Cu and Zn interactions with Aβ peptides: consequence of coordination on aggregation and formation of neurotoxic soluble Aβ oligomers

Abstract

The coordination chemistry of transition metal ions (Fe, Cu, Zn) with the amyloid-β (Aβ) peptides has attracted a lot of attention in recent years due to its repercussions in Alzheimer's disease (AD). Aβ peptide undergoes self-aggregation to form amyloid plaques and soluble oligomers, which are believed to play a central role in AD pathology. Metal ions and Aβ-metal adducts can generate toxic radical species capable of modifying biomolecules, ultimately causing death of the neuronal cells. The impact of these metal ions on Aβ aggregation and neurotoxic species formation is still not well understood. A large number of reports indicate that transition metal ions such as Cu and Zn have significant effects on Aβ peptide aggregation and stabilization of neurotoxic soluble Aβ aggregates; some of these reports are contradictory too. A review on the effects of Cu and Zn metal ions on Aβ peptide aggregation due to their coordination with these peptides is presented herein. The review includes brief discussions with regards to the levels of Cu and Zn in an AD affected brain, structures of Aβ peptides, and the coordination of Aβ peptides with Cu and Zn. In the current scenario, a consensus appears to be emerging regarding the coordination mode and the effect of metal ions (especially Cu and Zn) on the aggregation of Aβ peptides. Towards the end, a short discussion on current research trends in the field of bifunctional metal chelators for tackling the metal effect on Aβ aggregation and toxicity has also been presented. It is expected that this review would aid in gaining a better understanding of the current status of various aspects of AD from an inorganic chemistry perspective.

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Monika Rana

Monika Rana received her BSc (Honours) degree in Chemistry from Miranda House, University of Delhi in 2013 and her MSc degree in Chemistry from the Central University of Rajasthan in 2015 which included a dissertation entitled “Ascertaining the fluoride ions by selective and sensitive Chemosensors”. Currently, she is pursuing her PhD from the same university with Dr Anuj K. Sharma as her supervisor. Her research interest mainly includes synthesis of multifunctional chelators and studying their coordination chemistry and biological properties with relevance to metal-induced neurodegenerative disorders, specifically Alzheimer's disease.

Anuj Kumar Sharma

Dr Anuj K. Sharma obtained his MSc in Chemistry from the Indian Institute of Technology, Roorkee, in 2004 and PhD from the Indian Institute of Technology, Kanpur with Prof. Rabindranath Mukherjee in September 2009. Then he joined as postdoctoral fellow at Washington University in St. Louis, USA, with Prof. Liviu M. Mirica. During his post doc he worked on metal ion–amyloid interactions and to develop multifunctional compounds for controlling metal mediated problems (aggregation, neurotoxicity etc.) in Alzheimer's disease. He also worked on development of ^64Cu based PET imaging agents for early detection of Alzheimer's disease. Currently, he is an Assistant Professor of Chemistry at the Central University of Rajasthan, India.

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1. Introduction

Alzheimer's disease (AD), first described in 1906, is a degenerative brain disorder and the most common cause of dementia in elderly people.¹ AD has become a major socio-economic problem in many countries as the patients need constant attention from their caregivers. It is currently affecting more than 30 million people worldwide and more than 100 million people are estimated to be affected by 2050, mainly due to the rapidly escalating elderly population in developing countries (http://www.alz.co.uk). While many countries already have a significant level of ageing population, some of the developing countries like India have ∼70% of their population in the 30–40 years age bracket, and may be expected to face serious AD related problems in a few decades' time. The scientific community appears to have taken note of this enormous challenge as evidenced by the sharp increase in the number of publications in AD research in the recent past.

The causes of AD as well as other neurodegenerative disorders are not well understood. The disease is generally characterized by the presence of extracellular senile plaques, intracellular tangles and altered levels of neurotransmitters in the brain of those affected with it.^2,3 Senile plaques, commonly known as amyloid plaques, are composed of insoluble aggregated peptides called amyloid-β (Aβ), which are constituted of 39–43 amino acid residues.⁴ The Aβ peptides are formed after sequential cleavage of the APP by the proteolytic enzymes α-, β- and γ-secretase.^5,6 Formation of aggregates of these peptides into amyloid plaques (amyloid cascade hypothesis²) or smaller soluble oligomeric species (the oligomer hypothesis⁷) is believed to play a key role in AD pathology. It remains a topic of debate as to which of the two forms – the final form of aggregates, the amyloid plaques, or the initial smaller soluble oligomers – is more toxic. However, according to recent research, diffused smaller oligomeric aggregates are suggested to have higher neurotoxicity than the fully grown amyloid fibrils.^7–12

The inorganic chemistry aspects of Alzheimer's disease (AD) have certainly caught the attention of chemists in recent years.^4,13–15 Post-mortem examination of the brain of AD affected patients suggested that metal ions such as Cu(II), Fe(III), and Zn(II) were found in high concentrations (∼400, ∼950, and ∼1100 μM, respectively) within the core and periphery of senile plaque deposits.^16,17 Many of these transition metal ions can be found in healthy individuals too, as the human body has an elaborate system for managing and regulating the amount of these key metals circulating in the blood and stored inside the cells. Metal ions are believed to play an important role in the peptide aggregation and misfolding mechanisms.^18–20 However, these interactions and mechanisms are not completely understood even today.^8,9,21 Many amyloid forming proteins can interact with the transition metal ions such as zinc, copper and iron, and this binding can bring about changes to the structures and functions of these amyloidogenic proteins.^4,22,23 In an improperly functioning system, the ratios of these metal ions reach abnormal levels, a condition that is generally known as metal ion dyshomeostasis.⁴ Under these conditions, the levels of copper and iron, both redox metals, increase in the brain of the affected people, normally aged ones. Some reports have shown these metal ions to promote Aβ aggregation as well as take part in the formation of reactive oxygen species (ROS), leading to oxidative stress.²⁴ The redox nature of these metal ions and the presence of oxygen in our body lead to ROS formation in Fenton type reactions.^22,25,26 These ROS can cause oxidative modifications of biomolecules, especially in the brain, in the case of AD conditions.²⁷ The formation of misfolded protein aggregates and the role of metal ions in this misfolding process have led to the hypothesis in AD that interactions between metal ions and amyloidogenic protein are connected to metal dyshomeostasis.^28,29

Metal ions are believed to coordinate with the hydrophilic part of amyloid forming peptides. The coordination chemistry of these peptides with transition metal ions, especially Fe, Cu and Zn, is well documented. There are several well written research articles in the literature lucidly describing the Aβ–M (M = Fe, Cu, Zn) coordination modes studied using NMR and other spectroscopic techniques.^{19,20,30–34} Another aspect of metal–Aβ interaction is the effect that it has on aggregation processes and formation of metal-associated species. There have been concerted efforts by some research groups to understand the nature of these metal affected aggregates of Aβ peptides. In this regard, a careful study reveals that the experimental conditions and methods used to characterize the aggregates formed vary from one research group's report to another.⁹ Further, summarizing the studies on metal ions affecting the aggregation of Aβ peptides has not been as extensive in comparison to the reviews on other aspects related to AD research. We have therefore chosen to focus on this aspect and review the excellent efforts that have been carried out by researchers in this regard. The primary focus of this report shall thus revolve around metal ions affecting the aggregation process of Aβ peptides, especially the longer and more neurotoxic Aβ_1–42 peptide. Aβ_1–42 is established as more neurotoxic than Aβ_1–40 and is also more likely to generate hydrogen peroxide, which has been proposed to contribute to cytotoxicity.^35,36 In addition to this aspect, a brief discussion has also been presented on metal–Aβ coordination to set the stage for various explanations. Towards the end, the possible treatment/strategies suggested for combating the problems related to metal ions in Alzheimer's disease are also presented.

