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Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-141194 IOS Press

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Towards a Unified Vision of Copper Involvement in Alzheimer’s Disease: A Review Connecting Basic, Experimental, and Clinical Research

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Amit Pala,1 , Mariacristina Siottob,1 , Rajendra Prasada and Rosanna Squittic,d,∗

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a Department

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Handling Associate Editor: Roberta Ghidoni

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of Biochemistry, PGIMER, Chandigarh, India Carlo Gnocchi Foundation ONLUS, Milan, Italy c Fatebenefratelli Foundation, AFaR Division; Fatebenefratelli Hospital, Isola Tiberina, Rome, Italy d Laboratorio di Neurodegenerazione, IRCCS San Raffaele Pisana, Italy

Accepted 29 August 2014

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Keywords: Alzheimer’s disease, ceruloplasmin, copper, metal, subtype

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INTRODUCTION

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Abstract. Copper is an essential micronutrient for physiological cell functioning and central nervous system (CNS) development. Indeed, it is a cofactor of many proteins and enzymes in a number of molecular pathways, including energy generation, oxygen transportation, hematopoiesis, cellular growth and metabolism, and signal transduction. This is because it serves as a catalyst of reduction-oxidation (redox) reactions in these processes. When copper is kept under control, bound to special proteins, it yields key properties. However, when it spirals out of control, it is exchanged among small compounds (it is loosely bound to them), and its redox activity makes it dangerous for cell viability, promoting oxidative stress. Copper homeostasis in the CNS is securely synchronized, and perturbations in brain copper levels are known to underlie the pathoetiology of wide a spectrum of common neurodegenerative disorders, including Alzheimer’s disease. The main objective of this review is to provide some of the most relevant evidence gleaned from recent studies conducted on animal models and humans, and to discuss the evidence as it pertains to a new concept: Aberrant copper metabolism, which appears to have a genetic basis, is a modifiable risk factor accelerating Alzheimer’s disease and initiation/progression of cognitive deficits in a percentage of susceptible persons.

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As life expectancies in industrialized countries continue to increase, cognitive and brain aging are becoming key issues in human health. The cognitive 1 These

authors contributed equally to this work. ∗ Correspondence to: Rosanna Squitti, PhD, Department of Neuroscience, Fatebenefratelli Foundation, AFaR Division; Fatebenefratelli Hospital, Isola Tiberina 00186, Rome, Italy. Tel.: +39 66837385; Fax: +39 66837300; E-mail: [email protected].

decline that occurs during aging negatively impacts quality of life (albeit with varying degrees of severity) by disrupting learning and memory abilities. The present review specifically addresses three important topics. That is, it: 1) provides information about aberrant copper metabolism as a causative risk factor in the initiation/progression of Alzheimer’s disease (AD), connecting experimental results to clinical evidence; 2) summarizes the acquired evidence about a copper subtype of AD and discusses its estimated

ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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chondrial oxidative phosphorylation, neurotransmitter synthesis and denaturation, free radical detoxification, and pigment formation [6, 7]. Conversely, excess copper is highly cytotoxic, as it can readily interact with oxygen to produce extremely damaging free hydroxyl radicals unless homeostasis is tightly regulated [7, 8]. Copper toxicity occurs as a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber–Weiss reactions, which are responsible for lipid peroxidation in membranes, protein oxidation, and the cleavage of DNA and RNA molecules [9–12]. As the central nervous system (CNS) is mainly composed of polyunsaturated lipids (lipids are a major constituent of neuronal membranes), it is particularly susceptible to oxidative stress and cellular damage. In addition, the high flux of ROS produced during reduction-oxidation (redox) reactions is extremely damaging to CNS functioning [13–15]. It is intriguing to note that numerous investigations have implicated copper dyshomeostasis directly or indirectly in the pathogenesis of several neurological and neurodegenerative diseases, like AD, Parkinson’s disease, Wilson’s disease aceruloplasminemia, Menkes disease, amyotrophic lateral sclerosis, Huntington’s disease, occipital horn syndrome, and prion disease (reviewed in [4, 16–20]). Even though some of these are diseases characterized by true copper defects and others are diseases where copper influences only the course of the disease, they all show the relevance of copper physiology in humans. They also show that, in case of a dysmetabolism, copper eventually leads to a disorder in which the neurological and neurodegenerative connotation is strongly represented. Emblematic of copper toxicosis is Wilson’s disease [21]. Wilson’s disease (or hepatolenticular degeneration) is an autosomal recessive genetic disorder in which copper, in the form of a copper not bound to ceruloplasmin (Non-Cp-Cu) is increased in general circulation and accumulates in organs and tissues. Deposits in the cornea, named Kayser-Fleischer rings, are typical. Clinical symptoms can have a liver, neurological, or psychiatric presentation. Mutations in the Wilson’s disease protein (ATP7B) gene cause the disease. A single abnormal copy of the gene is present in 1 in 100 people. Nevertheless, the accumulated evidence is in agreement over the basic role copper toxicity plays in the initiation/development of neurodegenerative diseases, especially AD. AD is a multifactorial disease caused by host of genetic and environmental factors. It is the most common cause of dementia, and it is characterized by the progressive neurodegeneration associated with

