P.
      • Goldstein L.E.
      • Nunomura A.
      • Smith M.A.
      • Lim J.T.
      • Atwood C.S.
      • Huang X.
      • Farrag Y.W.
      • Perry G.
      • Bush A.I.
      Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc.
      ).
      As evidence for Zn2+ aggregating soluble Aβ in vivo, zinc accumulates in plaques in AD and may be as high as 1 mM in this vicinity (
      • Opazo C.
      • Huang X.
      • Cherny R.A.
      • Moir R.D.
      • Roher A.E.
      • White A.R.
      • Cappai R.
      • Masters C.L.
      • Tanzi R.E.
      • Inestrosa N.C.
      • Bush A.I.
      Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2).
      ,
      • Lovell M.A.
      • Robertson J.D.
      • Teesdale W.J.
      • Campbell J.L.
      • Markesbery W.R.
      Copper, iron and zinc in Alzheimer's disease senile plaques.
      ,
      • Religa D.
      • Strozyk D.
      • Cherny R.A.
      • Volitaskis I.
      • Haroutunian V.
      • Winblad B.
      • Naslund J.
      • Bush A.I.
      Elevated cortical zinc in Alzheimer disease.
      ). This is also seen in animal models of AD, where zinc is elevated in plaques of APP/PS1 mice determined by Timm’s stain (
      • Stoltenberg M.
      • Bush A.I.
      • Bach G.
      • Smidt K.
      • Larsen A.
      • Rungby J.
      • Lund S.
      • Doering P.
      • Danscher G.
      Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency.
      ) as well as X-ray fluorescence microscopy (
      • James S.A.
      • Churches Q.I.
      • de Jonge M.D.
      • Birchall I.E.
      • Streltsov V.
      • McColl G.
      • Adlard P.A.
      • Hare D.J.
      Iron, copper, and zinc concentration in abeta plaques in the APP/PS1 mouse model of Alzheimer's disease correlates with metal levels in the surrounding neuropil.
      ), plaques of Tg2576 mice determined by metallomic imaging mass spectrometry (
      • Craddock T.J.A.
      • Tuszynski J.A.
      • Chopra D.
      • Casey N.
      • Goldstein L.E.
      • Hameroff S.R.
      • Tanzi R.E.
      The zinc dyshomeostasis hypothesis of Alzheimer's disease.
      ), and plaques within the amygdala of aged (over 23-years-old) macaques (
      • Ichinohe N.
      • Hayashi M.
      • Wakabayashi K.
      • Rockland K.S.
      Distribution and progression of amyloid-beta deposits in the amygdala of the aged macaque monkey, and parallels with zinc distribution.
      ). Furthermore, chelators dissolve insoluble Aβ deposits while releasing Zn2+ from postmortem AD-affected brain tissue samples (
      • Cherny R.A.
      • Legg J.T.
      • McLean C.A.
      • Fairlie D.P.
      • Huang X.
      • Atwood C.S.
      • Beyreuther K.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion.
      ,
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ). Further evidence for extracellular Zn2+ inducing amyloid formation comes from the effects of ZnT3 knockout in suppressing interstitial and vascular amyloid pathology in APP transgenic mice (
      • Lee J.-Y.
      • Cole T.B.
      • Palmiter R.D.
      • Suh S.W.
      • Koh J.-Y.
      Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice.
      ,
      • Friedlich A.L.
      • Lee J.-Y.
      • van Groen T.
      • Cherny R.A.
      • Volitakis I.
      • Cole T.B.
      • Palmiter R.D.
      • Koh J.-Y.
      • Bush A.I.
      Neuronal zinc exchange with the blood vessel wall promotes cerebral amyloid angiopathy in an animal model of Alzheimer's disease.
      ).
      The significance of amyloid plaques themselves in the etiopathogenesis of AD is uncertain. It is understood from both postmortem and PET ligand studies that amyloid deposition commences 1 to 2 decades before the onset of functional impairments in the natural history of AD. However, 30 to 40% of people in the age of risk for AD have conspicuous amyloid pathology without cognitive impairment. Indeed, there is no association of amyloid burden with the rate of premortem cognitive decline (
      • Ayton S.
      • Wang Y.
      • Diouf I.
      • Schneider J.A.
      • Brockman J.
      • Morris M.C.
      • Bush A.I.
      Brain iron is associated with accelerated cognitive decline in people with Alzheimer pathology.
      ). With the failure of more than 30 phase 3 clinical trials that lower brain Aβ, it is possible that amyloid plaque pathology might be a biomarker of another process, such as zinc dyshomeostasis. Recent evidence has brought to light a mechanism that may explain amyloid deposition caused by the slow turnover of synaptic Zn2+ released during glutamatergic synaptic transmission (Fig. 1). This pool of Zn2+ is normally rapidly cleared by regional mechanisms that are still uncertain. Extracellular Zn2+ clearance from stimulated rat hippocampal slices is impaired by the advanced age of the donor and female sex, two prominent risk factors for extracellular amyloid pathology, which increase the average extracellular Zn2+ concentration over time and promote the aggregation of Aβ (
      • Datki Z.
      • Galik-Olah Z.
      • Janosi-Mozes E.
      • Szegedi V.
      • Kalman J.
      • Hunya A.G.
      • Fulop L.
      • Tamano H.
      • Takeda A.
      • Adlard P.A.
      • Bush A.I.
      Alzheimer risk factors age and female sex induce cortical Aβ aggregation by raising extracellular zinc.
      ). In mice, a drop in estrogen (induced by oophorectomy, recapitulating changes in human menopause) increases the levels of ZnT3 protein (
      • Lee J.-Y.
      • Kim J.-H.
      • Hong S.H.
      • Lee J.Y.
      • Cherny R.A.
      • Bush A.I.
      • Palmiter R.D.
      • Koh J.-Y.
      Estrogen decreases zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain.
      ).
      Clioquinol (5-chloro-7-iodoquinolin-8-ol) was originally identified as a copper/zinc chelator and ameliorated both amyloid pathology and cognitive loss in APP transgenic models of AD (
      • Cherny R.A.
      • Atwood C.S.
      • Xilinas M.E.
      • Gray D.N.
      • Jones W.D.
      • McLean C.A.
      • Barnham K.J.
      • Volitakis I.
      • Fraser F.W.
      • Kim Y.
      • Huang X.
      • Goldstein L.E.
      • Moir R.D.
      • Lim J.T.
      • Beyreuther K.
      • et al.
      Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.
      ,
      • Grossi C.
      • Francese S.
      • Casini A.
      • Rosi M.C.
      • Luccarini I.
      • Fiorentini A.
      • Gabbiani C.
      • Messori L.
      • Moneti G.
      • Casamenti F.
      Clioquinol decreases amyloid-beta burden and reduces working memory impairment in a transgenic mouse model of Alzheimer's disease.
      ). A 36-weeks phase 2 clinical trial of clioquinol for AD significantly slowed deterioration (
      • Ritchie C.W.
      • Bush A.I.
      • Mackinnon A.
      • Macfarlane S.
      • Mastwyk M.
      • MacGregor L.
      • Kiers L.
      • Cherny R.
      • Li Q.X.
      • Tammer A.
      • Carrington D.
      • Mavros C.
      • Volitakis I.
      • Xilinas M.
      • Ames D.
      • et al.
      Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial.
      ), but the drug was supplanted for development by PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol), which was more easily synthesized. Like clioquinol, PBT2 rescued the amyloid burden, lowered phosphorylated tau, and rapidly improved memory performance in the APP/PS1 transgenic model of AD (
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ).
      In a small phase 2a double-blind, placebo-controlled, randomized controlled trial (RCT) of PBT2 for AD (n = 29 placebo versus n = 29,250 mg/day), PBT2 caused significant executive function improvement in only 12 weeks of treatment (
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Batsman S.
      • Ames D.
      • Harrison J.
      • Masters C.L.
      • Targum S.
      • Bush A.I.
      • Murdoch R.
      • Wilson J.
      • Ritchie C.W.
      PBT2 EURO Study Group
      Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial.
      ,
      • Faux N.G.
      • Ritchie C.W.
      • Gunn A.
      • Rembach A.
      • Tsatsanis A.
      • Bedo J.
      • Harrison J.
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Ingelsson M.
      • Masters C.L.
      • Tanzi R.E.
      • Cummings J.L.
      • Herd C.M.
      • et al.
      PBT2 rapidly improves cognition in Alzheimer's Disease: additional phase II analyses.
      ,
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Batsman S.