2. Formation, structure and functions of Aβ peptides.

The Aβ peptides consist of a hydrophilic N-terminus and a hydrophobic C-terminus^2,13 and form after sequential cleavage of the Amyloid Precursor Protein (APP), the post-translationally modified protein containing 770 residues.^37–39 Three proteolytic enzymes α-, β- and γ-secretase cleave APP at different sites to produce Aβ peptides comprising 39–43 amino acid residues. The β-secretase cleaves APP to form the N-termini, whereas the γ-secretase does so to generate the C termini of Aβ leading to 39–43 variants (Fig. 1). These Aβ peptides undergo self-aggregation as well as metal induced aggregation, forming soluble Aβ oligomers and insoluble amyloid plaques. The normal function of Aβ is not well understood. Although some animal studies have shown that the absence of Aβ does not lead to any obvious loss of physiological functions,^40,41 several potential activities have been discovered for Aβ, including activation of kinase enzymes,⁴² protection against oxidative stress,^43,44 and anti-microbial activity that may potentially be associated with Aβ's pro-inflammatory activity.⁴⁵ Aβ is the main component of amyloid plaques, the extracellular deposits found in the brains of patients with Alzheimer's disease.^41,46 Aβ_1–40 is reported to be more abundant as compared to the longer version i.e., Aβ_1–42. The Aβ_1–40/Aβ_1–42 ratio in the soluble fractions was suggested to be ∼10 : 1 in the soluble fractions across the cortex in AD brains.⁴⁷ However, some recent reports describing the biochemical distribution and accumulation of Aβ are contradictory and indicate that Aβ_1–42 is more abundant than Aβ_1–40.⁴⁸ The ratio of Aβ_1–40/Aβ_1–42 in plaques was suggested to be ∼1 : 2; Aβ_1–42 is considered more toxic as compared to Aβ_1–40, displaying a higher propensity to aggregate, and initiate Aβ aggregation.^20,49–51 The increased proportion of Aβ_1–42, which is more prone than Aβ_1–40 to aggregate is thought to initiate the disease process.^52,53 Aβ_1–42 is known to form soluble neurotoxic oligomers, whereas it has been suggested that Aβ_1–40 does not form such oligomers.^9,54 The sites of action of α-, β, and γ-secretases on APP and the amino acid sequence for Aβ_1–42 peptide fragments are shown in Fig. 1. Amino acids which participate in metal binding are shown in blue and bold in the sequence of the full length Aβ_1–42.

Fig. 1 (top) Normal (α-, γ-secretases) and abnormal (β-, γ-secretases) cleavage pathways of APP. Aβ are formed by an abnormal mode of APP cleavage. (bottom) Showing the sites of action of α-, β, and γ-secretases on APP and amino acid sequence for Aβ_1–42 peptide fragments (in red); amino acids that participate in metal binding are shown in blue and bold.

3. Cu and Zn levels in Alzheimer's disease

Initial reports suggested that zinc (Zn), and copper (Cu) concentrations in cerebrospinal fluid (CSF) and serum were found increased in patients affected with neurodegenerative diseases. These concentrations were determined with atomic absorption spectrophotometry in 74 patients suffering from various neurological diseases, and in 28 healthy controls.⁵⁵ Another report by Basun et al. indicated no observable changes in CSF Zn concentrations; however a 2-fold increased level of Cu in CSF was reported.⁵⁶ Molina et al. reported that the CSF Zn level is significantly decreased in AD when compared to age-matched controls.⁵⁷ Deibel et al. studied the levels of Cu, Fe, and Zn in five different brain regions (amygdala, hippocampus, inferior parietal lobule, superior and middle temporal gyri, and cerebellum) by instrumental neutron activation analysis (INAA) in samples from Alzheimer's disease (AD) patients and age-matched control subjects. They found a significant decrease in Cu, and significant increases in Zn and Fe in AD hippocampus and amygdala, areas showing severe histopathologic alterations in AD.⁵⁸ A few other studies observed a significant low level of brain Cu in AD, including some recent reports.^58–60 X-ray fluorescence microprobe studies have indicated that deposits of copper and zinc co-localize with amyloid plaques in human AD brain tissue and amyloid plaque rim.^17,61 Other techniques like ICP-MS in combination of flow injection on amyloid plaques of Alzheimer's disease-affected brains suggested that the concentrations of Cr, Mn, Ni, Cu, and Pb were in the 0.2–0.8 mg L⁻¹ range, whereas that of Al, Fe, and Zn were 2–20 mg L⁻¹ in the plaque sample.⁶² Later in 2009, Rajendran et al. suggested that it is more accurate to combine the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe to determine the concentration of metal ions in amyloid plaques. Their data also suggested increased metal concentrations within the amyloid plaques compared with the surrounding tissues.⁶³ Roberts et al. described that Cu levels in AD-affected cortical tissues were decreased compared to normal age neuropil.⁶⁴ They suggested that due to Cu association in plaques, the remaining brain tissues have a lower metal concentration. Further, they also reported variations in Zn concentration in different tissues of the brain. Miller et al. used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to image the in situ secondary structure of the amyloid plaques in brain tissue of AD patients. They showed a strong spatial correlation between elevated β-sheet content in Aβ plaques and accumulated Cu and Zn ions, emphasizing an association of metal ions with amyloid formation in AD.⁶¹ The same group later compared the metal content in the plaques and the iron speciation in the cortex of three mouse models, two of which had significant neurodegeneration and one with very little neurodegeneration.⁶⁵ The content and speciation of Fe, Cu and Zn were determined by synchrotron X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS), respectively. The plaques from the mouse models with significant neurodegeneration contained ∼25% more Cu as compared to the one with little neurodegeneration, indicating that redox-active metal ions may be inducing oxidative damage and influencing neurodegeneration.⁶¹ In all these studies, the most common observation was the presence of a higher concentration of metal ions in amyloid plaques. The minor differences in results may be speculated as due to the source of samples, human AD brain tissues or tissues from mouse models, different techniques used or even due to the number of samples. Becker et al. has elegantly summarized the pros and cons of various techniques used for detection of metals in biological tissues.⁶⁶ The authors mentioned that “most analytical techniques for the visualization of inhomogeneous distributions of elements in tissues (e.g., histological or histochemical staining techniques) possess poor detection limits, that means, no element can be detected at trace and ultra-trace concentrations.” They suggested that other analytical techniques such as scanning electron microscopy with energy disperse X-ray analysis, microproton-induced X-ray emission and autoradiography also cannot be used for trace element detection due to their low detection power. Secondary ion mass spectrometry (SIMS) or laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) have been described as sensitive surface analytical techniques for the determination of element distribution on the surfaces of biological samples.^67,68 However, it is out of the scope of this review to compare each technique. In fact, we do not feel competent enough to comment on the pros and cons of some of the techniques. It is worth mentioning here that the content of metal ions in the brain is rigorously regulated, as is their movement across the BBB. The increased implications of metal ions in interactions with the major protein components of neurodegenerative diseases is not merely due to increased exposure to metals, but rather due to a breakdown in the homeostatic mechanisms that compartmentalize and regulate metals.⁶⁹

4. ROS production by redox metal ions in AD

Elevated levels of redox active metal ions (Cu/Fe) may result in the generation of reactive oxygen species (ROS), DNA damage, and mitochondrial dysfunction.⁷⁰ These metal ions are known to cause neuronal cell damage by promoting the production of reactive oxygen species (ROS)/nitrogenated species (RNS) species bearing unpaired electrons, mainly represented by superoxide anion radical, hydroxyl, alkoxyl and lipid peroxyl radicals, nitric oxide and peroxynitrite.^17,49,69 Oxidative stress is an imbalance in the generation and clearance of ROS, which are otherwise an inherent part of healthy metabolism, eventually ending up in the unusual degradation of biomolecules. Fenton type chemistry has been proposed to play roles in production of ROS.^71–74 Redox active metal ions, in both the free and loosely bound forms, are well known to be involved in the formation of ROS species via the Fenton reaction (Scheme 1: reactions (1) and (2)) and the Haber–Weiss reaction (Scheme 1: reaction (3)) as shown below.⁷⁵

