Copper Toxicity Links to Pathogenesis of Alzheimer’s Disease and Therapeutics Approaches

Alzheimer’s disease (AD) is an irreversible, age-related progressive neurological disorder, and the most common type of dementia in aged people. Neuropathological lesions of AD are neurofibrillary tangles (NFTs), and senile plaques comprise the accumulated amyloid-beta (Aβ), loaded with metal ions including Cu, Fe, or Zn. Some reports have identified metal dyshomeostasis as a neurotoxic factor of AD, among which Cu ions seem to be a central cationic metal in the formation of plaque and soluble oligomers, and have an essential role in the AD pathology. Cu-Aβ complex catalyzes the generation of reactive oxygen species (ROS) and results in oxidative damage. Several studies have indicated that oxidative stress plays a crucial role in the pathogenesis of AD. The connection of copper levels in AD is still ambiguous, as some researches indicate a Cu deficiency, while others show its higher content in AD, and therefore there is a need to increase and decrease its levels in animal models, respectively, to study which one is the cause. For more than twenty years, many in vitro studies have been devoted to identifying metals’ roles in Aβ accumulation, oxidative damage, and neurotoxicity. Towards the end, a short review of the modern therapeutic approach in chelation therapy, with the main focus on Cu ions, is discussed. Despite the lack of strong proofs of clinical advantage so far, the conjecture that using a therapeutic metal chelator is an effective strategy for AD remains popular. However, some recent reports of genetic-regulating copper transporters in AD models have shed light on treating this refractory disease. This review aims to succinctly present a better understanding of Cu ions’ current status in several AD features, and some conflicting reports are present herein.

Like other body parts, the brain contains many necessary transition metal ions, such as cobalt, copper, chromium, iron, zinc, and non-essential metals. Generally, the brain is the part of the body that contains the highest amount of transition metal ions content per weight. In comparison, the content of the copper ion in the brain is 0.004 g per kg [ 98 ]. It is an important chemical component of cell biology because it can receive and donate electrons. Once delivered and spread in a body, the cycle of Cu ions as the cupric ion (Cu 2+ ) in its higher oxidation state and cuprous (reduced) form (Cu + ), often joined to cuproenzymes with a small proportion as labile Cu, which was named as free or unbound Cu [ 99 ]. As a redox catalyst, Cu is necessary for many enzymes’ catalytic activity, regulating various cellular, biochemical, and regulatory processes. This metal plays an essential role in the catalytic centers of metalloproteins, electron transfer (ET) sites, and structural components. Many studies have provided information concerning high serum levels of non-Cp–Cu, which results in reduced cognitive function, and the rate of mild cognitive impairment (MCI) to AD increased [ 90 , 100 , 101 ]. Postmortem biochemical analyses of the AD brain have revealed the reduced total soluble Cu levels, while its content within insoluble neuritic plaques is raised [ 102 , 103 , 104 ]. However, despite the decreased total Cu level in the central nervous system (CNS), elevated levels of redox-active exchangeable Cu are found in the Brodmann (BA46) and the temporal lobe (BA22) areas. AD cortical tissue has an increased propensity to bind exchangeable Cu 2+ with increasing oxidative damage and neuropathological alterations, which have been seen in Alzheimer’s cases [ 105 ]. 2.1. Copper and Amyloid-Beta Precursor Protein Many authors have cited several models on copper implication in AD. Indeed, the most approved have put forward the “gain-of-function” of Aβ after binding Cu 2+ ions [ 106 ]. Alternatively, current hypotheses suggesting “loss-of-function” of Aβ as the pathology of this disorder [ 94 , 107 ]. However, APP exports metal from neurons and a lower level of the soluble, functional Aβ monomer may lead to copper accumulation in the cell [ 107 ]. APP can bind to Cu 2+ and reduce it to Cu 1+ through its copper-binding domain (CuBD). APP can strongly bind Cu 2+ to the N-terminal resulting in the decrement of copper ions [ 108 ]. Genetic studies of animal models have suggested that the APP-induced conversion of Cu 2+ to Cu + increases copper ion removal from the brain; this process could justify the point of why Alzheimer cases show lower brain level and higher Cu content in their serum-plasma [ 100 , 101 , 103 , 109 , 110 ]. However, Cu-binding with the N-terminal domain of APP may manage other