      • Ames D.
      • Harrison J.
      • Masters C.L.
      • Targum S.
      • Bush A.I.
      • Murdoch R.
      • Wilson J.
      • Ritchie C.W.
      Erratum: safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial.
      ). In other words, PBT2 did not just arrest boosted performance. How could a nootropic benefit from a purportedly disease-modifying drug emerge after only 12 weeks? While both clioquinol and PBT2 were developed to dissipate amyloid pathology through releasing Zn2+-bridged Aβ oligomers, this was on the presumption that Aβ aggregates were neurotoxic. A second, smaller, phase 2 RCT of PBT2 used changes in amyloid burden by PiB PET imaging as its primary readout. This exploratory study showed only a trend to decreasing amyloid burden after treatment with PBT2 (250 mg/d, n = 25) compared with placebo (n = 15) for 12 months (
      • Villemagne V.L.
      • Rowe C.C.
      • Barnham K.J.
      • Cherny R.
      • Woodward M.
      • Bozinosvski S.
      • Salvado O.
      • Bourgeat P.
      • Perez K.
      • Fowler C.
      • Rembach A.
      • Maruff P.
      • Ritchie C.
      • Tanzi R.
      • Masters C.L.
      A randomized, exploratory molecular imaging study targeting amyloid beta with a novel 8-OH quinoline in Alzheimer's disease: the PBT2-204 IMAGINE study.
      ), with no differences in cognitive performance. The study was underpowered for a cognitive readout, and the placebo group cognitive performance did not measurably deteriorate throughout the study (a confound of smaller studies of AD). Thus, the possible nootropic boost of PBT2 in 12 weeks at the first RCT could not be caused by a reduction of amyloid burden. Indeed, the PiB ligand detects fibrillar forms of Aβ, which were never the target of PBT2 (
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ,
      • Adlard P.A.
      • Bica L.
      • White A.R.
      • Nurjono M.
      • Filiz G.
      • Crouch P.J.
      • Donnelly P.S.
      • Cappai R.
      • Finkelstein D.I.
      • Bush A.I.
      Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease.
      ). As the clinical trials were underway, the mechanism of action of both clioquinol and PBT2 was further investigated, and it became appreciated that these compounds are not high-affinity chelators that lower brain metals, but rather they are copper/zinc ionophores that foster the uptake of Zn2+ and Cu2+ into cells with notable impact on multiple relevant neurochemical pathways (
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ,
      • Adlard P.A.
      • Bica L.
      • White A.R.
      • Nurjono M.
      • Filiz G.
      • Crouch P.J.
      • Donnelly P.S.
      • Cappai R.
      • Finkelstein D.I.
      • Bush A.I.
      Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease.
      ,
      • Crouch P.J.
      • Savva M.S.
      • Hung L.W.
      • Donnelly P.S.
      • Mot A.I.
      • Parker S.J.
      • Greenough M.A.
      • Volitakis I.
      • Adlard P.A.
      • Cherny R.A.
      • Masters C.L.
      • Bush A.I.
      • Barnham K.J.
      • White A.R.
      The Alzheimer's therapeutic PBT2 promotes amyloid-β degradation and GSK3 phosphorylation via a metal chaperone activity.
      ). Thus, it became apparent that these ionophores might be therapeutic not by clearing amyloid but by normalizing the bioavailability of these essential metal ions otherwise trapped in Aβ aggregates (Fig. 2). The Zn2+ released during glutamatergic neurotransmission must be recycled to maintain intracellular stores for various physiological events, including maintaining the expression of the N-methyl-D-aspartate (NMDA) receptor submits. The trapping of Zn2+ by extracellular Aβ aggregates can impair neuronal function by causing a deficiency of intracellular Zn2+ (
      • Datki Z.
      • Galik-Olah Z.
      • Janosi-Mozes E.
      • Szegedi V.
      • Kalman J.
      • Hunya A.G.
      • Fulop L.
      • Tamano H.
      • Takeda A.
      • Adlard P.A.
      • Bush A.I.
      Alzheimer risk factors age and female sex induce cortical Aβ aggregation by raising extracellular zinc.
      ), leading to deficiencies of ProSAP2/Shank3 postsynaptic density assembly (
      • Grabrucker A.M.
      • Schmeisser M.J.
      • Udvardi P.T.
      • Arons M.
      • Schoen M.
      • Woodling N.S.
      • Andreasson K.I.
      • Hof P.R.
      • Buxbaum J.D.
      • Garner C.C.
      • Boeckers T.M.
      Amyloid beta protein-induced zinc sequestration leads to synaptic loss via dysregulation of the ProSAP2/Shank3 scaffold.
      ), deviation of zinc from S100B and NMDA receptor targets (
      • Deshpande A.
      • Kawai H.
      • Metherate R.
      • Glabe C.G.
      • Busciglio J.
      A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation at excitatory synapses.
      ,
      • Hagmeyer S.
      • Cristovao J.S.
      • Mulvihill J.J.E.
      • Boeckers T.M.
      • Gomes C.M.
      • Grabrucker A.M.
      Zinc binding to S100B affords regulation of trace metal homeostasis and excitotoxicity in the brain.
      ), as well as interfering with the metabotropic zinc receptor, GPR39 (
      • Abramovitch-Dahan C.
      • Asraf H.
      • Bogdanovic M.
      • Sekler I.
      • Bush A.I.
      • Hershfinkel M.
      Amyloid beta attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca(2+) , ERK1/2 and Clusterin signaling in neurons.
      ). Drug candidates such as clioquinol and PBT2 may act, therefore, to normalize the distribution of Zn2+ by facilitating its reuptake and distribution to intracellular targets, as demonstrated in models of autism (
      • Lee E.-J.
      • Lee H.
      • Huang T.-N.
      • Chung C.
      • Shin W.
      • Kim K.
      • Koh J.-Y.
      • Hsueh Y.-P.
      • Kim E.
      Trans-synaptic zinc mobilization improves social interaction in two mouse models of autism through NMDAR activation.
      ), rather than acting as chelators or reversing Aβ aggregation.
      Figure thumbnail gr2
      Figure 2Potential mechanisms of PBT2 in Alzheimer's disease. Soluble interstitial Aβ reacts with extracellular Zn2+ and Cu2+ to form protease-resistant Aβ oligomers and aggregates, which are in dissociable equilibrium with the soluble Aβ species. PBT2 reacts with accessible Zn2+ and Cu2+, promoting dissolution or uptake and degradation of the aggregates. PBT2 also dissociates Zn2+ and Cu2+ from being trapped by Aβ, neutralizing the charge of the metal ion and allowing it to passively move across cell membranes. This promotes the recycling of Zn2+ and Cu2+ from the cleft, normalizing functional fluxes, and intracellular metal pools. Aβ, amyloid β; APP, amyloid precursor protein; PBT2, 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol.
      While ZnT3 is responsible for supplying the extracellular Zn2+ that promotes extracellular Aβ aggregates, its expression is essential for maintaining cognition with aging, as demonstrated with the accelerated decline in cognition in aging ZnT3 knockout mice (
      • Adlard P.A.
      • Parncutt J.M.
      • Finkelstein D.I.
      • Bush A.I.
      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ). Notably, cortical ZnT3 levels markedly decline with both mouse and human aging and decline even further in AD (
      • Adlard P.A.
      • Parncutt J.M.
      • Finkelstein D.I.
      • Bush A.I.
      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ). These changes are associated with decreases in essential components of the synaptic architecture, such as NMDA receptor subunits and PSD95 (
      • Adlard P.A.
      • Parncutt J.M.
      • Finkelstein D.I.
      • Bush A.I.
      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ), and are recapitulated in AD, AD models, and neuronal models treated with Aβ, where Zn2+ is trapped in the aggregates causing relative intracellular deficiency (
      • Datki Z.
      • Galik-Olah Z.
      • Janosi-Mozes E.
      • Szegedi V.
      • Kalman J.
      • Hunya A.G.
      • Fulop L.
      • Tamano H.
      • Takeda A.
      • Adlard P.A.
      • Bush A.I.
      Alzheimer risk factors age and female sex induce cortical Aβ aggregation by raising extracellular zinc.
      ,
      • Grabrucker A.M.
      • Schmeisser M.J.
      • Udvardi P.T.
      • Arons M.
      • Schoen M.
      • Woodling N.S.
      • Andreasson K.I.
      • Hof P.R.
      • Buxbaum J.D.
      • Garner C.C.
      • Boeckers T.M.