Scheme 1

Fe and Cu are both known to coordinate with Aβ peptide, and such Fe–Aβ and Cu–Aβ species have been implicated in the production of ROS. However, ROS production has been more commonly studied with Cu–Aβ, since Fe–Aβ has a lower redox activity.^71,76 Indeed, in vitro studies suggested that Cu–Aβ is able to catalyze the production of H₂O₂ and HO˙ in the presence of a biologically relevant reducing agent such as ascorbate and dioxygen.^8,77 There have also been discussions on whether Aβ is an anti-oxidant or pro-oxidant. It is considered as an antioxidant on the basis of the fact that free Cu catalyzes ROS production more efficiently than Cu–Aβ. However, “free” Cu concentrations in biology are extremely low; Nature has evolved a complex system of metal transporters and chaperones that tightly regulate metal homeostasis, and pools of ‘free’ metal ions, which in a biological context refers to metal ions bound by labile, low affinity, and therefore, readily exchangeable, ligands, are rare. Derreumaux et al. has suggested that an antioxidant activity of Aβ compared to “free” copper might be relevant only under particular conditions where free Cu reaches much higher concentrations.⁷⁸ However, chances for such a situation to occur are rare when taking into account the presence of high concentrations of potential ligands (such as glutathione, histidine, cysteine, etc.). When there is an imbalance of Cu, it would be more relevant to compare the efficiency of Cu–Aβ with that of the Cu pool from which Aβ obtains Cu in AD instead of free Cu.⁷⁸ A catalytic role of the Cu–Aβ complex in various redox reactions is depicted in Fig. 2. Naturally occurring reducing agents, i.e. ascorbate, glutathione etc., reduce these metal ions to their lower oxidation states e.g., Cu(I) and Fe(II). These reduced form of metal ions in amyloid plaques then trigger the initiation of ROS production by reacting with molecular oxygen.⁷⁵

Fig. 2 Depiction of the catalytic role of Cu–Aβ in ROS production by Fenton-type reactions.

In addition, it is proposed that production of ROS by Cu–Aβ adducts leads to the oxidative damage of Aβ peptide itself and also the surrounding biomolecules.⁷⁹ Sites for oxidative damage have been studied widely; Asp1, His13, His14, Try10 and phenylalanine have been identified as main targets which undergo oxidation leading to formation of various oxidative products.⁷¹ Dityrosine cross linking between two tyrosine residues is widely indicated as a marker for oxidative stress in different neurodegenerative disorders.⁸⁰ Furthermore, in the redox atmosphere in amyloid plaques, binding of heme to Aβ peptides has also been reported to play a key role in AD.^39,81–84

5. Coordination of Aβ with metal ions

Coordination chemistry of transition metal ions like Fe, Cu and Zn with Aβ peptide and its aggregates has been widely explored in recent years.⁸⁵ There are several divergent reports on the coordinating amino acid residues of Aβ peptides that form the coordination sphere of metal ions. The binding of different amino acids to metal ions depends on various experimental conditions such as pH, buffer medium and temperature etc.^86,87 Numerous studies have investigated the metal coordination environment of Aβ–Mⁿ⁺ complexes; very few reports have studied the full-length Aβ₄₀/Aβ₄₂ peptides,^88,89 shorter non-aggregating forms like Aβ_1–16 or Aβ_1–28 being used more commonly.^{20,32,34,89,90} The EPR spectra of the Cu(II) complexes with Aβ_{1–16/1–28} and Aβ_1–40 are similar, suggesting that the high affinity metal binding site lies on the N-terminus, and more specifically, in the first 16 amino acids.^91–94 Due to this, Aβ_1–16 has been considered as a decent model for studying coordination properties with metal ions.

5.1. Copper(II) interactions with Aβ peptides

Cu(II) has a d⁹ electronic configuration, which is Jahn–Teller distorted to produce a tetragonal geometry dominated by square planar coordination modes.^95,96 Cu(II) is known to bind Aβ using various coordination modes and the coordination sphere for these Aβ–M complexes is very dynamic.²⁰ Different models of coordination have been proposed and recent review articles have summarized such models.^{4,23,71,72,85,97–99} Typical models involve three N donor atoms and at least one O donor, but, precisely which of the N/O donors are involved in the coordination is still debatable. Among the very early reports, Huang et al. in 1999 proposed a 3N1O model for copper coordination to Aβ peptide using the computer simulations of the experimental EPR spectrum and correlations with other Cu protein spectra.³⁶ Curtain et al. in 2001 by means of NMR, EPR and CD spectroscopy supported the proposed 3N1O model for copper coordination to Aβ peptide with the involvement of three histidine units (His6, His13, His14). The fourth coordination by an oxygen could be a carboxyl or a hydroxyl side chain, water/hydroxo, or phosphate from the buffer, as suggested by EPR spectra of Aβ–Cu species.⁸⁸ Karr and co-workers also concurred with this model, as EPR parameters obtained were consistent with a characteristic Type2 Cu(II) centre with three nitrogen and one oxygen donor ligands.^{59,89,91,100,101} Another study shed light into the importance of the N-terminus in metal binding, where it was observed that even small changes in the N-terminus affected the metal coordination and also indicated that the O donor atom does not involve tyrosine.^89,91 Syme et al., using various spectroscopic techniques, studied the effect of pH on coordination of Cu(II) to Aβ and assigned a 4N coordination involving three histidine units and an N-terminus at physiological pH in a square planar geometry; by presumably deprotonating the amide nitrogen at a higher pH, they suggested that Tyr10 is not involved in coordination.⁸⁶ Later, XAS and EXAFS studies assigned a model in which Cu has a five coordinate geometry to Aβ_1–40, involving three nitrogen atoms from three histidine (His-6, His-13 and His-14) residues, one oxygen coordination from Tyr 10 and a second oxygen coordination possibly from water (or OH⁻) or from other amino acids.^90,102 In 2008, Streltsov et al. also ruled out Tyr as the oxygen donor ligand and reported that the Cu(II) binds to Aβ in a distorted six-coordinated (3N3O) geometry. The coordination sphere comprises three histidine residues (His-6, His-13 and His-14), an oxygen (from glutamic or aspartic acid) and axial ligands that come from a water molecule and a carboxylate oxygen from either Glu-11 or Asp-1, in an almost equatorial planar arrangement.¹⁰³ Drew and co-workers, who studied the site-specific labelling of amino acids together with continuous wave electron paramagnetic resonance (CW-EPR) and hyperfine sublevel correlation (HYSCORE), provided support for a 3N1O coordination environment. Their CW-EPR studies for Cu(II)/Aβ species at a comparatively lower pH (between 6 and 7), suggest two equally contributing forms in 3N1O coordinate modes intact upon the amino terminus, CO from Asp1–Ala2 and the imidazole side chain of His6, with the third nitrogen swapping between imidazole side chains of His13 and His14. The authors referred to these two forms as component Ia and component Ib. In component Ia, His13 provides the coordinating site, whereas His14 is the coordinating ligand for component Ib (Fig. 3, component I). At a higher pH of ∼8.0, a different coordination geometry was observed, which was referred to as component II. In this component, the coordination sphere was described comprising all three histidine residues, along with a C=O group from the Ala2–Glu3 peptide bond, while the amino terminus no longer coordinates. Tyr10 does not provide a phenolate oxygen ligand in any of the modes.^104–107 The proportions of the components I and II were described to be pH-sensitive;^86,108 as the pH increases, the intensity of component II also increases, while that of component I decreases. Ghosh et al. described that Cu–Aβ shows an equilibrium between these two species in the range of pH 6.5–9.5 with a pK_a of ∼8.1.¹⁰⁹ Another report by Furlan et al. suggested the N terminus, N⁻ (Asp1–Ala2), C=O (Ala2–Glu3), His6 and a water molecule to be the Cu(II) coordinating ligands at higher pH (Fig. 3, component II).¹¹⁰ Further reports were divergent in the involvement of different amino acids in the coordination sphere of Cu(II) in component II.^{15,32,85,105–107} Another study suggested that Cu(II) is bound to the –NH₂ group of Asp1 (amino terminus), the amidyl N⁻ from the Asp1–Ala2 peptide bond, carbonyl from Ala2–Glu3 and with only one of the imidazole units from the three His residues.¹⁰⁶ Silva et al. reported the presence of a single histidine by ESEEM analysis and determined that in component II, His13 and His14 (equal contribution) are more favoured as equatorial ligands compared to His6; they also quantified the contribution from each histidine, with His13 and His14 (each contributing 40% and His6 20% (Fig. 3)).¹¹¹ The above contradiction in the coordination sphere can be attributed to the varying experimental conditions, which comprise the use of various buffers, pH, temperature and concentration of peptides etc. However, in spite of the contradiction, the coordinating amino acids are now understood to a major extent for such complex systems.

