Targeting Metal Homeostasis as a Therapeutic Strategy for Alzheimer ’ s Disease

Trace metals play an important role in the pathophysiology of amyloid precursor protein, amyloid beta, and tau, the key molecules involved in Alzheimer’s disease. Altering trace metal concentrations in the brain has been explored as a therapeutic strategy for Alzheimer’s disease. It is not only the accumulation of metals that drives amyloid beta aggregation, but also the lack of sufficient trace metals for other biological processes created through sequestration by amyloid beta that affects brain health and function. Thus, balancing metal levels to achieve therapeutic effects is an intricate process. This chapter summarizes the role of trace metals in Alzheimer’s disease and highlights the preclinical and clinical studies targeting metal homeostasis in animal models and humans. It further discusses recent developments in pharmacological approaches targeting metals in Alzheimer’s disease and provides an outlook on possible future treatments based on current translational research, for example, nanomedicine. Targeting Metal Homeostasis as a Therapeutic Strategy for Alzheimer’s Disease Antonietta Vilella1* • Eleonora Daini1* • Chiara A. De Benedictis2* • Andreas M. Grabrucker2,3,4 1Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Cellular Neurobiology and Neuro-Nanotechnology Lab, Department of Biological Sciences, University of Limerick, Limerick, Ireland; 3Bernal Institute, University of Limerick, Limerick, Ireland; 4Health Research Institute (HRI), University of Limerick, Limerick, Ireland Author for correspondence: Andreas M. Grabrucker, Cellular Neurobiology and Neuro-Nanotechnology Lab, Department of Biological Sciences, Bernal Institute of University of Limerick, Limerick, Ireland. Email: andreas.grabrucker@ul.ie Doi: https://doi.org/10.36255/exonpublications.alzheimersdisease.2020.ch5 *These authors contributed equally.


METALS AND THE AMYLOID PRECURSOR PROTEIN
In AD, metal imbalances occur in the brain (3,19) as a result of the interactions between the metals and the critical proteins amyloid precursor protein (APP) and its products, amyloid beta (Aβ), and tau ( Figure 1). APP, which is mainly expressed Figure 1. Trace metals interactions with APP, Aβ, and tau. (A) APP can be considered to act as a metalloprotein. It has two putative metal-binding sides with which it binds Cu and Zn ions. Fe is involved in the regulation of APP translation, and APP may mediate Fe export of neurons. Aβ is derived from APP cleavage through β-and γ-secretases. Cu and Zn indirectly influence Aβ generation by interacting with all three secretases (α, β, and γ) involved in the cleavage of APP. (B) Aβ directly binds Zn and Cu ions after enzymatic cleavage. Aggregation of Aβ into insoluble fibrils is mediated by the interaction of Aβ with Zn and Cu. Chelation of Zn and Cu results in the enrichment of these ions in amyloid plaques. Aβ may also bind Fe, and changes in Fe metabolism occur after the development plaques. Al has been detected in Aβ plaques suggesting an interaction of Al with Aβ, and Al and Hg promote the accumulation of Aβ aggregates in vitro. (C) NFTs include trace metals such as Zn, Cu, Fe, and Mn, but also Pb, Cd, Hg, and Al have been detected. Trace metals may promote tau hyperphosphorylation and induce tau aggregation. A direct interaction between Zn and tau has been reported. Similarly, a selective binding between tau and Cu was found. Fe, as Zn and Cu, interacts with some isoforms of tau. Al, Cd, Pb, and Hg may not directly bind tau, but influence NFT formation through secondary processes.