functions of this protein, including synaptogenic function, stability, and metabolism [ 111 , 112 , 113 , 114 ]. Interestingly, the lower the copper content of the brain, the higher the ratio of endocytosed APP, and the generation of Aβ maybe works as a defense mechanism to stop the unnecessary loss of Cu [ 114 , 115 ]. Though newly produced intracellular Aβ can remove Cu, it probably leads to dyshomeostasis of copper ions and Aβ peptide deposition into plaques. This process results in neuritic plaques formation because Cu ions increase Aβ accumulation and cell damage due to the production of reactive oxygen species in AD [ 116 , 117 , 118 ]. Amyloid plaques or senile plaques mostly consist of the Aβ peptides, the essential peptide whose presence at a nanomolar concentration is shown by numerous studies in the cerebrospinal fluid (CSF) as well as in serum [ 119 , 120 ]. However, the TASTPM animal model study pointed out that the concentration of Cu in the Alzheimer’s brain does not link to plaques deposition [ 121 ]. The affinity of Cu 2+ ions for Aβ peptides is very high [ 122 , 123 ], and it also increases the portion of beta-sheet and alpha-helix in Aβ proteins, which may be the cause of its aggregation [ 124 ]. So, β-amyloid deposition is the reason behind pathological alterations in AD, and its clearance when patients are immunized does not stop this disorder [ 125 , 126 , 127 , 128 , 129 ]. However, some scientific studies reported the presence of neuritic plaques in the brains of cognitively healthy elderly [ 130 , 131 , 132 , 133 ]. The soluble oligomers obtained from the culturing of cells possess high chemical resistance and protect against its conversion into monomers via several degrading factors and maintain the presence of covalent cross-links in them [ 134 , 135 ]. Binding of Cu 2+ ions increases dityrosine-linked β-amyloid dimers as observed in vitro studies of this neurological disorder [ 136 , 137 , 138 , 139 ]. This dimer structure switches from parallel to anti-parallel in the presence of Cu 2+ and this process is regulated with the occupied binding sites of Cu [ 140 ]. Moreover, the same scholars later demons

Reactive oxygen species (ROS) generation is associated with a redox-active copper ion complex with aggregated Aβ, which has been identified to contribute to oxidative stress and damage to neuronal cells in AD [ 175 ].Copper-Aβ fibrils complex produces hydrogen peroxide (H 2 O 2 ) in the presence of ascorbic acid, a biological reductant [ 175 , 176 ]. Increment in the ratio of (Cu-Aβ) leads to the production of H 2 O 2 , hydroxyl radicals (OH•), and misfolding of proteins (aberrant aggregates) shifts from amyloid fibrils to amorphous aggregates [ 116 ]. While initial studies have shown ROS being dangerous to causing neurodegeneration, the recently gathered data suggest some ROS action is necessary for cognition function and memory development [ 177 , 178 , 179 , 180 , 181 ]. According to some results, it has been suggested that the Cu-Aβ complex generates less ROS than unbound Cu ions [ 182 ]. Cu ions interaction with Aβ results in its accumulation under a slightly acidic environment and promotes ROS production in Alzheimer’s patients [ 183 ]. The production of ROS due to metal ions such as Cu leads to oxidative damages to Aβ peptide. This oxidized β-amyloid has been seen in senile or amyloid plaques during in vivo studies [ 184 ]. Some in vitro data imply that oligomeric and fibrillar forms of Aβ prevent hydrogen peroxide production at high concentrations of Cu 2+ . Additionally, amyloid fibrils produce less hydrogen peroxide than that in the oligomeric state [ 185 ]. However, the pro-oxidant function of the Cu-Aβ complex is not confirmed yet, because this complex is more effective in ROS generation than several tested biological relevant Cu-peptides and Cu-binding proteins [ 186 ] but less efficient than loosely-bound Cu [ 182 , 187 , 188 , 189 ]. It is usually stated that hydrogen peroxide production is a two-electron oxidation process; however, current research has indicated the production of superoxide (O 2− ) as an intermediate in hydrogen peroxide formation via Cu-Aβ complex and oxygen [ 190 ]. Cu is redox-active and, when bound to Aβ, catalytically cycles between the Cu +1 and Cu +2 oxidative states to generate ROS such as O• −2 , OH•, and H 2 O 2 . Thus, the coordination of amyloid-β with Cu ions plays a significant role since ROS generation is a metal-catalyzed process, termed as a catalytic in-between state [ 191 ]. Therefore, computational studies have also examined Cu’s role in that state and its reactivity to the substrates such as oxygen or hydrogen peroxide [ 192 , 193 , 194 ]. Toxicity