Fig. 3 Representation of Cu(II) coordination sphere at low pH (component I) with Aβ_1–16 fragment (top) and at high pH (component II) (bottom).

5.2. Copper(I) interaction with Aβ peptides

As compared to Cu(II), only a few reports are available on the coordination of Cu(I) with different fragments of Aβ. Karlin and co-workers, using their model His–His dipeptide, hypothesised a His–His ligation of a Cu(I) ion in a linear two-coordinate geometry (N_His–Cu^I–N_His). The copper(I) complex of these ligands was observed as a linear, two-coordinate complex by X-ray absorption spectroscopy and found to be quite robust toward oxidation by O₂.¹¹² They further demonstrated the two-coordinate geometry of Cu(I) ions with N-terminal region fragments of Aβ even in the presence of potentially coordinating residues of histidine (His-6, His-13 and His-14) and other amino acids (Tyr10, Asp7, Glu11, Ser8, backbone carbonyl O and amide N).¹¹³ Simultaneously, Shearer et al. studied the X-ray absorption near-edge spectrum region of AβCu(I), which displayed an intense pre-edge feature at 8984.1(2) eV; carrying out a data-fitting operation yielded a two-coordinate geometry, with two imidazole ligands coordinated to Cu(I) at 1.877(2) Å in a linear geometry.¹¹⁴ In agreement with these results, tandem mass spectrometry studies also identified His13 and His14 as binding sites for Cu(I) with Aβ_1–16.¹¹⁵ However, an early computational study suggested contradictory results where Cu(I) forms a tricoordinated complex with two imidazole units and one carbonyl group.¹¹⁶ However, experimental evidence supports the linear two coordinate geometry around Cu(I). The above discussion indicates that Aβ binds to Cu(I) with two His residues, with a majority of the binding coming from His13 and His14. The commonly accepted binding model is shown in Fig. 4.

Fig. 4 Depiction of the chemical structure of an Aβ_1–16–Cu(I) complex. His13 and His14 are shown as coordinating residues.

5.3. Zinc(II) coordination with Aβ peptides

A zinc binding site for Aβ peptide is located in the N-terminus hydrophilic region (Asp1–Lys16), similar to Cu(II). It is relatively more challenging to define the exact coordination for Zn(II), considering its closed shell d¹⁰ electronic structure and its silent behaviour towards most of the usual spectroscopic techniques. Insights into the coordination mode have been mainly achieved by NMR and EXAFS spectroscopy. Early reports established that Zn(II) forms a 1 : 1 complex with Aβ with a mononuclear binding site.^117,118 Zn coordination was proposed to involve four to six ligands with Zn(II) at the centre.^28,97 By means of Raman spectroscopy, in 2000, Miura et al. demonstrated that Zn(II) binds to Aβ peptide with His–Zn–His bridges through an inter-peptide aggregation mode.¹⁰¹ In agreement with this, a four-coordinated Zn(II) model was proposed, with four histidine units coordinating simultaneously but from adjacent peptides. It favours an inter-peptide linkage due to lack of a fourth coordination site as histidine along the Aβ sequence.^90,119 Another coordination sphere was determined by the use of NMR spectroscopy, where Zn(II) was found to have a tetrahedral coordination environment with the involvement of three histidine residues (His-6, His-13 and His-14)⁸⁸ and an additional ligand. The identity of the fourth ligand is still under scrutiny, with Asp1, Arg5, Glu11, Try10 and an exogenous water molecule as probable candidates. Glu 11 as an oxygen donor with Aβ_1–16 at pH 6.5, as well as Arg 5 and Asp1 have been suggested, whereas Tyr10 has been ruled out.^118,120,121 Gaggelli et al. carried out further investigations on the structure and binding mode of Zn(II) metal with rat and human Aβ_1–28 using NMR spectroscopy. A tetrahedral coordinated complex was suggested for rat Aβ with Asp1-amine, His6, Glu11 and His13 being the coordinating amino acid residues. Whereas for human Aβ, a penta-coordination was suggested with amino acid residues His6, His13, His14, Asp1-amine and Glu11 as carboxylate in the Zn(II) coordination sphere.¹²² Recently, Alies et al. has reported a tetrahedral Zn(II) coordination site with the involvement of two histidine (His6, His13 or His14) residues along with two carboxylate side chains (Glu11 and Asp1 or Glu3 or Asp7); an equilibrium between different forms was observed with a marginal preference for Asp1.³⁰ In conclusion, different models of Zn(Aβ) coordination have been observed, where Zn is suggested to have a coordination number between four and six. In the first coordination mode, the involvement of three histidines (His6, His13 and His14) has been suggested, along with extra donor ligands (Asp1/Glu3/Asp7) (in Fig. 5, the coordination mode of Zn(II) with Aβ_1–16 is represented in line with this model). In the other coordination mode, it has been proposed that Zn coordinates to four histidines with inter-peptide linkage along with adjacent peptides. Also, in recent reports, a tetrahedral geometry has been proposed, involving two histidine residues and two carboxylate chains Asp1.^30,123

Fig. 5 Depiction of Zn(II) coordination with Aβ_1–16 peptide; only the coordination sphere is shown as inset for clarity.

6. Role of metal ions in Aβ aggregation

Altered concentrations of transition metal ions, mainly Cu, Zn and Fe, are found to be associated with amyloid plaques as discussed earlier. Particularly, Cu and Zn are well known to bind to the Aβ peptides and affect their self-aggregation.^17,61,124 These metal ions can coordinate with the Aβ peptides and bring structural and conformational changes as discussed and represented above in Fig. 3–5. The Aβ–metal supra–metal complexes are expected to alter the normal Aβ aggregation processes.^23,125 Various opinions have been offered regarding the exact role of metals in aggregation of these peptides. Metal induced effects are diverse and sometimes contradictory to each other.^9,97 In this section, selected studies on metal induced aggregation of Aβ peptides are discussed. Since the Aβ_1–40 does not form neurotoxic soluble species, the discussion focuses primarily on the aggregation of Aβ_1–42 peptide.^9,126 It is important to discuss that Aβ peptides are known to form polymorphic aggregates which differ significantly from each other: three types of aggregates are commonly observed, the most structured fibrillar aggregates, soluble oligomers, and large amorphous aggregates.¹²⁷ In some studies, metal ions are observed to increase the aggregation of Aβ peptides, expediting the formation of amyloid fibrils or amorphous aggregates.^128,129 In contrast, in other studies, the metal ions were observed to reduce the Aβ aggregation and stabilize the smaller aggregates, including soluble neurotoxic Aβ oligomers, especially with the Cu(II).^130,131 The molecular mechanisms of metal–Aβ species interactions are not completely understood, especially for the more neurotoxic Aβ_1–42 peptide, and the role of metal ions in the aggregation of Aβ is still under debate. In the next subsections, a detailed discussion is presented regarding the various studies that have been carried out on Cu and Zn induced Aβ aggregation.

