Metal Ions-Mediated Oxidative Stress in Alzheimer’s Disease and Chelation Therapy

Alzheimer’s disease (AD), ranked as the seventh leading cause of death worldwide, is one of the most incidental neurodegenerative disorders. AD patients experience irreparable damages to the brain, indicated as progressive, insidious, and degenerative. Past research has discovered that the amyloid cascade hypothesis best describes the pathophysiological etiology of AD, designating amyloid-β plaques and neurofibrillary tangles as the ‘hallmarks’ of AD pathology. Furthermore, accumulating evidence show that the oxidative stress state, the imbalance between reactive oxygen species (ROS) production and antioxidation, contributes to AD development. This chapter describes the oxidative stress process in AD. It mainly tackles the correlation of metal-catalyzed ROS production with amyloid-β and how it oxidatively damages both the amyloid-β itself and the surrounding molecules, potentially leading to AD. Additionally, both the role of metal chelation therapy as a treatment for AD and its challenges will be mentioned as well. This chapter specially focuses on how metal ions imbalance induces oxidative stress and how it affects AD pathology.


Introduction
Past research has tried to find the pathogenesis and etiologies regarding Alzheimer's disease (AD). Recent studies show that reactive oxygen species (ROS) are linked with the progression and development of AD, especially superoxide anion, hydrogen peroxide, and hydroxyl radical. Reactive oxygen species have been found as the by-products of metal-catalyzed oxidation associated with amyloid-β. These findings are crucial for the treatment of AD, as they provide the underlying mechanism for metal chelation therapy, which involves the use of metal chelators for metal removal.
This chapter discusses both past and current research with regards to AD pathology and treatment in the following order: Alzheimer's disease, reactive oxygen species, oxidative stress and Alzheimer's disease, metal chelation therapy, and challenges of metal chelation therapy.

Reactive oxygen species
Reactive oxygen species (ROS) are unstable, highly reactive molecules and radicals which are derived from molecular oxygen. ROS production takes place in aerobic organisms that utilize mitochondrial electron transport for respiration or undergo oxidation catalyzed by metals and intracellular enzymes. [12] In normal settings, ROS play a crucial role for cell signaling such as cell cycle regulation, enzyme activation and apoptosis. Yet, under oxidative stress conditions, the immoderate production of ROS has detrimental effects on cells causing protein, DNA, and lipids damage and eventually, cell death. [1] When a molecular oxygen goes through a monovalent reduction, superoxide anion radical (O ÁÀ 2 ), a precursor compound of ROS, is formed. [13] O ÁÀ 2 , due to its unstable state, react with other radicals such as nitric oxide (NOÞ, forming highly reactive peroxynitrite (ONNO À ). 0 ÁÀ 2 also propagates further oxidative chain reactions, producing hydrogen peroxide (H 2 O 2 Þ with the help of superoxide dismutase (SOD). H 2 O 2 are sequentially reduced either to hydroxyl radical (OH Á ), one of the most reactive oxidants, or fully reduced to water. [14,15] ROS generation, mainly in forms of O ÁÀ 2 , H 2 O 2 , OH Á , are induced by both endogenous and exogenous pathways. The endogenously produced ROS are mainly byproducts of mitochondrial respiratory chain and phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, circumstances in which the reduction of oxygen is enabled. [Figures 1 and 2] Transition metals and numerous intracellular enzymes such as, Xanthine oxidase (XO), Lipoxygenases (LXO), and Cyclooxygenase (COX) are also principal endogenous ROS generators (Figures 3-5) [16].
ROS are produced in response to exogenous or environmental factors as well, such as radiation, air pollutants, diet, tobacco smoke, drugs and xenobiotics, chemotherapy, and pesticides. [16,17] Exposure to UVR from solar radiation develops high concentrations of ROS, which causes an imbalance between ROS and cellular antioxidants, thus provoking oxidative stress. [17] Tobacco smoke, another notable factor of ROS production, consists of 10 14 -10 16 free radicals per puff which can potentially produce H 2 O 2 and OH Á . [16] The right duration, quantity, and location of ROS production is required for normal physiological processes. In cases where the appropriate conditions are not met, both insufficient and excessive ROS production, ROS-related diseases can arise. [15] Such medical conditions include glucolipotoxicity, insulin resistance, diabetes mellitus, mitochondrial dysfunction, cancer, autoimmune disorders, cardiovascular, neurological, and psychiatric disease. [15,18] Antioxidants work as the defense mechanism against ROS induced damage. Its role is to maintain the effective functions of ROS while at the same time, regulate its level. Oxidative stress is attenuated by both endogenous antioxidant system and the exogenous intake of antioxidants. [19,20] The former includes enzymes such as SOD, glutathione (GSH), catalase and glutathione peroxidase (GPx). [19] Meanwhile, the essential exogenous antioxidants are absorbed through vegetables, whole grains, fruits, and omega-3 fatty acid containing diet. Vitamin C, vitamin E, βcarotene, selenium, carotenoids, and polyphenols represent exogenous antioxidants. [19,20]