in the human brain, is involved in neuronal cell migration and neurite outgrowth (20). Copper binds to APP between residues 142 and 166 (21), playing essential roles in the structural stability of the protein, including folding stability and homodimerization, and expression levels (22). Zinc can also interact with APP using the site located between amino acid positions 170 and 188, regulating the homodimerization of APP (23,24). In addition, there is bidirectional regulation between iron and APP: iron can directly control APP translation, and APP levels control the export of iron in neurons (25). The Aβ protein is derived from APP in the process of sequential proteolysis realized by β and γsecretases. Insoluble amyloid fibrils of Aβ are associated with metals like Zn, Cu, and Fe and represent a pathological feature of AD (26). Zinc plays a role in the stabilization of amyloid fibrils (27), binding to Aβ at histidine (His)13 and His6 sites (28). High zinc concentrations have been found at the level of senile plaques in postmortem tissues of patients affected by AD and in plaques of genetic AD mouse models, underlining the critical role of zinc in this process (26). Copper is considered to play a significant role in the neurotoxicity of Aβ. The monomeric Aβ exhibits three highaffinity His Cu-binding sites: His6, His13, and His14. They are known to form a tetragonal complex with copper ions along with the N terminal amino group and aspartate (29). In addition to this, copper is involved in the formation of β-sheet structures, the precursors of the toxic aggregates of the fibrillary form of Aβ. Several studies have shown that, in transgenic AD mouse models, Cu chelators could inhibit the accumulation of Aβ (30,31). Iron also interacts with Aβ at the binding sites of Asp1, Glu3, and the three His residues: His6, His13, and His14. The consequence of this interaction is the release of free radicals via Fenton chemistry (32). Other metals such as Al and Hg have been identified in Aβ plaques. While the role of Al in the AD pathology is still not clear, Hg promotes the accumulation of Aβ deposits in vitro. Cd and Mn are also involved in increasing the Aβ accumulation (33).
Another pathological hallmark of AD is hyperphosphorylation of the protein tau that leads to the formation of abnormal intracellular structures known as neurofibrillary tangles (NFTs). Zn, Cu, Fe, Mg, Mn, Pb, Cd, Hg, and Al have been found in these structures. Zinc interacts with tau using the serine (Ser) and proline (Pro) binding sites, using two cysteine (Cys) residues (C291 and C322), or threonine (Thr) and Pro sites (34). Zinc also interacts indirectly with tau, through the activation of kinase and phosphatase pathways (35). The importance of copper in the hyperphosphorylation of protein tau via the activation of the cyclindependent kinase (CDK)5/p25 complex is still controversial, although high levels of Cu have been reported in amyloid plaques and NFTs (36,37). Iron (Fe 3+ ) is also reported to interact with NFTs through His residues (38). Like Cu, Fe can also indirectly contribute to hyperphosphorylation of tau, by activating the CDK5/ p25 complex and GSK-3β and MAP kinases (39).
The abundance of Aβ and the formation of NFTs lead to an imbalance in trace metals that interact with tau and Aβ. It is both the interaction with the hallmark proteins of AD and the loss of trace metals in their physiological processes due to excessive binding by Aβ and tau that contributes to the pathogenesis of AD. For example, in postmortem AD brains, increased Mn concentration, abnormal tau aggregation, and consequent hyperphosphorylation of tau caused by GSK-3β kinase, have been observed (40). The presence of toxic trace metals has also been reported in AD. For example, Cd is involved in the activation of GSK-3β kinase, inducing hyperphosphorylation of tau (41). Pb plays a role in the modulation of tau by regulating the activity of (CDK)5/p25 complex and GSK-3β kinase (42). To modify Aβ aggregation, plaques, and the formation of NFTs, and to return essential trace metals to their effector proteins, balancing levels of trace metals is necessary and has been pursued as therapeutic strategy with some success in the past (43).

NUTRITIONAL INTERVENTIONS
The essential metals (Na, K, Ca, Mg) and essential trace metals (Fe, Zn, Cu, Mn, Co, Mo) are obtained almost exclusively from the diet. Trace metal deficiencies may affect the elderly (44,45) and alter immune function (46). While some metaanalyses of studies analyzing the nutrient status of patients with AD found no significant differences in Fe, Zn, and Cu status (47,48), more recent meta-analyses revealed significantly lower brain Cu (49) and serum Mn levels (50). However, subclinical trace metal deficiencies are hard to diagnose (51) and may not be detected in many individuals. Considering this, nutritional supplementation studies may provide additional insights. A systematic review found that the amount of caloric intake and type of diet may influence AD onset and progression (52). For example, reports show that a Mediterranean diet, which is naturally rich in metals, such as Mg, K, Ca, and Zn, (53), can have a neuroprotective effect that may be relevant to AD (54). In mouse models of AD, zinc supplementation prevented cognitive deficits (55). In humans, despite case-control and postmortem studies showing decreased systemic Zn levels in AD patients (47,56), studies investigating the effects of zinc supplementation provided inconclusive results. Zinc supplementation also reduces copper uptake due to an antagonistic relationship between the two metals (57). A diet low in Cu has been suggested as beneficial for AD (58-60), but a recent study does not support this hypothesis (61).