due to Cu ions in AD has been linked with the oxidant form of Cu ions such as Cu 2+ [ 65 , 195 ]. Considerable studies found that the elimination of Cu + from Aβ inhibits the production of Aβ oligomers and oxidative damage [ 196 ], and Cu 1+ has a stronger affinity to monomeric Aβ peptide than Cu 2+ , which leads us to propose that Cu 1+ cation is principal in the oxidation state in vivo [ 197 ]. Contrarily, the same metal ions also exist as catalytic metal ions, like Cu in SOD1, where they stop producing the H 2 O 2 . This also proves the value of coordination compounds. Copper can be in both pro-oxidants and antioxidants, which depends on its coordination position in compounds. However, in AD, higher production of ROS or less activity of the enzymes which degrade ROS results in an imbalance of pro-oxidants and antioxidants form, which cause oxidative damage on biomolecules [ 176 ]. Therefore, it is now clear how important it is to regulate copper ions’ metabolism in terms of content, transportation, storage, and association with active sites. Abnormal Cu homeostasis increases the levels of free or loosely bound copper, which often produces ROS [ 98 ]. These ions can also attach to off-target biomolecules and disrupt their functional roles, leading to higher chances of oxidative damage. Perhaps the Cu-Aβ complex is directly linked with ROS generation, so most of the studies have been associated with Cu-Aβ complex, suggesting a direct link between AD and oxidative damage [ 182 ]. Remarkably, all of the above data highlights the point that Cu is highly toxic in excess amounts and responsible for its participation in a redox-cycling reaction, which produces ROS that results in much damage to biomolecules such as carbohydrates, nucleic acids, lipids, and proteins. So, a higher level of free Cu ions causes more toxicity to the cells and eventually leads to cell death. Therefore, cellular Cu should be tightly controlled ( Figure 3 ) [ 99 ].

In the body cells, Cu is absorbed through a high-affinity copper transporter Ctr1, incorporating cuprous (Cu + ) ions from the intestinal microvilli’s surface. Little is known about Cu 2+ absorption, which is probably absorbed by divalent metal transporter 1 (DMT1) or other shared metal transporters [ 218 ]. Ctr1is responsible for the majority (~70%) of Cu import into mammalian cells, from which Cu is passed to glutathione, which carries Cu through the cytoplasm [ 219 ]. The absorbed copper ions will be targeted to Cu-binding chaperones and enzymes in different cell compartments such as cytosolic, mitochondrial, and Golgi. In the cytosol, Cu chaperone for superoxide dismutase 1 (SOD1), CCS, mediates Cu + loading. A recent study suggested that the direct transfer of copper from Ctr1 to chaperones and then passing it to SOD1 is via forming a Ctr1-CCS-SOD1 complex [ 218 ]. Besides CCS, soluble copper chaperones such as Atox1 and Cox17 can also escort Cu + from Ctr1 in the cytosolic pool to facilitate copper supply to their specific target compartments [ 220 ]. Consequently, in the absence of Ctr1, other pathways to absorbed Cu ions are unavailable to the organism because of the sequestration of copper in the sub-apical vesicles. This has been confirmed by making the intestinal epithelial cell-specific knockout of the Ctr1 (Ctr1int/int) mice, which manifested severe Cu deficiency, and the majority died within three weeks of post-birth [ 221 ]. A considerable portion of ingested cuprous ions are passed into circulation in enterocytes to reach different tissues by Atox1/ATPase routes. The mouse model with inactivated ATOX1/ATP7A routes showed defects in Cu distribution, which leads to pathological variations in many organs, especially the brain [ 222 ]. In the CNS, Cu deficiency has been found in the hippocampus and amygdala regions of Alzheimer’s patients, which causes severe histopathologic alterations in AD. Additionally, scientific research has put forward that the frontal cortex tissue of Alzheimer’s patients had an increased susceptibility for exchangeable copper (CuEXC), which is associated with the overproduction of free radicals (ROS) in AD [ 223 ]. In the CSF of the AD patients, there is no significant change in Cu concentration as compared to that of the healthy cases (HC) [ 224 ]. Furthermore, within peripheral fluids, abnormal homeostasis of copper ions has been intensively investigated. The relevant data point to increased [ 224 , 225 ], decreased [ 88 , 226 ], or unchanged [ 227 ] serum or plasma Cu in Alzheimer’s patients. Many other scientific analyses have also reported excessive free or diffusible copper in serum [ 224 , 226 , 228 ]. However, Rembach (2013) has suggested the possibility