6.1. Aβ aggregation in the presence of copper(II); stabilization of neurotoxic soluble Aβ₄₂ oligomers

In this section, we shall be focusing majorly on the influence of Cu(II) on the aggregation of Aβ peptides. According to some studies, Cu(II) has been suggested to be involved in disrupting the larger aggregates or reducing Aβ_1–42 aggregation,^131–135 whereas, in stark contrast, other studies report a dramatic enhancement of aggregation in the presence of Cu(II).^129,136,137 The cell toxicity studies in the presence of transition metal ions also showed both increased^35,138–140 as well as decreased^43,133 toxicity of Aβ peptides. It was believed from very early on that Cu(II) ions stabilize the toxic early-stage aggregates of Aβ.¹⁰¹ In 2001, Zou et al. studied the effect of Cu(II) on Aβ_1–42 aggregation by means of ThT fluorescence and AFM imaging and observed an inhibitory effect of Cu(II) on Aβ_1–42 aggregation.¹³² Exley et al. reported that Al(III), Fe(III), Zn(II), and Cu(II) can influence the in vitro formation of amyloid fibrils of Aβ_1–42.^130,141 In this study, Cu(II) was shown to inhibit the formation of amyloid fibrils in both freshly prepared and aged solutions. They monitored the fibrillization of Aβ_1–42 in the presence of Al(III), Fe(III), Zn(II), and Cu(II) over a period of 32 weeks. The research group also investigated the dissolution of these aged peptide aggregates in the presence of both desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA), the two well-known metal chelators. Aβ_1–42 aggregation was followed by the methods of ThT fluorescence and TEM images. In 2007, Park et al. studied the morphological behaviour of Aβ_1–42 aggregates formed on a solid template incubated at pH 7.4, 6.0, or 4.6 in the presence of different metal ions. The authors wrote about a synthetic template by immobilizing Aβ oligomers onto an N-hydroxysuccinimide ester-activated solid surface. The methods of ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and ThT fluorescence spectroscopy were used in this study. The authors observed that Cu(II) and Zn(II) ions accelerated both Aβ_1–40 and Aβ_1–42 deposition but resulted only in the formation of “amorphous” aggregates. In contrast, Fe(III) induced the deposition of “fibrillar” amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work.¹⁴² In 2008, Barnham et al. reported that the presence of Cu(II) significantly promotes and stabilizes the formation of the soluble oligomeric β-sheet structures. However, in the absence of added Cu(II), more amyloid plaques were formed.⁵⁴

In 2009, Exley et al. wrote that Cu(II) is bound by monomeric Aβ_1–42 and, upon precipitation of the copper–peptide complex, prevents Aβ_1–42 from adopting a β-sheet secondary structure. In this report, they investigated the influence of this interaction on the conformation of the precipitated peptide and suggested that copper significantly reduced the thioflavin T fluorescence of aged Aβ_1–42 samples. Transmission electron microscopy also showed that copper significantly reduced the quantities of amyloid fibrils. Congo red staining and polarized light demonstrated a copper-induced abolition of apple-green birefringence. The authors wrote that “while we can agree that fibrillar Aβ_1–42 binds Cu(II) with great avidity, we can now add that binding actually results in the abolition of its β-sheet structure.”¹³¹ The same group in 2013 reported that methods of fluorimetry and transmission electron microscopy provide unequivocal evidence that, under near-physiological conditions, both sub- and super-stoichiometric concentrations of Cu(II) prevented the assembly of Aβ_1–42 into ThT-positive β-sheet rich amyloid fibrils.¹⁴¹ Another study by Garai et al. showed that low concentrations of Cu(II) (1 μM) and Zn(II) (4 μM) selectively eliminate the oligomeric population (within ∼2 h) of Aβ, by using fluorescence correlation spectroscopy to resolve the various soluble species.¹⁴³ The authors suggested that physiological concentrations of Cu(II) and Zn(II) ions can critically alter the stability of the toxic Aβ oligomers and can potentially control the course of neurodegeneration. This was in stark contrast to the reports related to the stabilization of Aβ_1–42 oligomers by Cu(II).¹⁴³ Zatta et al. studied the aggregation of Aβ_1–42 in the presence of Al, Fe, Cu and Zn; they found that all the four metals differentially altered the aggregation properties of Aβ_1–42 peptide. Their studies suggested that Aβ_1–42–Cu and Aβ_1–42–Zn show a similar profile and form amorphous aggregates, whereas Aβ_1–42–Al and Aβ_1–42–Fe promoted the formation of oligomers.¹⁴⁴ Pederson et al. reported that the Cu(II) : Aβ ratio in solution has a major influence on the aggregation kinetics/mechanism of Aβ and the morphology of the aggregates, which shifted from fibrillar to non-fibrillar with increasing Cu(II) : Aβ ratios. The authors suggested that the presence of a slow-aggregating soluble Aβ–Cu(II) oligomer could act as a rate-limiting step for the overall aggregation process and may help in understanding the Aβ–Cu(II)-mediated neurotoxicity in AD.¹³⁵

In 2013, Mirica et al. employed a wide range of techniques to study the effect of metal ions on the oligomerization and aggregation of the more neurotoxic Aβ_1–42 peptide.^9,21 ThT fluorescence, native gel electrophoresis and Western blotting, transmission electron microscopy (TEM), and cellular toxicity studies were used to determine the formation of both soluble and insoluble Aβ_1–42 aggregates in the presence as well as absence of Cu(II) and Zn(II). Most importantly, they correlated these in vitro results with neurotoxicity studies, in order to address the potential in vivo role of metal–Aβ_1–42 interactions in AD. A significantly low ThT fluorescence was observed for Cu(II) and Zn(II) containing Aβ aggregates. TEM and native gel/Western blotting studies suggested that Cu(II) and Zn(II) both inhibit fibrillization. Interestingly in this report, Zn(II) was shown to form amorphous aggregates, whereas Cu(II) stabilized the soluble Aβ_1–42 species. The cellular toxicity studies suggested that while Zn(II) reduces the Aβ_1–42 toxicity by formation of non-toxic insoluble amorphous aggregates, Cu(II) significantly increases Aβ_1–42 neurotoxicity due to the formation of neurotoxic soluble Aβ_1–42 oligomers. These studies ruled out a preventive role of Cu(II), and suggested that Cu(II) may lead to an increased Aβ_1–42 neurotoxicity in vivo due to the formation of Aβ_1–42 oligomers. The authors proposed that this Cu(II) promoted increased neurotoxicity of soluble Aβ_1–42 species may play an important role in the aetiology of AD (see the schematic representation in Fig. 6).

Fig. 6 Depiction of metal effects on Aβ_1–42 aggregation and proposed role of bifunctional chelators in metal chelation therapy.