      Amyloid beta protein-induced zinc sequestration leads to synaptic loss via dysregulation of the ProSAP2/Shank3 scaffold.
      ). Decreased cortical ZnT3 levels have also been reported in Parkinson’s disease dementia and Lewy Body Disease (
      • Whitfield D.R.
      • Vallortigara J.
      • Alghamdi A.
      • Hortobágyi T.
      • Ballard C.
      • Thomas A.J.
      • O'Brien J.T.
      • Aarsland D.
      • Francis P.T.
      Depression and synaptic zinc regulation in Alzheimer disease, dementia with Lewy bodies, and Parkinson disease dementia.
      ,
      • Whitfield D.R.
      • Vallortigara J.
      • Alghamdi A.
      • Howlett D.
      • Hortobágyi T.
      • Johnson M.
      • Attems J.
      • Newhouse S.
      • Ballard C.
      • Thomas A.J.
      • O'Brien J.T.
      • Aarsland D.
      • Francis P.T.
      Assessment of ZnT3 and PSD95 protein levels in Lewy body dementias and Alzheimer's disease: association with cognitive impairment.
      ). Higher levels of ZnT3 were associated with slower antecedent cognitive decline in an unbiased large-scale proteomic analysis of postmortem brain from two tissue banks, even when adjusted for AD pathology (
      • Wingo A.P.
      • Dammer E.B.
      • Breen M.S.
      • Logsdon B.A.
      • Duong D.M.
      • Troncosco J.C.
      • Thambisetty M.
      • Beach T.G.
      • Serrano G.E.
      • Reiman E.M.
      • Caselli R.J.
      • Lah J.J.
      • Seyfried N.T.
      • Levey A.I.
      • Wingo T.S.
      Large-scale proteomic analysis of human brain identifies proteins associated with cognitive trajectory in advanced age.
      ). Critically, treatment of ZnT3 knockout mice (with no amyloid) with the zinc/copper ionophore, clioquinol, for 6 weeks corrects the early onset cognitive deficits and normalizes changes in synaptic proteins (
      • Adlard P.A.
      • Parncutt J.
      • Lal V.
      • James S.
      • Hare D.
      • Doble P.
      • Finkelstein D.I.
      • Bush A.I.
      Metal chaperones prevent zinc-mediated cognitive decline.
      ). Similarly, treatment with PBT2 of normal old (22 months) C57Bl6 mice (also without amyloid) restores age-dependent cognitive deficits within 12 days, while rejuvenating synaptic architecture and markers, decreasing phosphorylated tau and significantly increasing zinc (but not copper) in the CA1 hippocampal region but not in any other cortical region (
      • Adlard P.A.
      • Sedjahtera A.
      • Gunawan L.
      • Bray L.
      • Hare D.
      • Lear J.
      • Doble P.
      • Bush A.I.
      • Finkelstein D.I.
      • Cherny R.A.
      A novel approach to rapidly prevent age-related cognitive decline.
      ). The regional selectivity of the zinc elevation in PBT2-treated old mice probably reflects the greater dynamic zinc release and uptake physiology in this region, where zinc turnover is impaired with age (
      • Datki Z.
      • Galik-Olah Z.
      • Janosi-Mozes E.
      • Szegedi V.
      • Kalman J.
      • Hunya A.G.
      • Fulop L.
      • Tamano H.
      • Takeda A.
      • Adlard P.A.
      • Bush A.I.
      Alzheimer risk factors age and female sex induce cortical Aβ aggregation by raising extracellular zinc.
      ). These results strongly argue that the benefits of zinc ionophore treatment of amyloid-bearing transgenic mice with clioquinol or PBT2 (
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ,
      • Cherny R.A.
      • Atwood C.S.
      • Xilinas M.E.
      • Gray D.N.
      • Jones W.D.
      • McLean C.A.
      • Barnham K.J.
      • Volitakis I.
      • Fraser F.W.
      • Kim Y.
      • Huang X.
      • Goldstein L.E.
      • Moir R.D.
      • Lim J.T.
      • Beyreuther K.
      • et al.
      Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.
      ) are mediated by restoring cortical zinc homeostasis and that the amyloid aggregates are a proxy for perturbed zinc trafficking that may exaggerate the problem by trapping more zinc. Therefore, the significant cognitive improvement in a strikingly rapid time frame, 12 weeks, observed in trials (
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Batsman S.
      • Ames D.
      • Harrison J.
      • Masters C.L.
      • Targum S.
      • Bush A.I.
      • Murdoch R.
      • Wilson J.
      • Ritchie C.W.
      PBT2 EURO Study Group
      Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial.
      ,
      • Faux N.G.
      • Ritchie C.W.
      • Gunn A.
      • Rembach A.
      • Tsatsanis A.
      • Bedo J.
      • Harrison J.
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Ingelsson M.
      • Masters C.L.
      • Tanzi R.E.
      • Cummings J.L.
      • Herd C.M.
      • et al.
      PBT2 rapidly improves cognition in Alzheimer's Disease: additional phase II analyses.
      ,
      • Lannfelt L.
      • Blennow K.
      • Zetterberg H.
      • Batsman S.
      • Ames D.
      • Harrison J.
      • Masters C.L.
      • Targum S.
      • Bush A.I.
      • Murdoch R.
      • Wilson J.
      • Ritchie C.W.
      Erratum: safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial.
      ) is consistent with the time frame in cognitive improvement in each report of these animal models treated with zinc ionophores and therefore most likely reflects the treatment benefits of correcting cortical zinc homeostasis.
      Disruption of cortical zinc homeostasis in AD has not been reflected in reports of bulk zinc levels from postmortem tissue (
      • Grabrucker A.M.
      • Schmeisser M.J.
      • Udvardi P.T.
      • Arons M.
      • Schoen M.
      • Woodling N.S.
      • Andreasson K.I.
      • Hof P.R.
      • Buxbaum J.D.
      • Garner C.C.
      • Boeckers T.M.
      Amyloid beta protein-induced zinc sequestration leads to synaptic loss via dysregulation of the ProSAP2/Shank3 scaffold.
      ,
      • Graham S.F.
      • Nasaruddin M.B.
      • Carey M.
      • Holscher C.
      • McGuinness B.
      • Kehoe P.G.
      • Love S.
      • Passmore P.
      • Elliott C.T.
      • Meharg A.A.
      • Green B.D.
      Age-associated changes of brain copper, iron, and zinc in Alzheimer's disease and dementia with Lewy bodies.
      ,
      • Danscher G.
      • Jensen K.B.
      • Frederickson C.J.
      • Kemp K.
      • Andreasen A.
      • Juhl S.
      • Stoltenberg M.
      • Ravid R.
      Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material.
      ,
      • Schrag M.
      • Mueller C.
      • Oyoyo U.
      • Smith M.A.
      • Kirsch W.M.
      Iron, zinc and copper in the Alzheimer's disease brain: a quantitative meta-analysis. Some insight on the influence of citation bias on scientific opinion.
      ,
      • Panayi A.E.
      • Spyrou N.M.
      • Iversen B.S.
      • White M.A.
      • Part P.
      Determination of cadmium and zinc in Alzheimer's brain tissue using inductively coupled plasma mass spectrometry.
      ). However, factors including the accuracy of diagnosis, statistical power, methods of sample preparation, and detection limits may have made changes inconsistent between studies. Also, the total tissue zinc levels might not rise until the plaque burden is severe (
      • Religa D.
      • Strozyk D.
      • Cherny R.A.
      • Volitaskis I.
      • Haroutunian V.
      • Winblad B.
      • Naslund J.
      • Bush A.I.
      Elevated cortical zinc in Alzheimer disease.
      ). In the brain of the aged macaque monkey, the difference in zinc concentrations of district brain regions could account for the density of plaques in that region, regardless of the total Aβ level (
      • Ichinohe N.
      • Hayashi M.
      • Wakabayashi K.
      • Rockland K.S.
      Distribution and progression of amyloid-beta deposits in the amygdala of the aged macaque monkey, and parallels with zinc distribution.
      ). This is reminiscent of the spatial association of plaque burden in APP transgenic mice with cortical layers that are most rich in exchangeable zinc (
      • Stoltenberg M.
      • Bush A.I.
      • Bach G.
      • Smidt K.
      • Larsen A.
      • Rungby J.
      • Lund S.
      • Doering P.
      • Danscher G.
      Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency.
      ).