Oxidative stress and Alzheimer's disease
Majority of current research show that oxidative stress, the imbalanced state of ROS production level and antioxidative level, is related to the pathogenesis of neurodegenerative diseases, representatively AD. [21] This chapter approaches mainly the association of oxidative stress with AD, mostly regarding the correlation between Aβ and ROS production and how it affects the neighboring neural molecules.
As previously stated above in the Alzheimer's Disease section, amyloid plaques and NFTs are regarded as the 'hallmarks' of AD. Overwhelming evidence show that amyloid plaques are highly concentrated in metal ions, such as copper Cu ð Þ, iron Fe ð Þ,  zinc Zn ð Þ and calcium Ca ð Þ, which are present in the synaptic areas. Such metal ions are interconnected with the amyloid cascade reaction and NFT formation. [22] Metal ions imbalance induces oxidative stress which triggers ROS production. Increased production of ROS leads to secretases imbalance and phosphatases imbalance, each interconnected with the formation of Aβ and P-tau. Accordingly, Aβ and P-tau production increases, which eventually leads to neurodegenerative diseases including AD. [23] Thus, the Aβ toxicity, NFTs, oxidative stress, and ultimately neuronal cell death depend on the existence of redox metals. [24] This  chapter mainly discusses the correlation of metal-catalyzed ROS production with Aβ ( Figure 6).

Copper
Among the metal ions, copper is considered the most redox reactive. The association of copper ions with Aβ can be described as a three-step process. First, endogenous reductants bind with the copper, followed by the reduction of Cu(II) to Cu(I). The reductive state of copper triggers the reduction of molecular oxygen as well, producing ROS. [25] Copper directly interacts with Aβ, promoting increased aggregation of Aβ and the toxicity of amyloid oligomers and plaques. [22,26] Histidine His6, His13, His14 ð Þ and Tyrosine (Tyr10) amino acid residues modulate the binding of copper to Aβ:Cu(II) is reduced to Cu(I), after its chemically binding to Aβ(higher affinity to Aβ1-42 compared to Aβ1-40), generating hydrogen peroxide as a byproduct which has high potential to be reduced to hydroxyl radical. Accordingly, the complexation of copper in Aβ elevates the neurotoxicity, now endowed with enlarged reduction potential. [24] The Cu-Aβ couple correspondingly assists the further process of ROS production. The copper-Aβ-mediated oxidation of reductant species such as ascorbate, which are abundant in the brain, induces generation of ROS: hydrogen peroxide, hydroxyl radical and superoxide anion. [25]