In another study by Viles et al. in 2015, it was reported that substoichiometric Cu(II) was observed to affect the misfolding pathway of Aβ_1–40, and Aβ_1–42, by distinctly different mechanisms. Cu(II) was observed to accelerate Aβ_1–40 fibrils formation; however, with Aβ_1–42, substoichiometric levels of Cu(II) exclusively promote the formation of oligomeric and protofibrillar assemblies. In addition, the mature Aβ_1–42 fibrils were disassembled into oligomers when Cu(II) was added. The authors also reported that these Cu(II) stabilized oligomers of Aβ_1–42 interact with the lipid bilayer, disrupting the membrane and increasing permeability. In addition, investigation of Aβ_1–40/Aβ_1–42 mixtures with Cu(II) revealed that Aβ_1–40 neither contributed to nor perturbed the formation of Aβ_1–42 oligomers, although Cu(II)–Aβ_1–42 does frustrate Cu(II)–Aβ_1–40 fibre growth. The authors also mentioned that small amounts of Cu(II) accentuated the differences in the propensity of Aβ_1–40 and Aβ_1–42 to form toxic oligomers, providing an explanation for the connection between disrupted Cu(II) homoeostasis and elevated Aβ_1–42 neurotoxicity in Alzheimer's disease.¹⁴⁵

6.2. Aβ aggregation in the presence of Zn(II) and neuroprotective role of Zn

Although there are several studies that report the effect of both Cu and Zn on Aβ aggregation, there are few reports exclusively on the effect of Zn(II) ions on aggregation of Aβ_1–40 and/or Aβ_1–42. Zinc was shown to specifically bind Aβ_1–40 with a 1 : 1 stoichiometry, displaying a high affinity binding with K_d = 107 nM and another low affinity binding with K_d = 5.2 μM.^128,146 Early reports from Bush et al. showed that human Aβ_1–40 peptide rapidly forms amyloid in the presence of more than 300 nM concentrations of Zn(II).^128,147 Some reports suggested the involvement of specific amino acids that interact with Zn(II) in the rapid precipitation of Aβ peptides.¹⁴⁸ Zn(II) at a concentration of a few micromolar, which is too dilute to affect the precipitation equilibrium of Aβ, can destabilize these aggregates.¹⁴³ Nevertheless, a growing number of reports indicate that zinc inhibits Aβ-mediated cytotoxicity. Garai et al. observed reduced Aβ toxicity in the presence of Zn(II) by selectively precipitating aggregation intermediates.¹⁴⁹ In the same report, it was shown that the larger soluble aggregates (size > 10 nm) form only in supersaturated Aβ solutions, implying that they are intermediates in the pathway toward fibril formation. Zn(II) was observed to destabilize these intermediates by accelerating their aggregation kinetics. The resulting change in the size distribution of the Aβ solution is sufficient to eliminate its toxicity to cultured mammalian neurons, lending support to the observations that at a concentration of a few micromolar, Zn(II), significantly reduces Aβ toxicity.¹⁴⁹

Zinc and copper may also exert a combined effect. A report by Lee et al. systematically characterized Zn–Aβ aggregates by incubating equimolar Aβ with Zn. The authors reported that Zn–Aβ_1–40 and Zn–Aβ_1–42 both form spherical oligomers with a diameter of ∼12–14 nm composed of reduced β-sheet content. In addition, Zn–Aβ and Aβ derived diffusible ligands (ADDLs) were found to be distinct as examined by analytical ultracentrifugation, hydrophobic exposure by BisANS spectra, and their immunoreactivity. Moreover, Zn–Aβ oligomers were shown to exhibit stronger toxicity than ADDLs by cytotoxicity assays.¹⁵⁰ Very recently, Zhang et al. made use of a combination of analytical ultracentrifugation (AUC), circular dichroism (CD) spectroscopy, thioflavin T (ThT) assay and atomic force microscopy (AFM) imaging to analyze the impact of stoichiometric Zn(II) on the aggregation process of Aβ_1–42. It was observed that the Aβ_1–42 aggregates found in the presence of Zn(II) were smaller in size, non-fibrillary and showed less β-sheet structures than aggregates formed in the absence of Zn(II). Their studies showed that Zn(II) was capable of retaining monomeric Aβ_1–42 in solution. The authors wrote that “our results provide further evidence that Zn(II) shifts the self-association of Aβ_1–42 toward a non-fibrillary pathway by interfering with the aggregation process at multiple levels”.¹⁵¹ Exley et al. in 2004 showed that fibrilization observed in the presence of Zn(II) was very slow and suggested that the fibrillar structures formed slowly in the presence of Zn(II) were less “ThT-reactive”. The authors suggested that this lack of reactivity may be due to either a conformational state which was specific to Zn(II) binding or a competition between Zn(II) and ThT for a specific binding region.¹⁴¹ In contrast, studies by Mirica et al., as discussed above, suggested that Zn induces precipitation of amorphous aggregates of Aβ_1–42 as well as of Aβ_1–40. It was also found that the Zn induced aggregates were not toxic to the Neuro2A cells.⁹

7. Discussion on sample preparation and techniques used to study Aβ aggregation

Overall, the above discussion gives an overview of the current status of metal effects on Aβ aggregation. In this regard, sample preparation and techniques used for carrying out the studies are important factors to be taken into consideration. Metal-induced aggregation has been widely studied under different pH conditions, temperature, concentration/metal to peptide ratio, etc. A few important factors which can potentially affect the Aβ aggregation results may also be brought into the discussion. Aβ peptide available from commercial sources is normally not 100% pure. Aβ peptide is either chemically synthesized or a recombinant peptide. Chemically synthesized peptides contain counter ions, typically trifluoroacetate (TFA) from HPLC purification. Purification at lower pH (∼2) results in protonation of histidine and carboxylates. If such a peptide is dissolved in water at higher concentrations (mM), the presence of counter anions will be resulting in quite low pH.¹⁵² There can be a significant drop in pH even when the peptide is dissolved in a high concentration of buffer; hence, it is important that the buffer used and the pH be chosen with care. Aggregation is generally studied under the so-called fibrillation conditions.^9,77 The most common methods use Aβ monomeric films, which are prepared by dissolving the peptides in HFIP to break the preformed aggregates and obtain the monomeric form.¹⁵³ The aliquots of this solution after evaporation and vacuum centrifugation form monomeric films, which are generally stored at −80 °C and used for months. The aggregation of these Aβ films is initiated by first dissolving them in DMSO or dilute aq. NH₄OH, followed by diluting into the appropriate buffer medium and incubating for a sufficiently long time at 25–37 °C with or without continuous agitation.^8,77 The concentration of the peptide is usually evaluated using UV spectral data, as Tyr absorbs at 275 nm with an extinction coefficient of 1400 M⁻¹ cm⁻¹. UV-vis is not always a valuable method to determine the peptide concentration, since metals bound to the peptide could also absorb in the same region as that of the aromatic amino acids. For metal-containing fibrils, the corresponding metal ions are normally mixed before the initiation of the fibrillation conditions. The methods employed for fibrillization may vary marginally in the reports from different research groups. For example, another method by Dahlgren et al. that has been used for fibrillar conditions is using 10 mM HCl to bring the peptide to a final concentration of 100 μM and incubated for 24 h at 37 °C.¹⁰ The same reports also described the method for the oligomer preparation as follows: monomeric film of Aβ_1–42 was dissolved in anhydrous DMSO and diluted in DMEM : F12 media (1 : 1 v : v, without phenol red, Invitrogen). After that the Aβ_1–42 solution (50–100 μM) is incubated at 4 °C for 24 h and then centrifuged at 10 000g for 10 min. The supernatant can be used as a solution of soluble Aβ_1–42 oligomers.^10,153

Techniques used for monitoring the metal–Aβ interaction and aggregation are wide-ranging and have been documented in detail elsewhere.¹⁵² A brief summary of selected methods is presented here for the benefit of the reader. Fluorescence spectroscopy has emerged as one of the most important diagnostic tools for analysis and imaging of amyloids. The most widely used method to monitor the extent of fibrillation is thioflavin-T (ThT) fluorescence assay. ThT was first introduced as a fluorescent marker for amyloid fibrils in 1965 by Levine and has been in use ever since.¹⁵⁴ However, the exact mechanism, information regarding specific interactions, and stoichiometry have not yet been completely understood.¹⁵⁵ The dye is weakly fluorescent in an aqueous environment but increases by several folds upon binding to amyloid fibrils. This protocol has been largely unchanged and is perhaps the most widely employed method for monitoring Aβ aggregation. ThT is the most commonly used dye but lacks the specificity for various amyloids and detects only the final aggregates, the amyloid fibrils. Early stage aggregates, which are suggested to be much more neurotoxic, cannot be determined by ThT assay. The presence of paramagnetic metal ions like Cu(II) may affect the emission.⁹ Hence, ThT fluorescence assay should not be used in isolation to determine the nature and extent of aggregation.