      The blood–brain barrier prevents passive fluctuations of plasma zinc from being transduced into the brain. Nevertheless, some reports have explored the impact of dietary zinc on Aβ transgenic models, with inconsistent results reported. Prenatal and postnatal zinc-enriched diets in Tg2576 and TgCRND8 were described to induce accelerated cognitive impairment in these mice (
      • Linkous D.H.
      • Adlard P.A.
      • Wanschura P.B.
      • Conko K.M.
      • Flinn J.M.
      The effects of enhanced zinc on spatial memory and plaque formation in transgenic mice.
      ,
      • Flinn J.M.
      • Bozzelli P.L.
      • Adlard P.A.
      • Railey A.M.
      Spatial memory deficits in a mouse model of late-onset Alzheimer's disease are caused by zinc supplementation and correlate with amyloid-beta levels.
      ). Zinc supplementation to APP/PS1 mice was reported to induce Aβ deposition as well as impaired spatial memory (
      • Wang C.Y.
      • Wang T.
      • Zheng W.
      • Zhao B.L.
      • Danscher G.
      • Chen Y.H.
      • Wang Z.Y.
      Zinc overload enhances APP cleavage and Abeta deposition in the Alzheimer mouse brain.
      ) but did not affect Tg2576 mice (
      • Akiyama H.
      • Hosokawa M.
      • Kametani F.
      • Kondo H.
      • Chiba M.
      • Fukushima M.
      • Tabira T.
      Long-term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AbetaPP and AbetaPP/tau transgenic mice.
      ). Conversely, dietary zinc deficiency enlarged plaque size in APP/PS1 mice (
      • Stoltenberg M.
      • Bush A.I.
      • Bach G.
      • Smidt K.
      • Larsen A.
      • Rungby J.
      • Lund S.
      • Doering P.
      • Danscher G.
      Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency.
      ). A drosophila model of AD overexpressing Aβ was reported to express eye damage that was exaggerated by dietary supplements of zinc or copper but rescued by zinc/copper chelators (
      • Hua H.
      • Munter L.
      • Harmeier A.
      • Georgiev O.
      • Multhaup G.
      • Schaffner W.
      Toxicity of Alzheimer's disease-associated Abeta peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability.
      ). Nutritional zinc deficiency is common in old age and exacerbates age-dependent cognitive loss in rodents, which can be rescued by zinc supplementation (
      • Sandusky-Beltran L.A.
      • Manchester B.L.
      • McNay E.C.
      Supplementation with zinc in rats enhances memory and reverses an age-dependent increase in plasma copper.
      ). Zinc supplementation to 3xTg-AD mice in adulthood was reported to delay hippocampal-dependent memory deficits and reduce both Aβ and tau (
      • Corona C.
      • Masciopinto F.
      • Silvestri E.
      • Viscovo A.D.
      • Lattanzio R.
      • Sorda R.L.
      • Ciavardelli D.
      • Goglia F.
      • Piantelli M.
      • Canzoniero L.M.
      • Sensi S.L.
      Dietary zinc supplementation of 3xTg-AD mice increases BDNF levels and prevents cognitive deficits as well as mitochondrial dysfunction.
      ). However, a meta-analysis of clinical trials of zinc supplementation found no evidence of benefit in treating AD (
      • Loef M.
      • Stillfried N.v.
      • Walach H.
      Zinc diet and Alzheimer's disease: a systematic review.
      ).
      Changes in the expression of several zinc transporting proteins have also been reported in studies of postmortem brain tissue from AD cases and models (
      • Xu Y.
      • Xiao G.
      • Liu L.
      • Lang M.
      Zinc transporters in Alzheimer's disease.
      ), although it is difficult to know whether these changes are, like ZnT3, potentially upstream in the pathological process or whether they represent homeostatic responses. The message RNA levels of several ZnT family proteins such as ZnT1, ZnT4, and ZnT6 are increased in AD tissue and correlate with Braak pathological staging (
      • Beyer N.
      • Coulson D.T.
      • Heggarty S.
      • Ravid R.
      • Hellemans J.
      • Irvine G.B.
      • Johnston J.A.
      Zinc transporter mRNA levels in Alzheimer's disease postmortem brain.
      ). ZnT10 was reported reduced in the frontal cortex of AD subjects and APP/PS1 mice (
      • Bosomworth H.J.
      • Adlard P.A.
      • Ford D.
      • Valentine R.A.
      Altered expression of ZnT10 in Alzheimer's disease brain.
      ). The protein level of ZnT6 has been reported to be elevated in the hippocampus/parahippocampal gyrus region of pathologically confirmed AD cases, but the level of ZnT1 was significantly decreased in the same region (
      • Lyubartseva G.
      • Smith J.L.
      • Markesbery W.R.
      • Lovell M.A.
      Alterations of zinc transporter proteins ZnT-1, ZnT-4 and ZnT-6 in preclinical Alzheimer's disease brain.
      ). An inhibitor of cellular zinc export, 4-hydroxynonenal, is induced by lipid peroxidation, a feature of ferroptosis (
      • Smith J.L.
      • Xiong S.
      • Lovell M.A.
      4-Hydroxynonenal disrupts zinc export in primary rat cortical cells.
      ). Cytoplasmic-free Zn2+ might also be elevated in AD from a depletion of metallothionein III (
      • Uchida Y.
      • Takio K.
      • Titani K.
      • Ihara Y.
      • Tomonaga M.
      The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein.
      ,
      • Yu W.H.
      • Lukiw W.J.
      • Bergeron C.
      • Niznik H.B.
      • Fraser P.E.
      Metallothionein III is reduced in Alzheimer's disease.
      ,
      • Tsuji S.
      • Kobayashi H.
      • Uchida Y.
      • Ihara Y.
      • Miyatake T.
      Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer's disease.
      ), which is the main zinc storage protein in neurons.
      Interestingly, zinc may regulate the production of Aβ via affecting the secretases that are responsible for its production. The activity of β-secretase responsible for APP cleavage into the nonamylogenic pathway, a disintegrin and metalloprotease 10, is a zinc metalloproteinase, and mutation of its zinc-binding site abolishes its activity (
      • Lammich S.
      • Kojro E.
      • Postina R.
      • Gilbert S.
      • Pfeiffer R.
      • Jasionowski M.
      • Haass C.
      • Fahrenholz F.
      Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease.
      ). Zinc is also reported to inhibit β-secretase activity in vitro (
      • Hoke D.E.
      • Tan J.L.
      • Ilaya N.T.
      • Culvenor J.G.
      • Smith S.J.
      • White A.R.
      • Masters C.L.
      • Evin G.M.
      In vitro gamma-secretase cleavage of the Alzheimer's amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc.
      ) and cell culture by induction of APP-C99 fragment dimerization (
      • Gerber H.
      • Wu F.
      • Dimitrov M.
      • Garcia Osuna G.M.
      • Fraering P.C.
      Zinc and copper differentially modulate amyloid precursor protein processing by gamma-secretase and amyloid-beta peptide production.
      ), indicating that increased zinc may limit Aβ production. At similar concentrations to those that inhibit β-secretase activity in vitro, zinc is also described to promote the production of Aβ43 (
      • Gerber H.
      • Wu F.
      • Dimitrov M.
      • Garcia Osuna G.M.
      • Fraering P.C.
      Zinc and copper differentially modulate amyloid precursor protein processing by gamma-secretase and amyloid-beta peptide production.
      ).
      There are several reports of zinc interacting also with the other major proteins implicated in AD. Zinc is reported to increase presenilin 1 expression (
      • Greenough M.A.
      • Volitaskis I.
      • Li Q.-X.
      • Laughton K.M.
      • Evin G.
      • Ho M.
      • Dalziel A.H.
      • Camakaris J.
      • Bush A.I.
      Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity.
      ) and to affect the stability of apolipoprotein E (ApoE), particularly ApoE4 (
      • Xu H.
      • Gupta V.B.
      • Martins I.J.
      • Martins R.N.
      • Fowler C.J.
      • Bush A.I.
      • Finkelstein D.I.
      • Adlard P.A.
      Zinc affects the proteolytic stability of Apolipoprotein E in an isoform-dependent way.
      ). Conversely, presenilin 1 and ApoE expression have been reported to play essential roles in maintaining cellular and neuronal zinc trafficking (
      • Greenough M.A.
      • Volitaskis I.
      • Li Q.-X.
      • Laughton K.M.
      • Evin G.
      • Ho M.
      • Dalziel A.H.
      • Camakaris J.
      • Bush A.I.
      Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity.
      ,
      • Lee J.Y.
      • Cho E.
      • Kim T.Y.
      • Kim D.K.