Trace metal homeostasis in AD is affected at cellular and sub-cellular levels in the brain and, thus, therapeutic strategies involving their manipulation may require more targeted approaches than dietary supplementation/restriction. However, trace metal supplementation in AD is still understudied. In addition to their direct effects on physiological processes in the brain, they may exert indirect effects through the microbiota composition of the gut (62). Regardless, maintaining adequate trace metal status will affect general health positively and may modify AD phenotypes, for example, by lowering pro-inflammatory responses and oxidative stress (63).

TARGETED INTERVENTIONS: DELIVERY OF METAL-ATTENUATING COMPOUNDS
Altered homeostasis of certain essential and non-essential trace metals can be the cause, as well as the consequence of AD pathology. Abnormal trace metal levels have been associated with neuroinflammation, mitochondrial dysfunction, oxidative stress, and protein aggregation (19). The AD hallmark proteins Aβ and tau are metal-binding proteins. Therefore, metal chaperones and/or metal chelators may help modulate and counteract AD progression. Metal chaperones or metallochaperones are a class of compounds that shuttle metal ions to specific intracellular targets. In contrast, metal chelators or buffers function to exclude or deplete metals from discrete cellular compartments to limit biological or pathological interactions of essential metal ions. Cumulatively, these processes serve to maintain tight regulatory control over cellular metal ion homeostasis such that the intracellular concentration of freely available metal ions (like Cu and Zn) is close to zero, and sufficient metals are available for crucial metal-binding proteins. Among the metalfocused interventions (Table 1), metal protein attenuating compounds (MPACs) have been employed in both preclinical animal studies and human clinical trials. MPACs are metal chelators with a moderate affinity for metal ions and can cross the BBB. They correct abnormal metal interactions and have subtle effects on metal homeostasis. Therefore, they may limit metal binding to plaques, inhibit Zn and Cu-induced oligomerization of Aβ, and consequently reduce Aβ aggregation and toxicity (64). Clioquinol (CQ, 5-chloro-7-iodo-8-hydroxyquinoline, MW = 305.5) is a hydrophobic 8-hydroxy (OH) quinoline derivative and an archetype of MAPCs. CQ freely crosses the BBB and is a moderate chelator of Fe, Cu, and Zn. Additionally, with its ionophore activity, it works as a metal chaperon relocating metal ions into cells (65). Originally developed as an oral antiparasitic agent, CQ treatment significantly prevents the cognitive decline of patients with moderately severe AD, as demonstrated in a phase II clinical trial (64). The slowed cognitive decline was also accompanied by a transient decrease in Aβ 1-42 plasma levels. These results indicate the potential of CQ and related compounds as treatments for AD and have sparked further interest in MPACs. Also, in animal models of AD such as Tg2576 mice, chronic treatment with CQ can decrease Aβ loads without adverse effects (30). Treated mice showed significant improvements in general behavior and neurotoxicity, in accordance with the results obtained in severely affected AD patients (64).
DP-109 is a diester derivative of BAPTA (1,2-bis(2-aminophenyloxy)ethane-N,N,N′,N′-tetra acetic acid), a widely-used calcium chelator, that is able to cross the BBB and is designed to selectively chelate transition metals such as Zn, Cu, and Fe within membrane compartments (increased efficacy for divalent metals). Like CQ, prolonged administration of DP-109 to female Tg2576 mice markedly reduced the amyloid plaque load and the degree of cerebral amyloid angiopathy (CAA) (66).