of decreased non-CP copper levels - copper that is not bound to ceruloplasmin in mild cognitive impairment (MCI) and AD, which leads to a decline of copper-dependent biochemical activities in AD [ 229 ], such as reducing SOD1 activity of erythrocytes [ 88 ]. Cu association for AD is ambiguous as some substantial researches showed Cu deficiency in AD and, hence, it is required to increase Cu levels [ 86 , 87 , 88 ]. In contrast, many different scientific pieces of evidence demonstrated Cu overload, and thus it is necessary to reduce it [ 90 , 91 , 92 , 93 , 94 , 95 ]. The main updated explanation so far is that the abnormal Cu homeostasis is due to an increment in the labile Cu ions and a reduced attachment to proteins [ 107 , 174 ]. Until 2012, the published contradictory scientific researches fueled the debate of copper concentrations in AD. So far, to check Cu levels in various biological specimens of AD patients, such as serum, plasma, and CSF, six meta-analyses have been done during the past six years. Studies published from 1984 to 2017 have been included in these meta-analyses [ 100 , 101 , 224 , 230 , 231 , 232 ], which give unambiguous results: overall and unbound Cu both are present in higher concentrations in the serum-plasma samples of the AD patients compared to that in the healthy cases [ 230 ]. According to the very recent meta-analysis, which includes a total of 35 pieces of research: eighteen report an increase, fourteen show no change, and one reports a decrease in Cu level in the serum-plasma of this disorder [ 232 ]. Subsequently, three more studies have been published, stating increased Cu 2+ ions level in Alzheimer’s compared to that in the healthy controls [ 233 , 234 , 235 ]. These recent researches have contributed considerably to the explanation of the previous controversy. In blood, a higher level of free plasma Cu, which has been identified in 50–60% of Alzheimer’s patients, can explain the higher level of serum Cu in AD [ 145 , 174 , 233 , 236 ]. Another earlier research also observed an increased concentration of serum copper ions in a special kind of AD (Alzheimer’s disease epsilon four apolipoprotein E allele carriers) [ 237 ]. According to some scientific investigation, a genetic

Because multiple pathological variables are involved in the pathology of AD, therapeutics or drugs target a single mechanism that is not enough to treat this disorder. New therapeutics or medicines that can target multiple factors at the same time can be beneficial for patients suffering from neurodegenerative disorders. It is now undeniable that the next generation of therapies should have the ability to target the different causes of disease progression at the same time [ 287 ]. Neurodegenerative disorder drugs must have more than one of the following properties to be useful for these diseases such as control of the production of ROS, the ability of metal chelating, and greater BBB permeability, a decrease of β-Amyloid peptide deposition ( Figure 5 ), and last but not least, of regulating enzymes associated with the mechanism of the disease, for example, acetylcholinesterase [ 288 , 289 ]. Bifunctional metal chelators (BFCs) were suggested to treat multifactorial AD because they have the ability of both metals chelating and binding with amyloids. Substantial research has been made in this field in the last decade [ 259 , 263 , 290 , 291 ]. The fluorescent dye thioflavin T (ThT), also known as Basic Yellow 1 or CI 49005, has been widely used to detect amyloid fibrils [ 292 ] in both in vivo and in vitro studies. The first bifunctional chelator designed was XH1 ( Figure 6 ), connecting various molecular fragments of different specificities to make a hybrid molecule [ 293 ]. Its structure is composed of two-terminal thioflavin-T-derived moieties, which are attached by a DTPA (diethylene triamine penta-acetic acid) binding unit. Besides metal ions’ chelation role in Alzheimer’s, new chelating molecules such as phenyl benzotriazole followed by the same design principle of thioflavin-T(ThT), correlating them with dipicolylamine or pyrinophane type metal chelators have been reported by the studies [ 263 , 287 , 291 , 294 ]. Several studies have analyzed the deposition of Aβ1-42 in the deficiencies and presence of essential metal ions [ 291 , 295 ]. Franz and co-workers have used persuasive strategies to design prochelators in 2006. These chelators only work in the presence of oxidative stress and inhibit essential ion loss like Cu and Zn of metalloproteins. Followed by the same approach, boronic ester (BSIH), an excellent first-generation prochelator metal affinity group, was composed. It works as an iron chelation with salicylaldehyde