      • Palmiter R.D.
      • Volitakis I.
      • Kim J.S.
      • Bush A.I.
      • Koh J.Y.
      Apolipoprotein E ablation decreases synaptic vesicular zinc in the brain.
      ). Free Zn2+ is reported to promote tau phosphorylation and aggregation (Fig. 1). Several kinases and phosphorylases were suggested as mediators of zinc-induced tau hyperphosphorylation in cell culture and mice, including glycogen synthase kinase 3β, cyclin-dependent kinase 5, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and protein phosphatase 2A (PP2A) (
      • An W.-L.
      • Bjorkdahl C.
      • Liu R.
      • Cowburn R.F.
      • Winblad B.
      • Pei J.-J.
      Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells.
      ,
      • Pei J.-J.
      • An W.-L.
      • Zhou X.-W.
      • Nishimura T.
      • Norberg J.
      • Benedikz E.
      • Götz J.
      • Winblad B.
      P70 S6 kinase mediates tau phosphorylation and synthesis.
      ,
      • Kim I.
      • Park E.J.
      • Seo J.
      • Ko S.J.
      • Lee J.
      • Kim C.H.
      Zinc stimulates tau S214 phosphorylation by the activation of Raf/mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase pathway.
      ,
      • Sun X.Y.
      • Wei Y.P.
      • Xiong Y.
      • Wang X.C.
      • Xie A.J.
      • Wang X.L.
      • Yang Y.
      • Wang Q.
      • Lu Y.M.
      • Liu R.
      • Wang J.Z.
      Synaptic released zinc promotes tau hyperphosphorylation by inhibition of protein phosphatase 2A (PP2A).
      ,
      • Xiong Y.
      • Jing X.P.
      • Zhou X.W.
      • Wang X.L.
      • Yang Y.
      • Sun X.Y.
      • Qiu M.
      • Cao F.Y.
      • Lu Y.M.
      • Liu R.
      • Wang J.Z.
      Zinc induces protein phosphatase 2A inactivation and tau hyperphosphorylation through Src dependent PP2A (tyrosine 307) phosphorylation.
      ,
      • Harris F.M.
      • Brecht W.J.
      • Xu Q.
      • Mahley R.W.
      • Huang Y.
      Increased tau phosphorylation in apolipoprotein E4 transgenic mice is associated with activation of extracellular signal-regulated kinase: modulation by zinc.
      ,
      • Lei P.
      • Ayton S.
      • Bush A.I.
      • Adlard P.A.
      GSK-3 in neurodegenerative diseases.
      ). Alternatively, zinc may facilitate the bridging between Cys-291 and Cys-322 of tau for aggregation, evidenced by point mutation of these sites preventing zinc-induced tau aggregation (
      • Mo Z.-Y.
      • Zhu Y.-Z.
      • Zhu H.-L.
      • Fan J.-B.
      • Chen J.
      • Liang Y.
      Low micromolar zinc accelerates the fibrillization of human tau via bridging of Cys-291 and Cys-322.
      ). This may as well lead to toxicity, as zinc can also directly bind to tau protein to promote neurotoxicity independent of hyperphosphorylation in a drosophila hTauR406W model (
      • Huang Y.
      • Wu Z.
      • Cao Y.
      • Lang M.
      • Lu B.
      • Zhou B.
      Zinc binding directly regulates tau toxicity independent of tau hyperphosphorylation.
      ). Zinc supplementation also has been reported to facilitate the neurodegeneration and tangle formation in P301L mice, a model of tauopathy (
      • Craven K.M.
      • Kochen W.R.
      • Hernandez C.M.
      • Flinn J.M.
      Zinc exacerbates tau pathology in a tau mouse model.
      ).
      The slowing of zinc synaptic turnover with normal aging could lie upstream of the amyloid pathology of AD as well as some facets of cognitive impairment. This warrants more in-depth research, especially because the nootropic benefits of correcting in aging mice without amyloid are provocative and could be the basis of drug interventions for which there is already some clinical trial evidence. The mechanisms for clearing the synapse of zinc released during neurotransmission need to be elaborated urgently. While zinc dyshomeostasis may hamper neuronal function, its connection to neurodegeneration is still unclear, as is the mechanism for synaptic loss in ZnT3 knockout mice that is corrected by zinc ionophore treatment (
      • Adlard P.A.
      • Parncutt J.M.
      • Finkelstein D.I.
      • Bush A.I.
      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ,
      • Adlard P.A.
      • Cherny R.A.
      • Finkelstein D.I.
      • Gautier E.
      • Robb E.
      • Cortes M.
      • Volitakis I.
      • Liu X.
      • Smith J.P.
      • Perez K.
      • Laughton K.
      • Li Q.X.
      • Charman S.A.
      • Nicolazzo J.A.
      • Wilkins S.
      • et al.
      Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta.
      ,
      • Adlard P.A.
      • Bica L.
      • White A.R.
      • Nurjono M.
      • Filiz G.
      • Crouch P.J.
      • Donnelly P.S.
      • Cappai R.
      • Finkelstein D.I.
      • Bush A.I.
      Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease.
      ,
      • Adlard P.A.
      • Parncutt J.
      • Lal V.
      • James S.
      • Hare D.
      • Doble P.
      • Finkelstein D.I.
      • Bush A.I.
      Metal chaperones prevent zinc-mediated cognitive decline.
      ,
      • Adlard P.A.
      • Sedjahtera A.
      • Gunawan L.
      • Bray L.
      • Hare D.
      • Lear J.
      • Doble P.
      • Bush A.I.
      • Finkelstein D.I.
      • Cherny R.A.
      A novel approach to rapidly prevent age-related cognitive decline.
      ). Insights into this could be very relevant to understanding cognitive dysfunction in AD where there is a marked loss of ZnT3 in cortical tissue despite the accumulation of zinc in plaques (
      • Adlard P.A.
      • Parncutt J.M.
      • Finkelstein D.I.
      • Bush A.I.
      Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic and memory deficits of Alzheimer's disease?.
      ).

      Copper

      Copper is a redox-active metal that is involved in multiple metabolic activities in the brain, and it serves as the active site for a range of cuproenzymes such as ceruloplasmin (Cp), superoxide dismutase 1 (SOD1), tyrosinase, cytochrome oxidase, etc (
      • Barnham K.J.
      • Masters C.L.
      • Bush A.I.
      Neurodegenerative diseases and oxidative stress.
      ). The regulation of copper transport within all cells is mediated by Ctr1 for uptake and ATP7A/B for efflux (
      • Scheiber I.F.
      • Mercer J.F.
      • Dringen R.
      Metabolism and functions of copper in brain.
      ) (Fig. 3). Mutation of ATP7B (K832R) increases the risk for AD and causes loss of ATP7B function (
      • Mercer S.W.
      • Wang J.
      • Burke R.
      In Vivo modeling of the pathogenic effect of copper transporter mutations that cause Menkes and Wilson diseases, motor neuropathy, and susceptibility to Alzheimer's disease.
      ). Like zinc, copper concentrations in the synapse elevate transiently during neurotransmission (to 15 μM, from basal levels of ≈0.5 μM based on cerebrospinal fluid (CSF) values [
      • Strozyk D.
      • Launer L.J.
      • Adlard P.A.
      • Cherny R.A.
      • Tsatsanis A.
      • Volitakis I.
      • Blennow K.
      • Petrovitch H.
      • White L.R.
      • Bush A.I.
      Zinc and copper modulate Alzheimer Abeta levels in human cerebrospinal fluid.
      ]), but instead of being released from the bouton, is released postsynaptically upon stimulation of the NMDA receptor (
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ,
      • Schlief M.L.
      • West T.
      • Craig A.M.
      • Holtzman D.M.
      • Gitlin J.D.
      Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity.
      ). Copper can dose-dependently affect LTP, where a low concentration of copper (1 μM) inhibits hippocampal LTP (
      • Doreulee N.
      • Yanovsky Y.
      • Haas H.L.
      Suppression of long-term potentiation in hippocampal slices by copper.
      ), while a high concentration (10 μM) promotes LTP through activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (
      • Peters C.
      • Munoz B.
      • Sepulveda F.J.
      • Urrutia J.
      • Quiroz M.
      • Luza S.
      • De Ferrari G.V.
      • Aguayo L.G.
      • Opazo C.
      Biphasic effects of copper on neurotransmission in rat hippocampal neurons.
      ). Several studies have reported that copper supplementation in cultured neurons inhibits the activation of receptors for NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and gamma aminobutyric acid (
      • Vlachova V.