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annual incidence; and 3) discuss human copper intake in terms of its content in drinking water, as well as in foods. To address the first topic: evidence from milestone studies in in vitro and animal models of copper toxicity have been provided. They focus on copper toxicity, which induces AD-like pathology in diverse animal models via drinking water supplemented with trace levels of copper. It is important to define the terminology that we have used throughout the review. Copper in the diet represents the exclusive way copper comes into the body, since that from inhalation or contact is generally negligible. Food, supplements, and, in a minor percentage, drinking water are considered as sources for intake of copper in humans [1]. In experimental models, the most used approach to increase copper in the diet is to add it in drinking water. In this review, we were interested in providing a unified vision of copper involvement in AD, merging data from experimental models, to those coming from clinical studies, which are data referred to humans, in which copper ingestion is truly from the diet and not merely through drinking water. So, with the expression ‘copper in the diet’ we want to determine the amount of copper intake into the body and we consider copper ingested with drinking water as a part of the copper from diet. This terminology appears to be relevant when comparing evidence from experimental models and data from humans, which need a more holistic interpretation, which is included in the expression ‘copper in the diet’. Subsequently, addressing the second topic, we will show data on humans who demonstrate the association of copper with cognitive worsening. This is not a generalized feature of AD, but it is exhibited by a percentage of patients, who can be distinguished in a subtype of AD [2–5]. These people, who have an aberrant metabolism in handling copper that precedes the clinical diagnosis of AD, are at a higher risk with respect to copper intake. To address the third topic, information about water quality in terms of copper content, as well as copper in foods will be provided. We will try to discuss this data in the framework of recent evidence from large study data-set.

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A. Pal et al. / Non-Cp-Cu in AD

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COPPER TOXICITY AND CENTRAL NERVOUS SYSTEM Copper is an essential trace element that plays a key role in the growth and development of the body. Copper takes part in diverse aspects of metabolism, including connective tissue synthesis, iron metabolism, mito-

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EVIDENCE SUPPORTING A CAUSATIVE ROLE OF COPPER IN ALZHEIMER’S DISEASE: IN VITRO AND ANIMAL STUDIES Salient studies conducted in the late 1990 s demonstrated the strict interaction of the A␤ peptide with the metals copper, iron and zinc, which react as catalysts. It has been demonstrated that copper reacts with A␤ in reduction-oxidation (redox) cycles, which have as by-products H2 O2 and oxidative stress (Fenton’s and Haber Weiss chemistry). A␤ oligomer formation and precipitation within plaques is prompted in these reactions, along with lipid peroxidation [30–35] amyloid-␤ protein precursor (A␤PP) binds and reduces copper from Cu(II) to Cu(I), triggering oxidative chain reactions that produce H2 O2 and OH•. An additional milestone study demonstrated that A␤ and metals are packed together in plaques within the brain, and chelating agents, which sequester metals, can dissolve these plaques in autoptic brain samples [36]. Results from these studies have provided the basics of the potential effects of ‘copper toxicity’ in AD. Plentiful and diverse animal studies support this role of copper as a toxic agent in AD pathology. In an innovative study of late 1990 s, which suggested direct involvement of the APP gene in copper metabolism, hepatic and cerebral cortex (brain region particularly involved in AD) copper content were shown to be significantly increased in APP−/− and amyloid precursorlike protein (APLP2−/− ) knockout mice [35]. Thus, it is conceivable that APP gene expression modulates