Another routinely employed technique to quantify fibril content and inhibitory ability of small molecules toward amyloids is Congo Red (CR) spectral shift assays.¹⁵⁶ It is known that CR has two binding sites in amyloid, parallel and antiparallel to the β-sheet and the binding ratio may depend on the type of amyloid under investigation. Binding of CR to the extensive β-sheet structures results in an enhanced absorption as well as a bathochromic shift in its absorption spectrum from ∼480 nm to 540 nm. When crossed polarizers are used (polarized light), green birefringence is observed upon binding to the fibrils and has been typically used as a qualitative measure of fibril formation.¹⁵⁷ There are several reports indicating CR as an inhibitor of amyloid aggregation.¹⁵⁸

A more quantitative analysis of the Aβ aggregation studies is provided by native gel electrophoresis/Western blot analysis and transmission electron microscopy (TEM) or atomic force microscopic (AFM) techniques. While the former type of analysis reveals the presence of smaller, soluble Aβ aggregates and their molecular weight distribution, the latter method allows the characterization of the larger, insoluble Aβ aggregates that cannot be analyzed by gel electrophoresis. In general, TEM images should be reported with great care as the image taken from just one region of the surface of TEM grid may not represent the entire sample. Therefore, images which are representative of the entire surface of TEM grids that show a homogeneous distribution of the Aβ species should be presented. The use of all these methods together provides a more comprehensive picture of the extent and pathways of Aβ aggregation under various conditions.^{131,132,134,136,141,159} However, only a few reports have employed all these techniques together under similar experimental conditions to provide a detailed picture of the neurotoxicity implications of the Cu(II) and Zn(II) mediated formation of soluble and/or insoluble Aβ_1–42 aggregates. More comprehensive studies on the effect of metal ions on Aβ_1–42 aggregation and neurotoxicity are required for a better understanding of their role in AD progression.

8. Rational design of therapeutics to tackle metal ions in AD

Research into the field of neurodegenerative diseases has rapidly advanced in the last two decades as is evident from an exponential growth in research articles published in this domain. Despite the continuous efforts of a large body of researchers in the field of neurodegenerative diseases and more specifically Alzheimer's disease, the exact cause for the pathology of AD is still not known, and as such, there is no treatment available for AD to date. The five FDA approved medications for Alzheimer's disease help only in alleviating the symptoms or slow them down from worsening. Out of the five available medications, donepezil, galantamine and rivastigmine belong to the “cholinesterase inhibitors” class of drugs.^160–162 These drugs help by increasing the levels of acetylcholine, an important neurotransmitter for learning and memory. Considering the involvement of multiple factors in the pathogenesis of AD, therapies or drugs targeting a single cause are inadequate to address disease progression. It is becoming more evident from the trends in recent research that the next generation of therapies must have multiple functions to fight the various mechanisms of disease succession. Individuals with neurodegenerative diseases can be benefited by these new drug molecules being developed, which can target multiple problems.^163,164 To function as an effective drug for such diseases, these molecules should possess more than one of the following properties: control over ROS formation, metal chelating ability, affinity for their uptake to cross the blood–brain barrier, antioxidant capabilities, lower Aβ peptide aggregation, or inhibit enzymes associated with disease pathways such as acetylcholinesterase and monoamine oxidase, etc.^165,166 Current AD therapies primarily focus on various underlying factors like modulating neurotransmission, tau based therapies, amyloid based strategies, modulating intracellular signalling cascades, oxidative stress reduction and metal chelation therapy. Considering the crucial role played by metal ions, metal chelation therapy is expected to play a pivotal role in AD therapeutic research. Keeping the essential role of metal ions in biology in the background, designing selective and specific metal chelators is highly challenging. 2,3-Dimercaptopropanol (British anti-Lewisite, BAL) was the first metal chelator which was developed in the 1940's against the toxicity associated with arsenic.^14,167 It was followed by the use of ethylenediaminetetraacetic acid (EDTA), introduced in the 1950s for the treatment of lead toxicity.¹⁴ Much later, the sequence of diethylenetriaminepentaacetic acid (DTPA), triethylenetetraamine (TETA) and standard iron chelator desferrioxamine (DFO) emerged in the late 1980s and 1990s.¹⁴ It was followed by the discovery of the first generation of metal chelators towards the end of 1990s and the introduction of a small lipophilic molecule clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ).^168,169 It significantly reduced Aβ plaque deposits, leading to improved cognitive behavior, in early phase II clinical trials.^16,22,26,137 Unfortunately, the long-term use of CQ is limited due to its adverse side effects, especially the sub-acute myelo-optic neuropathy.^169,170 However, it has led to the development of several CQ based metal chelators extensively studied in the context of metal chelation therapy for AD.¹³

Bifunctional metal chelators (BFC) having functionalities that have not just metal chelating ability but are also capable of binding with amyloids were proposed to tackle the multifactorial nature of AD (Fig. 6). Considerable progress has been made in this field in the last decade.^8,13,171,172 Thioflavin-T (ThT) is well known to bind most amyloids and, as discussed earlier, has been used as the most important fluorescent dye indicator to detect the presence of amyloids.¹⁵⁴ The ThT molecular framework has been extensively explored for multifunctional molecule design.^8,163,173 XH1 was the first bifunctional molecule based on this design principle to link different molecular fragments with different functions to form a hybrid molecule (Fig. 7).¹⁷⁴ Its structure comprised two terminal neutral ThT-derived moieties linked via a diethylenetriaminepentaacetic acid (DTPA) binding unit.

Fig. 7 Structures of selected bifunctional chelators/prochelators reported from different research groups as discussed in the text.

Mirica et al. reported the role of metal ions in AD and new bifunctional compounds based on phenylbenzothiazole (inspired by ThT), linking them with small metal chelators such as dipicolylamine or pyrinophane type metal chelators (Fig. 7).^{8,163,175,176} In addition to metal binding and Aβ fibril binding properties, the effects on Aβ_1–42 aggregation in the presence and absence of metal ions were also investigated.⁸ Bifunctional compounds having ThT and CQ type chelation properties, which have the capability of converting neurotoxic soluble oligomers into less toxic fibrils, were reported by the same group. A novel and effective strategy was introduced by Franz and co-workers in 2006 to design prochelators. The rationale in the strategy was that the chelators get activated only in the presence of oxidative stress and that they do not chelate metal ions until a protective mask is selectively removed by oxidative stress. This approach proved to be excellent in addressing the selective removal of metal ions and preventing loss of essential metal ions from metalloproteins. Boronic ester (BSIH), a first generation prochelator containing a boronic ester as a mask in place of phenolic oxygen, an excellent metal affinity group, was designed following this approach. It gets activated to salicylaldehyde isonicotinoyl hydrazone (SIH), a known iron chelator in the presence of H₂O₂.¹⁷⁷ Different analogues for BSIH with various substitutions as boronic esters as well as acids were explored with respect to their ability as metal chelators.¹⁷⁸ Later, the same group also utilized this strategy for various promising molecular scaffolds and investigated them for their effect on multiple factors responsible for AD.^179–182