      • Zemkova H.
      • Vyklicky Jr., L.
      Copper modulation of NMDA responses in mouse and rat cultured hippocampal neurons.
      ,
      • Trombley P.Q.
      • Shepherd G.M.
      Differential modulation by zinc and copper of amino acid receptors from rat olfactory bulb neurons.
      ,
      • Weiser T.
      • Wienrich M.
      The effects of copper ions on glutamate receptors in cultured rat cortical neurons.
      ). However, the in vivo relevance of these findings is yet to be investigated.
      Figure thumbnail gr3
      Figure 3Copper dysregulation in Alzheimer's disease. Cu+ is taken up into neurons by CTR1 and exported by ATP7A/B. Aβ oligomers can trap extracellular Cu2+ and then embed into the membrane, forming a catalytic complex that generates H2O2. H2O2 is freely permeable and can migrate to deplete antioxidants like GSH and denature SOD1. In bulk tissue, copper levels are decreased, consistent with a decrease in the activity of ceruloplasmin. But the fraction of cytoplasmic-free Cu+ increases in AD-affected tissue, which might contribute to tau hyperphosphorylation by activation of CDK5 or GSK3β. Aβ, amyloid β; AD, Alzheimer's disease; APP, amyloid precursor protein; CDK5, cyclin-dependent kinase 5; GSH, glutathione; GSK3β, glycogen synthase kinase 3β; SOD1, superoxide dismutase 1.
      Copper concentrations in AD-affected brain tissue are reported to be lower compared with healthy control tissue (
      • Deibel M.A.
      • Ehmann W.D.
      • Markesbery W.R.
      Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress.
      ,
      • Magaki S.
      • Raghavan R.
      • Mueller C.
      • Oberg K.C.
      • Vinters H.V.
      • Kirsch W.M.
      Iron, copper, and iron regulatory protein 2 in Alzheimer's disease and related dementias.
      ,
      • Xu J.
      • Church S.J.
      • Patassini S.
      • Begley P.
      • Waldvogel H.J.
      • Curtis M.A.
      • Faull R.L.M.
      • Unwin R.D.
      • Cooper G.J.S.
      Evidence for widespread, severe brain copper deficiency in Alzheimer's dementia.
      ,
      • Plantin L.O.
      • Lying-Tunell U.
      • Kristensson K.
      Trace elements in the human central nervous system studied with neutron activation analysis.
      ,
      • James S.A.
      • Volitakis I.
      • Adlard P.A.
      • Duce J.A.
      • Masters C.L.
      • Cherny R.A.
      • Bush A.I.
      Elevated labile Cu is associated with oxidative pathology in Alzheimer disease.
      ,
      • Rembach A.
      • Hare D.J.
      • Lind M.
      • Fowler C.J.
      • Cherny R.A.
      • McLean C.
      • Bush A.I.
      • Masters C.L.
      • Roberts B.R.
      Decreased copper in Alzheimer's disease brain is predominantly in the soluble extractable fraction.
      ) and accompanied by decreased concentrations of cuproproteins such as SOD1 (
      • Bayer T.A.
      • Schafer S.
      • Simons A.
      • Kemmling A.
      • Kamer T.
      • Tepest R.
      • Eckert A.
      • Schussel K.
      • Eikenberg O.
      • Sturchler-Pierrat C.
      • Abramowski D.
      • Staufenbiel M.
      • Multhaup G.
      Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Abeta production in APP23 transgenic mice.
      ). Despite the decrease in total copper in AD-affected tissue, the proportion of “labile” or loosely bound exchangeable copper ions was increased, indicating a disruption of the average coordination environment of cellular copper ions in the tissue (
      • James S.A.
      • Volitakis I.
      • Adlard P.A.
      • Duce J.A.
      • Masters C.L.
      • Cherny R.A.
      • Bush A.I.
      Elevated labile Cu is associated with oxidative pathology in Alzheimer disease.
      ). Furthermore, there is evidence that copper concentrates with other metals in amyloid plaques (vide infra). Thus, there is a change in the distribution of copper in the AD brain tissue where it is deficient in the cells but trapped in the extracellular plaques. This complex picture is consistent with experimental results, reviewed here, that intracellular copper deficiency promotes Aβ production, whereas extracellular Cu2+ pooling can promote Aβ precipitation (under acidic conditions) and oxidative cross-linking and modification. Therefore, neither copper chelation nor copper supplementation are likely to have unopposed benefits, and the theoretical ideal agent would mobilize extracellular Cu2+ to be taken back into the cell. Here, we review the evidence for this.
      Lowering cellular copper has been shown to increase Aβ production (
      • Gerber H.
      • Wu F.
      • Dimitrov M.
      • Garcia Osuna G.M.
      • Fraering P.C.
      Zinc and copper differentially modulate amyloid precursor protein processing by gamma-secretase and amyloid-beta peptide production.
      ,
      • Acevedo K.M.
      • Hung Y.H.
      • Dalziel A.H.
      • Li Q.X.
      • Laughton K.
      • Wikhe K.
      • Rembach A.
      • Roberts B.
      • Masters C.L.
      • Bush A.I.
      • Camakaris J.
      Copper promotes the trafficking of the amyloid precursor protein.
      ,
      • Cater M.A.
      • McInnes K.T.
      • Li Q.-X.
      • Volitaskis I.
      • La Fontaine S.
      • Mercer J.F.B.
      • Bush A.I.
      Intracellular copper deficiency increases amyloid-beta secretion by diverse mechanisms.
      ,
      • Treiber C.
      • Quadir M.A.
      • Voigt P.
      • Radowski M.
      • Xu S.
      • Munter L.M.
      • Bayer T.A.
      • Schaefer M.
      • Haag R.
      • Multhaup G.
      Cellular copper import by nanocarrier systems, intracellular availability, and effects on amyloid beta peptide secretion.
      ,
      • Wang Z.
      • Zhang Y.H.
      • Zhang W.
      • Gao H.L.
      • Zhong M.L.
      • Huang T.T.
      • Guo R.F.
      • Liu N.N.
      • Li D.D.
      • Li Y.
      • Wang Z.Y.
      • Zhao P.
      Copper chelators promote nonamyloidogenic processing of AbetaPP via MT1/2/CREB-dependent signaling pathways in AbetaPP/PS1 transgenic mice.
      ,
      • Borchardt T.
      • Camakaris J.
      • Cappai R.
      • Masters C.L.
      • Beyreuther K.
      • Multhaup G.
      Copper inhibits beta-amyloid production and stimulates the non-amyloidogenic pathway of amyloid-precursor-protein secretion.
      ), as does a deficiency in the copper chaperone of SOD1 (
      • Gray E.H.
      • De Vos K.J.
      • Dingwall C.
      • Perkinton M.S.
      • Miller C.C.
      Deficiency of the copper chaperone for superoxide dismutase increases amyloid-beta production.
      ). Copper (and zinc) added to human CSF promotes the degradation of Aβ, also consistent with the inverse association between levels of these metals in the CSF and levels of Aβ (
      • Strozyk D.
      • Launer L.J.
      • Adlard P.A.
      • Cherny R.A.
      • Tsatsanis A.
      • Volitakis I.
      • Blennow K.
      • Petrovitch H.
      • White L.R.
      • Bush A.I.
      Zinc and copper modulate Alzheimer Abeta levels in human cerebrospinal fluid.
      ). Earlier studies found that APP can bind to and reduce Cu2+ through a site on its N-terminal ectodomain, remote from the Aβ sequence (
      • Multhaup G.
      • Schlicksupp A.
      • Hesse L.
      • Beher D.
      • Ruppert T.
      • Masters C.L.
      • Beyreuther K.
      The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I).
      ,
      • White A.R.
      • Multhaup G.
      • Maher F.
      • Bellingham S.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      The Alzheimer's disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures.
      ,
      • Leong S.L.
      • Young T.R.
      • Barnham K.J.
      • Wedd A.G.
      • Hinds M.G.
      • Xiao Z.
      • Cappai R.
      Quantification of copper binding to amyloid precursor protein domain 2 and its Caenorhabditis elegans ortholog. Implications for biological function.
      ,
      • Young T.R.
      • Pukala T.L.
      • Cappai R.
      • Wedd A.G.
      • Xiao Z.
      The human amyloid precursor protein binds copper ions dominated by a picomolar-affinity site in the helix-rich E2 domain.
      ). This may subserve a physiological purpose, possibly in copper homeostasis, because APP expression is upregulated by copper (
      • Bellingham S.A.