isonicotinoyl hydrazone (SIH), in the presence of H 2 O 2 [ 296 ]. Various analogs of boronic ester, such as boronic esters (BSIH, BSBH) and acids (BASIH), have been investigated to check their performance as metal chelators [ 297 ]. Currently, many promising molecular scaffolds using the same strategy are exploring their effect on the multifactorial AD [ 298 , 299 , 300 , 301 ]. Choi (2011) and Hindo (2009) [ 302 , 303 ] reported the derived compounds and analyzed their impact on metal-binding properties, β-amyloid deposition both in deficiency and the presence of metal ions. Currently, small novel compounds such as 2,2-bipyridine (bpy) derivatives (1–4) and other N,N-dimethylaniline including novel N-bidentate ligands, have been described to show good results for the treatment of multifactorial AD [ 304 , 305 ]. The effects of several flavonoids such as myricetin and EGCG (epigallocatechin gallate) have also been tested for this disorder [ 306 , 307 , 308 ]. While Orvig described salen-type Schiff-bases in addition to other chelating agents for the first time, it has also been connected with carbohydrate moieties [ 259 , 309 , 310 , 311 , 312 ]. In vivo investigation of the diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(ATSM)) ( Figure 6 ) compound has seen its protection against nitrosative damage of peroxynitrite and an increase of survival in the ALS mouse models [ 313 ]. Unfortunately, it was not studied much for metal chelation and protection against oxidative damage in AD. Many multifunctional molecules have been rationally designed to simultaneously resist and target various pathological hallmarks of the brain disorders, and their effects have been checked and reviewed by many studies [ 290 , 314 ]. Finally, despite all tremendous efforts that have been made for the success of multifunctional molecules as a potential therapy, only a handful of them show promising results in human clinical trials. Indeed the usage of these compounds to target the multi-mechanisms of the neurodegenerative diseases, its potential causes of failure also need to be understood, which has been reviewed elsewhere [ 122 , 265 ]. Overall, it is an effective strategy and will hopefully provide a better therapeutic option for Alzheimer’s cases when the compounds made become more tissue targeting specific.

Collectively, studies strongly advocate that dyshomeostasis of Cu, leads to the onset and progression of AD. Earlier researches have recognized amyloid plaques as toxic factors in AD. Though 20–40% of healthy cases have amyloid plaques, as illustrated by some studies [ 333 ]. Furthermore, cell death often leads to amyloid plaque formation in the brain. While mounting evidence implicates ROS in the AD etiology, loosely bound copper ions are very efficient catalysts for ROS generation by a copper-amyloid complex [ 105 , 334 ]. Some studies indicate an increased liable pool of Cu in the brain [ 105 , 230 ] responsible for Cu deficiency. The reason of Cu deficiency seems to be an essential factor in AD. Copper deficiency leads to Cu enrichment in lipid rafts, so maybe an elevation in lipid raft domains could be the reason for Cu deficiency in the brain; thus, lipid raft domains could be an efficient drug target. Studies indicated that the disrupted lipid rafts (by omega-3 fatty acids) slowed the progression of AD [ 215 , 335 ]. Another direction for research depends on the feasibility of developing novel therapeutic approaches to work against this disease. The proposal for direct chelation therapy of Cu ions to work in this disorder is still in discussion [ 265 ]. Support for the lowering cellular Cu levels comes from the Drosophila model of AD, where although copper chelation or genetic knockdown of copper transporters (Ctr1C) decreased the expression of Aβ degrading proteases but rescued the toxic phenotype [ 336 ]. Similar results were also observed by silencing the expression of Ctr1B, or when copper exporter DmATP7 [ 336 ] and dMTF-1 or MtnA [ 94 ] were overexpressed in the nervous system of the Aβ transgenic flies. These flies exhibited improved neurodegeneration, locomotion, longevity, and a reduction in Cu-Aβ complex-induced oxidative stress. Furthermore, in parallel, antibody-based treatment for Aβ aggregation is now developing and providing safe results as well [ 337 ]. Research organizations should come to the same standpoint regarding the experimental requirements and procedures to be used, to avoid different and ambiguous results for such serious matters. In this context, all the struggles for a better understanding of AD pathology’s molecular mechanisms and developing innovative therapeutic approaches should be appreciated.