Iron
Iron, as a redox active metal, is also significantly linked with AD pathology. However, unlike copper, iron ions do not directly interact with and bind to Aβ. [27]  Metal ions imbalance, increased ROS production, and neurodegeneration imbalance of metal ions, such as copper, iron, zinc, and calcium, creates oxidative stress condition. This is followed by increased production of ROS, and consequently Aβ and NFTs, which eventually provokes neurodegenerative diseases including AD.
Iron exists in both in redox-inactive forms Fe 3þ À Á and redox-active forms Fe 2þ À Á within the brain. They are also found in zero-oxidation-state Fe 0 À Á or as ionic compounds such as magnetite Fe 3 O 4 ð Þas well. All forms are possible inducers for Aβ aggregation, prompting the iron redox cycle and ROS production. [28,29] Iron concentration and increased free radical production had been noticed in the cerebellum and glia cells of AD patients. [23] After iron's indirect interaction with Aβ, the redox cycle of Haber-Wiess and Fenton reaction is triggered, yielding ROS in forms of hydrogen peroxide, hydroxyl radical and superoxide anion, as in the process of copper-mediated oxidation. The resulting ROS effects Aβ aggregation and other oxidative damages in local organelles as well. Research results based on high-resolution transmission electron microscopy (HR-TEM) and synchrotron-based X-ray absorption studies support the storage of iron within Aβ and the iron-catalyzed ROS production. [27,29] During the process of the metal-catalyzed ROS production in correlation with Aβ, both the Aβ peptide itself and the surrounding molecules undergo oxidative damages. The amino acid residues of Aβ, cysteine, methionine, arginine, histidine, lysine, phenylalanine, tryptophan, and tyrosine, are oxidated as well, chemically changed, and impaired. The ROS produced through metal-mediated oxidation also cause protein carbonylation and nitration, lipid peroxidation, and protein modification. The mitochondria of nearby cells also experience oxidation, leading to increased mitochondrial and nuclear DNA &RNA damages which all potentially lead to the etiology of AD. [30]

Zinc
The impact of zinc Zn 2þ À Á in AD is rather controversial. [23] Some research suggests irregularly high concentration of Zn 2þ have been investigated in AD patients' brains, inferring the linkage between imbalance of Zn 2þ homeostasis with AD pathogenesis. [31] One study indicated that Zn 2þ promotes both Aβ40 and Aβ42 aggregation, but only at the early stage. [32] In another study, high concentration of Zn 2þ was shown to induce NADPH-oxidase reaction and ROS production (especially mitochondrial ROS production) in AD pathological state. Excessive zinc therefore prompted Aβ cascade reaction. [23] On the contrary, other research analysis show significant decrease of Zn 2þ in AD patients. [33]

Calcium
Calcium Cu 2þ À Á elevation also significantly contributes to Aβ production in AD patients. Sequentially, increased Aβ level in turn promotes an increase in Cu 2þ level by triggering the opening of voltage-dependent Cu 2þ channels. Moreover, high degree of Cu 2þ provokes further influx of Cu 2þ by enabling overexpression of L-type calcium channel subtype (Cav1.2). Excessive Cu 2þ consecutively stimulateAβ production and aggregation. [23]

Metal chelation therapy, a potential treatment for Alzheimer's disease
Based on the thesis that AD pathology relates to the interplay between metal ions and Aβ, treatments for AD have been proposed established on this characteristic. Metal chelation therapy has been raised as a method to agitate metal-Aβ interactions to treat AD in a lot of research. [27] Metal chelation therapy is initiated by injection of chelators (chelating agents) into the bloodstream which bind to the targeted metals and excrete them. [34] Studies show that metal chelating agents must satisfy the following conditions to manipulate as prospective treatments for AD.
1. Low molecular weight 2. Target certain: must be able to selectively attach to targeted metal ions bound to Aβ 3. Free or poor charge: must be able to cross blood brain barrier (BBB) 4. Low toxicity