      • Lahiri D.K.
      • Maloney B.
      • La Fontaine S.
      • Multhaup G.
      • Camakaris J.
      Copper depletion down-regulates expression of the Alzheimer's disease amyloid-beta precursor protein gene.
      ) and increased APP expression lowers brain, neuronal, and tissue copper levels (
      • White A.R.
      • Reyes R.
      • Mercer J.F.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Multhaup G.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice.
      ,
      • Maynard C.J.
      • Cappai R.
      • Volitakis I.
      • Cherny R.A.
      • White A.R.
      • Beyreuther K.
      • Masters C.L.
      • Bush A.I.
      • Li Q.X.
      Overexpression of Alzheimer's disease amyloid-beta opposes the age-dependent elevations of brain copper and iron.
      ,
      • Bellingham S.A.
      • Ciccotosto G.D.
      • Needham B.E.
      • Fodero L.R.
      • White A.R.
      • Masters C.L.
      • Cappai R.
      • Camakaris J.
      Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts.
      ). Also, APP trafficking is sensitive to copper load (
      • Acevedo K.M.
      • Hung Y.H.
      • Dalziel A.H.
      • Li Q.X.
      • Laughton K.
      • Wikhe K.
      • Rembach A.
      • Roberts B.
      • Masters C.L.
      • Bush A.I.
      • Camakaris J.
      Copper promotes the trafficking of the amyloid precursor protein.
      ,
      • Acevedo K.M.
      • Opazo C.M.
      • Norrish D.
      • Challis L.M.
      • Li Q.X.
      • White A.R.
      • Bush A.I.
      • Camakaris J.
      Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.
      ).
      In contrast to the background parenchymal brain tissue where copper levels are decreased in AD, amyloid plaques concentrate copper in AD and mouse models of AD (
      • Lovell M.A.
      • Robertson J.D.
      • Teesdale W.J.
      • Campbell J.L.
      • Markesbery W.R.
      Copper, iron and zinc in Alzheimer's disease senile plaques.
      ,
      • James S.A.
      • Churches Q.I.
      • de Jonge M.D.
      • Birchall I.E.
      • Streltsov V.
      • McColl G.
      • Adlard P.A.
      • Hare D.J.
      Iron, copper, and zinc concentration in abeta plaques in the APP/PS1 mouse model of Alzheimer's disease correlates with metal levels in the surrounding neuropil.
      ,
      • Miller L.M.
      • Wang Q.
      • Telivala T.P.
      • Smith R.J.
      • Lanzirotti A.
      • Miklossy J.
      Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease.
      ,
      • Lanzirotti A.
      • Miller L.M.
      Amyloid plaques in PSAPP mice bind less metal than plaques in human Alzheimer's disease.
      ,
      • Dong J.
      • Atwood C.S.
      • Anderson V.E.
      • Siedlak S.L.
      • Smith M.A.
      • Perry G.
      • Carey P.R.
      Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence.
      ), supporting the possibility that copper co-aggregates with Aβ (Fig. 3). While APP has separate ectodomain copper (
      • Multhaup G.
      • Schlicksupp A.
      • Hesse L.
      • Beher D.
      • Ruppert T.
      • Masters C.L.
      • Beyreuther K.
      The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I).
      ) and zinc (
      • Bush A.I.
      • Multhaup G.
      • Moir R.D.
      • Williamson T.G.
      • Small D.H.
      • Rumble B.
      • Pollwein P.
      • Beyreuther K.
      • Masters C.L.
      A novel zinc(II) binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer's disease.
      ,
      • Bush A.I.
      • Pettingell W.H.
      • de Paradis M.
      • Tanzi R.E.
      • Wasco W.
      The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily.
      ) binding sites remote from the Aβ sequence, the copper/zinc binding site in Aβ is overlapping and only emerges once the carboxyl terminus of Aβ is cleaved from full-length APP through the activity of the presenilins. Copper-Aβ interaction was first described in 1994, where Cu2+ was observed to strikingly induce soluble dimer formation of Aβ1–40 at neutral pH (
      • Bush A.I.
      • Pettingell W.H.
      • Paradis M.D.
      • Tanzi R.E.
      Modulation of Aβ adhesiveness and secretase site cleavage by zinc.
      ), although little precipitation was noted under these conditions (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      Rapid induction of Alzheimer Aβ amyloid formation by zinc.
      ). Subsequently, Cu2+ was found to induce dramatic aggregation of Aβ1-40 under mildly physiologically acidic conditions (e.g., pH 6.8) (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.
      ) with highest apparent affinities of Cu2+ for the peptide aggregates being measured as ≈50 pM for Aβ1-40 and ≈6 aM for Aβ1-42, with the aggregates binding up to three equivalents of Cu per Aβ peptide (
      • Atwood C.S.
      • Scarpa R.C.
      • Huang X.
      • Moir R.D.
      • Jones W.D.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      Characterization of copper interactions with Alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42.
      ). The very high apparent affinity of Aβ1-42 for Cu2+ may be a product of the perturbed equilibrium of the peptide–metal complex coming out of solution, but nonetheless the peptide aggregation is reversible with chelation, evidencing proof of principle of pharmacological targeting of the metal center for reversing amyloid formation, which was recapitulated by the solubilization of Aβ from the insoluble fraction of AD-affected brain tissue by copper chelation (
      • Cherny R.A.
      • Legg J.T.
      • McLean C.A.
      • Fairlie D.P.
      • Huang X.
      • Atwood C.S.
      • Beyreuther K.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion.
      ).
      These interactions have been extensively studied since (
      • Tõugu V.
      • Karafin A.
      • Zovo K.
      • Chung R.S.
      • Howells C.
      • West A.K.
      • Palumaa P.
      Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.
      ,
      • James S.A.
      • Volitakis I.
      • Adlard P.A.
      • Duce J.A.
      • Masters C.L.
      • Cherny R.A.
      • Bush A.I.
      Elevated labile Cu is associated with oxidative pathology in Alzheimer disease.
      ,
      • Jiao Y.
      • Han D.X.
      • Yang P.
      Molecular modeling of the inhibitory mechanism of copper(II) on aggregation of amyloid beta-peptide.
      ,
      • Jiao Y.
      • Yang P.
      Mechanism of copper(II) inhibiting Alzheimer's amyloid beta-peptide from aggregation: a molecular dynamics investigation.
      ,
      • Hindo S.S.
      • Mancino A.M.
      • Braymer J.J.
      • Liu Y.
      • Vivekanandan S.
      • Ramamoorthy A.
      • Lim M.H.
      Small molecule modulators of copper-induced Abeta aggregation.
      ,
      • Folk D.S.
      • Franz K.J.
      A prochelator activated by beta-secretase inhibits Abeta aggregation and suppresses copper-induced reactive oxygen species formation.
      ,
      • Gomes L.M.
      • Vieira R.P.
      • Jones M.R.
      • Wang M.C.
      • Dyrager C.
      • Souza-Fagundes E.M.
      • Da Silva J.G.
      • Storr T.
      • Beraldo H.
      8-Hydroxyquinoline Schiff-base compounds as antioxidants and modulators of copper-mediated Abeta peptide aggregation.
      ,
      • Zhang W.
      • Huang D.
      • Huang M.
      • Huang J.
      • Wang D.
      • Liu X.
      • Nguyen M.
      • Vendier L.
      • Mazeres S.
      • Robert A.
      • Liu Y.
      • Meunier B.
      Preparation of tetradentate copper chelators as potential anti-Alzheimer agents.
      ,
      • Faller P.
      • Hureau C.
      • Berthoumieu O.
      Role of metal ions in the self-assembly of the Alzheimer's amyloid-beta peptide.
      ,
      • Pedersen J.T.
      • Ostergaard J.
      • Rozlosnik N.
      • Gammelgaard B.
      • Heegaard N.H.
      Cu(II) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-beta peptides.
      ). It is now understood that Cu2+ binds to Aβ residues His6, His13, and His14, and under different pH conditions, Asp1, Ala2, Glu3, and Phe4 can also be involved (
      • Alies B.
      • Eury H.
      • Bijani C.
      • Rechignat L.
      • Faller P.
      • Hureau C.
      pH-Dependent Cu(II) coordination to amyloid-beta peptide: impact of sequence alterations, including the H6R and D7N familial mutations.
      ,
      • Dorlet P.
      • Gambarelli S.
      • Faller P.
      • Hureau C.