PBT2 belongs to the second-generation MPACs and is a highly soluble derivative of 8-OH quinoline that lacks iodine. With increased BBB permeability and ionophore activity compared to CQ, PBT2 reduced Aβ levels and improved cognitive abilities in Tg2576 and APP/PS1 AD mouse models (67,68). In these mice, both acute and chronic dosing resulted in a significant decrease in Aβ plaque burden and interstitial fluid Aβ levels, a significant increase in synaptophysin levels, and restoration of hippocampal dendritic spine density (69). In a 12-week phase-II clinical trial of early AD patients, PBT2 was also found to lower cerebrospinal fluid Aβ 42 concentrations and reverse frontal lobe functional deficits significantly, reducing executive dysfunction; however, no significant effects on cognition and memory were reported (70,71).
The ionophore activity (the selective Aβ-metal-ion binding) of these compounds seems more promising than metal-chelation (metal-depletion) in counteracting AD pathology. While CQ and PBT2, possessing both features, are effective in reducing sedimented Aβ, triethylenetetramine (TETA), a classic hydrophilic Cu chelator, is not (72). TETA, a well-characterized anti-diabetic molecule, acts as a highly selective divalent copper (Cu(II)) chelator that prevents or reverses diabetic copper overload, thereby suppressing oxidative stress. Recent data showed that TETA disuccinate or trientine treatment also reduces Aβ production and deposition in APP/PS1 mice but not in Tg2576 mice in the same experimental paradigm in which CQ was effective (73). Given the hybrid action of many of these compounds, the original proposed name shifted from MAPCs to "ionophores" and finally to "metallochaperones" (74). Other molecules with metal ion chaperone properties, like pyrrolidine dithiocarbamate (PDTC) and copper-bis(thiosemicarbazonoto) complexes, have also proved to ameliorate cognitive functions in AD mouse models, though they did not affect Aβ (75,76). On the contrary, d-penicillamine chelates Cu by forming a stable complex with thiol groups and allowing renal copper excretion. A clinical pilot study indicates that d-penicillamine can reduce oxidative stress, but not the clinical progression of AD (77).
While Zn, Fe, and Cu mainly bind to Aβ (78), Al and Fe accumulate and bind to hyperphosphorylated tau in neurons with NTFs (79). As Fe binds to both misfolded proteins, Fe is a potential target for chelation therapy. A 2-year long, single-blind clinical study showed that sustained intramuscular administration of desferrioxamine mesylate (DFO), an Al/Fe metal chelator, slows the clinical progression of AD-associated dementia (80). Unfortunately, its hydrophilic nature and large molecular size limit its absorption across the gastrointestinal tract and prevent it from penetrating the BBB (80), inducing undesired side effects such as systemic metal depletion. All these features have prevented its final use in the clinics (81). More recent evidence showed that intranasal administration of DFO is accompanied by a reduction in Aβ burden/ load as well as improved learning and memory abilities in APP/PS1 mice (82) confirming, again, the beneficial effects of this drug on AD pathology.
Other Fe chelators have been developed in the last 20 years. Feralex-G, for instance, showed enhanced chelating activity for Al/Fe associated with hyperphosphorylated tau compared to DFO (79).
Chelators such as TETA, penicillamine, and DFO are routinely used in the treatment of metal overload disorders, such as Wilson' s disease. However, these molecules are hydrophilic and exert their effects by systemic depletion of metals and are poorly absorbed through the BBB with limited bioavailability. The efficacy of chelation therapy is still a matter of debate (74). Several metal-binding proteins are currently being tested as AD treatment. Metallothionein 3 (MT-3), a key regulator of metal homeostasis in the brain, has been found to decrease in AD patients. Intraventricular injection of Zn-loaded MT-3 (Zn/MT3) attenuates behavioral deficits, Aβ aggregation, oxidative damage, and neurodegeneration in the Tg2576 AD mouse model. This drug allows the exchange of Aβ Cu with Zn making Aβ ROS inert (83).