Low possibility of side effects
Metal chelators content with above properties will successfully affix to aimed metal ions associated with Aβ, engendering their break-up and removal. [27] Among the various chelator drugs, only a few are suitable for AD; drugs that fulfill the properties stated above. The common chelator drugs adopted for AD treatments that have shown favorable results include desferrioxamine (DFO), bathophenanthroline, bathocuproine (BC), trientine, penincillamine, bis (thiosemicarbazone), tetrathiomolybdate (TTM). [35][36][37] In one clinical trial in 48 patients with AD, DFO has shown its positive effects. Using trace-metal analysis, the research team confirmed that DFO decreased the aluminum level in neocortical brains of AD patients dosed with DFO;125 mg per injection, twice a day, five days a week. [38] Although it showed outcomes regarding aluminum, one research insisted that, considering the affinity DFO has for iron, the result might have also been due to the elimination of iron. [39] In addition to iron, DFO also shows binding affinity towards copper. [40] Penincillamine, bathophenanthroline, bathocuproine (BC), and trientine have also been proven to be effective copper chelators. In one research test, these agents showed interaction with Cu-Aβ couple, deleting copper and improving Aβ solubility. Furthermore, BC has been proved to be the most efficacious, showing constant results across the broad range of AD brain tissue samples. [37] It has been suggested that the bis(thiosemicarbazone) compounds can regulate the concentration of copper in Aβ as well. [41] In one study, chemical compounds of the bis(thiosemicarbazone) metal complex family have shown successful treatment for animal models with AD. [42] Similar results have been noticed in another study using APP/PS1 transgenic AD mice model as well. Bis(thiosemicarbazone) enhanced the soluble Aβ level by deleting copper and led to the restoration of cognitive activity. [43] The effect of tetrathiomolybdate (TTM) as a copper chelator has been demonstrated as well. In one experiment, TTM was applied to Tg2576 transgenic mice model for five months. Positive effects were derived, showing that TTM lowered both the level of Aβ and Aβ plaques present in the brain. [44]

Challenges of metal chelation therapy
Although the above-stated metal chelating agents have shown positive effects in reducing Aβ levels in AD patients, there are still challenges surrounding the metal chelation therapy.
First, in addition to the originally aimed effects, metal chelating agents can induce undesirable outcomes as well. One study revealed that the application of divalent chelators, such as Cu, Fe and Zn, to severe AD patients lessened the requisite divalent metals that were already in their appropriate levels, as well as the targeted metal ions. Accordingly, the depletion of essential metals aggravated rather than treated AD pathology. [45] Furthermore, as stated in Metal Chelation Therapy, a Potential Treatment for Alzheimer's Disease, metal chelating agents have been proved to lowerAβ level through solubilization. However, it is still rather controversial whether metal chelators can not only solubilize but reverse the Aβ plaques to any forms of intermediates such as monomers, oligomers, protofibrils, short fibrils, or extended fibrils. [27,45] Finally, there are remaining questions concerning the efficacy of certain metal chelating agents. For instance, clioquinol (CQ), aCu -Zn chelator capable of agitating Aβ aggregation has been used in numerous clinical trials. However, the clinical and experimental results show that the effectiveness of CQ is yet contentious. [45,46] In one experiment, the utilization of CQ perturbed Cu and Zn homeostasis which elevated metal ion concentrations, which is contradictory to the predicted results. CQ also showed side effects, arising astrogliosis, spongiosis, and brain edema to the mice model. [47] For further development of metal chelation therapy, such disadvantages should be improved.

Conclusion
Aβ and amyloid plaques are determining symbols of AD. Metal ions, especially copper and iron, interact with Aβ in AD patient's brain which generates ROS, such as superoxide anion, hydrogen peroxide, and hydroxyl radical. This process promotes the aggregation Aβ and increases the toxicity of Aβ plaques. ROS induces damages to both Aβ itself and the surrounding molecules leading to protein, lipid, DNA, and RNA impairment. Metal chelation therapy has been proposed as method to agitate metal-Aβ interactions for AD treatment. Metal chelators injected into the bloodstream will target metals associated with Aβ and eliminate them, cutting off the activity of causative substances. The metal chelating agents that have shown positive effects towards AD so far include desferrioxamine (DFO), bathophenanthroline, bathocuproine (BC), bis(thiosemicarbazone), tetrathiomolybdate (TTM), trientine, and penicillamine. However, there are ongoing challenges facing the metal chelation therapy. The remaining questions regarding the efficacy of chelating agents and the precise mechanism of chelation therapy should be solved.