      Pulse EPR spectroscopy reveals the coordination sphere of copper(II) ions in the 1-16 amyloid-beta peptide: a key role of the first two N-terminus residues.
      ,
      • Syme C.D.
      • Nadal R.C.
      • Rigby S.E.J.
      • Viles J.H.
      Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques.
      ,
      • Hou L.
      • Zagorski M.G.
      NMR reveals anomalous copper(II) binding to the amyloid Abeta peptide of Alzheimer's disease.
      ,
      • Kim D.
      • Bang J.K.
      • Kim S.H.
      Multi-frequency, multi-technique pulsed EPR investigation of the copper binding site of murine amyloid beta peptide.
      ,
      • Girvan P.
      • Miyake T.
      • Teng X.
      • Branch T.
      • Ying L.
      Kinetics of the interactions between copper and amyloid-beta with FAD mutations and phosphorylation at the N terminus.
      ,
      • Summers K.L.
      • Schilling K.M.
      • Roseman G.
      • Markham K.A.
      • Dolgova N.V.
      • Kroll T.
      • Sokaras D.
      • Millhauser G.L.
      • Pickering I.J.
      • George G.N.
      X-ray absorption spectroscopy investigations of copper(II) coordination in the human amyloid beta peptide.
      ). Mouse Aβ lacks His13 that coordinates Cu2+ binding (
      • Eury H.
      • Bijani C.
      • Faller P.
      • Hureau C.
      Copper(II) coordination to amyloid beta: murine versus human peptide.
      ) and Zn2+ binding (vide supra). This is important because mice and rats are exceptional for lacking brain amyloid deposition with age. Zinc (and copper under low pH) induces Aβ oligomerization that favored by greater α-helix content in the peptide. In contrast to metal-free aggregation that proceeds by β-sheet–mediated hydrophobic attraction, zinc-induced aggregation is reversible by dissociating the metal ion (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.P.
      • Tanzi R.E.
      • Bush A.I.
      Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors.
      ,
      • Tõugu V.
      • Karafin A.
      • Zovo K.
      • Chung R.S.
      • Howells C.
      • West A.K.
      • Palumaa P.
      Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.
      ,
      • Jiao Y.
      • Han D.X.
      • Yang P.
      Molecular modeling of the inhibitory mechanism of copper(II) on aggregation of amyloid beta-peptide.
      ,
      • Jiao Y.
      • Yang P.
      Mechanism of copper(II) inhibiting Alzheimer's amyloid beta-peptide from aggregation: a molecular dynamics investigation.
      ,
      • Gu M.
      • Bode D.C.
      • Viles J.H.
      Copper redox cycling inhibits abeta fibre formation and promotes fibre fragmentation, while generating a dityrosine abeta dimer.
      ). Even the trace contaminant metal concentrations (nM) found in neutral buffers is sufficient to promote the seeding and profibrillar aggregation of Aβ peptide solutions and is important to consider in experimental studies (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer's Abeta peptides.
      ). Whereas Zn2+ induces rapid precipitation of Aβ at neutral pH, Cu2+ induces minimal precipitation at neutral pH but profound precipitation under physiologically acidic conditions (pH ≤6.8) (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.
      ,
      • Atwood C.S.
      • Scarpa R.C.
      • Huang X.
      • Moir R.D.
      • Jones W.D.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      Characterization of copper interactions with Alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42.
      ). The structural basis for this reaction and the pathophysiological significance of this dramatic difference in response to these metal ions has not yet been resolved. Mildly acidic conditions where Cu2+ could precipitate Aβ are thought to be present in the synapse, but this view has been challenged (
      • Stawarski M.
      • Hernandez R.X.
      • Feghhi T.
      • Borycz J.A.
      • Lu Z.
      • Agarwal A.B.
      • Reihl K.D.
      • Tavora R.
      • Lau A.W.C.
      • Meinertzhagen I.A.
      • Renden R.
      • Macleod G.T.
      Neuronal glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission.
      ) and remains to be investigated in AD.
      Importantly, copper–Aβ interaction can form a catalytic redox-cycling complex that embeds in lipid membrane and recruits substrates like cholesterol to produce hydrogen peroxide and promote oxidative stress that causes neurotoxicity in cell culture (
      • Huang X.
      • Cuajungco M.P.
      • Atwood C.S.
      • Hartshorn M.A.
      • Tyndall J.D.
      • Hanson G.R.
      • Stokes K.C.
      • Leopold M.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Saunders A.J.
      • Lim J.
      • Moir R.D.
      • Glabe C.G.
      • et al.
      Cu(II) potentiation of Alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.
      ,
      • Opazo C.
      • Huang X.
      • Cherny R.A.
      • Moir R.D.
      • Roher A.E.
      • White A.R.
      • Cappai R.
      • Masters C.L.
      • Tanzi R.E.
      • Inestrosa N.C.
      • Bush A.I.
      Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2).
      ,
      • Huang X.
      • Atwood C.S.
      • Hartshorn M.A.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Cuajungco M.P.
      • Gray D.N.
      • Lim J.
      • Moir R.D.
      • Tanzi R.E.
      • Bush A.I.
      The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.
      ,
      • Jiang D.
      • Men L.
      • Wang J.
      • Zhang Y.
      • Chickenyen S.
      • Wang Y.
      • Zhou F.
      Redox reactions of copper complexes formed with different beta-amyloid peptides and their neuropathological [correction of neuropathalogical] relevance.
      ,
      • Balland V.
      • Hureau C.
      • Saveant J.M.
      Electrochemical and homogeneous electron transfers to the Alzheimer amyloid-beta copper complex follow a preorganization mechanism.
      ,
      • Reybier K.
      • Ayala S.
      • Alies B.
      • Rodrigues J.V.
      • Bustos Rodriguez S.
      • La Penna G.
      • Collin F.
      • Gomes C.M.
      • Hureau C.
      • Faller P.
      Free superoxide is an intermediate in the production of H2O2 by copper(I)-Abeta peptide and O2.
      ,
      • Puglielli L.
      • Friedlich A.L.
      • Setchell K.D.
      • Nagano S.
      • Opazo C.
      • Cherny R.A.
      • Barnham K.J.
      • Wade J.D.
      • Melov S.
      • Kovacs D.M.
      • Bush A.I.
      Alzheimer disease β-amyloid activity mimics cholesterol oxidase.
      ,
      • Wu W.H.
      • Lei P.
      • Liu Q.
      • Hu J.
      • Gunn A.P.
      • Chen M.S.
      • Rui Y.F.
      • Su X.Y.
      • Xie Z.P.
      • Zhao Y.F.
      • Bush A.I.
      • Li Y.M.
      Sequestration of copper from beta-amyloid promotes selective lysis by cyclen-hybrid cleavage agents.
      ,
      • Perrone L.
      • Mothes E.
      • Vignes M.
      • Mockel A.
      • Figueroa C.
      • Miquel M.-C.
      • Maddelein M.-L.
      • Faller P.
      Copper transfer from Cu-Abeta to human serum albumin inhibits aggregation, radical production and reduces Abeta toxicity.
      ,
      • Zhao L.
      • Wang J.L.
      • Wang Y.R.
      • Fa X.Z.
      Apigenin attenuates copper-mediated beta-amyloid neurotoxicity through antioxidation, mitochondrion protection and MAPK signal inactivation in an AD cell model.
      ,
      • Hu X.
      • Zhang Q.
      • Wang W.
      • Yuan Z.
      • Zhu X.
      • Chen B.
      • Chen X.
      Tripeptide GGH as the inhibitor of copper-amyloid-beta-mediated redox reaction and toxicity.
      ,
      • Curtain C.C.
      • Ali F.
      • Volitakis I.
      • Cherny R.A.
      • Norton R.S.
      • Beyreuther K.
      • Barrow C.J.
      • Masters C.L.
      • Bush A.I.
      • Barnham K.J.
      Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits.
      ,
      • Curtain C.C.
      • Ali F.E.
      • Smith D.G.
      • Bush A.I.
      • Masters C.L.
      • Barnham K.J.
      Metal ions, pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid.
      ). This redox activity is abrogated in the rat/mouse Aβ, which is not only less able to promote the catalytic cycling of Cu2+ (and Fe3+) but also lacks the Tyr at position 10 (which becomes Phe) to permit dityrosine modification (
      • Atwood C.S.
      • Perry G.
      • Zeng H.
      • Kato Y.
      • Jones W.D.
      • Ling K.Q.
      • Huang X.
      • Moir R.D.
      • Wang D.
      • Sayre L.M.
      • Smith M.A.
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