A series of rationally designed bifunctional compounds from several families with various frameworks have been reported by Lim et al. IMPY and stilbene derived compounds were studied for their metal binding properties, and effect on Aβ₄₀ aggregation in the presence and absence of metal ions.^77,183 Very recently, novel small molecules (2,2′-bipyridine (bpy) derivatives and other N,N-dimethylanalines (DMA) with bidentate ligands) were reported as chemical tools and were shown to be effective in targeting multiple pathological factors associated with Alzheimer's disease.^184,185 The effects of various flavonoids including myricitin and epigallocatechin gallate (EGCG) molecules have also been reported.^186–189 SALEN type Schiff-bases linked with carbohydrate moieties were reported for the first time by Orvig et al. in addition to other metal chelators by the same group.^{13,164,190–194} The most advanced chelator in terms of reaching the patients has been PBT2, a second generation 8-hydroxyquinoline based scaffold which is inspired from CQ but lacking iodine. It is a metal binding agent which has a binding affinity for copper and zinc, while also exhibiting good antioxidant properties.^13,69,195 PBT2 also has a good blood brain barrier permeability, is more effective than CQ in cognition effects and is a superior ionophore. In vitro, PBT2 significantly reduces H₂O₂ production and prevents Cu- and Zn-induced formation of Aβ aggregates. Treatment with PBT2 displayed inhibition to metal-induced damage, and importantly, did not lead to any changes in the concentration of metal ions in other parts such as the brain, kidney, liver or plasma.¹⁹⁶ PBT2 reverses the Alzheimer phenotype within days by significantly decreasing insoluble Aβ levels by ∼30% and affects both Tau phosphorylation and synaptophysin protein levels. Upon PBT2 treatment, total insoluble tau levels were decreased while the soluble tau level was increased.¹³ Another important ligand to be mentioned here is diacetylbis(N(4)-methylthiosemicarbazonato), commonly known as ATSM (see the structure in Fig. 7). The Cu(ATSM) compound was found to play a protective role against peroxynitrite-induced nitrosative damage and prolongs survival in the Amyotrophic Lateral Sclerosis (ALS) mouse model.¹⁹⁷ However, ATSM has not been much evaluated for metal chelation or protection from ROS in the context of AD. It is important to mention that numerous multifunctional molecules capable of simultaneously targeting and combating multiple pathological factors of neurodegenerative disorders have been rationally designed and evaluated for their efficacies employing different relevant studies; such molecular frameworks have been well summarized elsewhere.^13,171,198 Finally, it is important to mention that, despite the enormous progress that has been made in the development of multifunctional molecules as therapeutics, very few have made it to human clinical trials. Although the above strategy of using multifunctional-molecules to tackle the multifactorial nature of these disorders seems quite promising, the potential issues causing clinical failure have to be understood; a summary of probable causative factors are reviewed elsewhere.^199,200 Overall, it is an attractive strategy that is making progress and hopefully delivers better therapeutics for AD in the near future.

9. Conclusions

In summary, most aspects of Cu and Zn interactions with Aβ peptides have been reviewed. A brief discussion on the formation, structure and functions of Aβ peptide as well as the levels of metal ions in AD affected brains and related studies has been presented. A discussion on the redox nature of metal ions and role of the Cu–Aβ complex in ROS formation has also been presented to highlight this chemistry. Whereas excellent articles in great detail are available in the literature, a concise summary of Cu(II), Cu(I) and Zn(II) coordination chemistry has been presented here. In addition, the rarely reviewed topic of aggregation of Aβ peptides in the presence of metal ions has been a major focus of this review. In general, it has been observed that while most of the initial studies were carried out with Aβ_1–40 peptide, it was understood that it has a limited role in the toxicity involved in AD. It is the longer version, the Aβ_1–42 that forms more soluble neurotoxic oligomeric aggregates, and studies with Aβ_1–42 are therefore becoming more common. In this review, the effect of Cu(II) on Aβ aggregation and especially on the stabilization of neurotoxic soluble oligomerization has been presented. Similarly, the effect of Zn(II) on Aβ aggregation and its proposed neuroprotective roles has also been reviewed. It is comprehensible from the above discussion that, although there were conflicting results obtained by various groups regarding the coordination of metal ions to Aβ, a consensus appears to be emerging as of today. Similarly, the aggregation of Aβ peptide and especially Aβ_1–42 aggregation in the presence of metal ions has led to several contradictory studies. However again, in the present day scenario, researchers evidently concur that Cu(II) stabilizes the smaller soluble oligomers, while, on the other hand, Zn(II) has been suggested to form larger, amorphous less toxic aggregates. The reason for these conflicting studies may be attributed to the inherent nature of Aβ peptides to form different conformations even upon minimal changes in the experimental conditions, leading to different results; the environmental factors in the studies comprise the pH, metal ions, ionic strength and buffer used, presence of metal ions, choice of peptide (Aβ_1–40vs. Aβ_1–42), concentration of peptide or concentration of metal ions etc. In addition, the methods of study and the techniques used to monitor the aggregation also differ from one study to another. Research groups have to come on the same platform with regards to the experimental conditions and techniques to be employed, so as to avoid conflicting results for such important issues.

Abbreviations

Aβ	Amyloid-β
AD	Alzheimer's disease
ADDLs	Aβ derived diffusible ligands
AFM	Atomic force microscopy
ALS	Amyotrophic lateral sclerosis
ANS	1-Anilinonaphthalene-8-sulphonate
APP	Amyloid precursor protein
AUC	Analytical ultracentrifugation
BAL	British anti-Lewisite
BBB	Blood brain barrier
BFC	Bifunctional chelators
CD	Circular dichroism
CQ	5-Chloro-7-iodo-8-hydroxyquinoline
CR	Congo red
CSF	Cerebrospinal fluid
CW-EPR	Continuous wave electron paramagnetic resonance
DFO	Desferrioxamine
DMEM	Dulbecco's modified eagle's medium
DTPA	Diethylenetriaminepentaacetic acid
EGCG	Epigallocatechin gallate
EPR	Electron paramagnetic resonance
ESEEM	Electron spin-echo envelope modulation
EXAFS	Extended absorption fine structure
HFIP	Hexafluoroisopropanol
HYSCORE	Hyperfine sublevel correlation
ICP-MS	Inductively coupled plasma – mass spectrometry
FTIRM	Fourier transform infrared micro-spectroscopy
INAA	Instrumental neutron activation analysis
LA-ICPMS	Laser ablation inductively coupled plasma mass spectrometry
ROS	Reactive oxygen species
RNS	Reactive nitrogenated species
SIH	Salicylaldehyde isonicotinoyl hydrazone
TEM	Transmission electron microscopy
TFA	Trifluoroacetate
TETA	Triethylenetetraamine
ThT	Thioflavin T
NMR	Nuclear magnetic resonance
SIMS	Secondary ion mass spectrometry
XFM	X-ray fluorescence microscopy
XAS	X-ray absorption spectroscopy

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

A. K. S. acknowledges the Department of Science and Technology (DST, Govt. of India) and Science and Engineering Research Board (SERB) for financial support (grant reference numbers EMR/2016/001452). The authors acknowledge the help and suggestions from Prof. Liviu M. Mirica and Dr Easwar Srinivasan in improving the manuscript. We thank the reviewers of this manuscript for their valuable comments and advice. Authors appreciate the help of Dr Anurag Prakash Sunda in preparation of figures of the manuscript.

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