Finally, another group of molecules has been characterized for their ability to modulate APP expression, leading to a novel therapeutic approach aimed at reducing the amyloidogenic process (84). A common feature of these molecules, to which the previously mentioned DFO belongs, is their ability to recognize an IRE stem-loop upstream to the start codon of the APP transcript at the APP-5'UTR region (84,85). Like tetrathiomolybdate (TM) (a Cu chelator) and dimercaptopropanol (a Pb and Hg chelator), DFO reduces Aβ secretion and restrains the expression of APP holo-protein in vitro (84,86). Similarly, XH1, a bifunctional compound capable of both APP binding and metal-chelation, was able to lower APP expression in the SH-SY5Y cell line and reduce Aβ burden in APP/PS1 mice (87). Several other chelators with multifunctional properties with potential applications in AD treatment exist (88). However, so far, they have mainly been tested in vitro, and the beneficial effects they exert cannot all be attributed to their interaction with metals.

TARGETED INTERVENTIONS: DELIVERY OF TRACE METALS
Although many studies support the efficacy of chelation therapy in vitro, the translation of metal chelators into clinical studies is currently limited due to their inability to cross the BBB easily: they are hydrophilic, and many are neurotoxic at high concentrations. The use of innovative drug delivery systems (DDSs) such as nanoparticles designed to overcome the BBB could be considered as a strategic approach in the prevention and treatment of neurological and neurodegenerative diseases such as AD (89)(90)(91). Several sustained-release nanocarriers have been studied for the treatment of AD; in particular, polymeric nanoparticles (NPs) allow a significant increase in bioavailability and effectiveness of the administered drugs. Although the efficacy and advantage of these systems have been supported by several experimental studies (Table 2), the translational efficacy is yet to be proven.

CONCLUSION
The role of trace metals in AD is complicated as they are involved in both pathological and physiological processes in the brain. In addition, trace metals such as zinc, which binds to more than 10% of proteins encoded in the human genome (96), regulate a plethora of processes whose effects are neither fully understood nor assessable in preclinical and clinical studies. Furthermore, levels of one trace metal affect the levels of other metals (19). Therefore, any therapeutic strategy targeting trace metal levels in AD needs to be specific in terms of drug delivery and consider its effect on the concentration of a particular metal. Preclinical and clinical studies attempting to manipulate trace metals, although not entirely successful, were among the most promising approaches for the treatment for AD. Metal chelation and chaperoning successfully reduced Aβ generation and aggregation, synaptic dysfunction, neuroinflammation, oxidative stress, and induced degradation of senile plaques ( Figure 2). In addition, effects on cognitive abilities were observed in some studies. To further develop treatment strategies targeting metal homeostasis, it is necessary to clearly understand the role of trace metals in AD. It is important to explore the role of trace metals, such as zinc, in physiological processes that could be exploited as therapeutic targets apart from Aβ aggregates, such as synaptic plasticity mediated by zinc-dependent SHANK proteins (97) or reducing excitotoxicity and Aβ burden by zinc-binding S100B proteins (98)(99)(100)(101). A re-distribution and balancing of metals may be necessary between neurons, astrocytes, and microglial cells to optimize effects on synapses and inflammatory processes. This could be achieved by targeted delivery of trace metals to Aβ aggregates and NFTs. To this end, novel compounds, combinations of novel metal chelators and chaperones, and novel delivery platforms will be needed.

Conflict of Interest:
The authors declare no potential conflict of interest with respect to research, authorship and/or publication of this chapter. Metal chelators and ionophores can prevent metal binding to Aβ, inhibit oligomerization of Aβ, and consequently reduce its aggregation and toxicity. Altering metal homeostasis by depletion, delivery, and chaperoning was reported to decrease Aβ plaque burden and interstitial fluid Aβ levels, positively affect synaptic spine density, and suppress oxidative stress. Zinc delivery further reduces inflammation, while iron chelation modifies tau phosphorylation, and APP expression.

Copyright and Permission Statement:
To the best of our knowledge, the materials included in this chapter do not violate copyright laws. All original sources have been appropriately acknowledged and/or referenced. Where relevant, appropriate permissions have been obtained from the original copyright holder(s).