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hepatic and cerebral cortex copper levels, but the mechanism leading to increased brain copper content (due to APP gene knockout) is not currently known. Serum copper levels were not significantly more altered in APP−/− and APLP2−/− mice than in wild-type mice. This suggests that a failure of copper excretion from the brain to the blood or, more likely, that an imbalance of copper distribution between the fractions bound and unbound to Cp could take part in the deposition of excessive copper levels in the brain in this knockout model. This will be explained better below. Several findings in experimental animal models account for the observation that the ingestion of inorganic copper has some deleterious effects on CNS functioning. Along with seminal studies demonstrating that the A␤PP is an important regulator of brain copper homeostasis [35], it was demonstrated that it also potentiates the A␤ mediated neurotoxicity by augmenting the oxidative stress [32]. A review of the animal models provides the most direct evidence in favor of the toxic burden brought by copper in AD etiology. In a seminal study published in 2003, Sparks and Schreurs demonstrated that, in a cholesterol fed rabbit model of AD, adding trace amounts of 0.12 ppm (0.12 mg/L) copper to distilled drinking water resulted in significantly enhanced cognitive waning. It also exacerbated amyloid plaque deposition to that of control animals [37]. This finding was somehow provocative, as it led to concerns about the content of copper in drinking water leached from copper pipe-lines. Although cholesterol is vital for neuronal transmission, synaptic plasticity, and cell function, it is also a well-established risk factor for development of atherosclerosis and AD [30]. Cholesterol to 7-hydroxy cholesterol oxidation, caused by A␤, is extremely toxic for neurons [38]. In the cholesterol fed rabbit model, plasma Cp levels, measured using o-dianisidine dihydrochloride as a substrate, suggest an increase in Cp levels, though statistical significance was not attained drinking distilled water supplemented with copper. This suggests that a Non-Cp-Cu increase is a vehicle of copper within the brain. In a different investigation, Sparks reconfirmed his earlier finding, reporting that other animal models, like spontaneously hypercholesterolemic Watanabe rabbits, cholesterol-fed beagles and rabbits, and PS1 and APP transgenic mice showed considerably increased brain levels of A␤ when exposed to drinking water with added copper (0.12 ppm) [39]. Notably, noncholesterol fed PS1 and APP transgenic mice models of AD demonstrated significantly enhanced levels of A␤ due to copper exposure via drinking water. Thus,

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senile plaque deposition and neurofibrillary tangles in the brain. Although the current focus of AD research is on the accumulation of amyloid-␤ (A␤) and tau [22, 23], numerous studies argued that a deregulation of copper metabolism contributes to these pathogenetic pathways and can be a risk factor accelerating the disease cascade [24–26]. We have juxtaposed these two diseases, as they seem to share some common features. As we will detail later, a percentage of AD patients have Non-Cp-Cu higher than normal reference values, have some genetic variants in the ATP7B gene which associates with an increased risk of the disease, and also have increased levels of Cp apo-form in general circulation [27, 28]. A recently published case report notes the presence of Kayser-Fleisher rings in an AD diagnosed patient, studied with [18 F]fluorodeoxyglucose positron emission tomography (PET), and 11 C-labelled Pittsburgh Compound-B PET [29].

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from the gastro-intestinal tract to general circulation, there is no doubt that Non-Cp-Cu is the fraction of blood copper bioavailable for tissues and organs, since it is not structurally bound to Cp (which is actually a master protein in iron metabolism), and it is a low molecular weight copper that can easily be bound and exchanged among the micronutrients, peptide and albumin, for cell uptake. Although this physiological knowledge gives a primary role to Non-Cp-Cu as a causative agent for the modification induced by copper ingestion on A␤ toxicity shown in all the animal models described above, none of these studies ever searched for and provided evidence of an increase of Non-CpCu as a vehicle of this metal, able to pass through the blood-brain barrier (BBB) filter, reach the brain, and partake in the AD cascade. In their recent study, Singh and colleagues [43] demonstrated exactly this simple path for copper, showing what was expected but never proven in AD research by actually connecting two pieces of evidence: one resulting from experimental models investigating the causation of copper in models of AD cascade, and one coming from clinical observational studies and showing Non-Cp-Cu involvement in the clinical picture, prognosis, and prediction of clinical worsening in humans. Ultimately, this yielded unique information: Non-Cp-Cu is a causative risk factor for AD. Indeed, Singh et al. [43] studied normal mice (wild type) and a mouse model of AD (A␤PP transgenic mice) exposed to 0.13 ppm of copper sulfate for 90 days levels of copper via drinking water, which doubled plasma concentrations of Non-Cp-Cu. This caused either a reduction of cerebrospinal fluid A␤ clearance across the BBB in wild-type mice, or an identical effect, along with an increase in A␤ production in the transgenic mice. Thus, we are proposing the reevaluation of a concept already expressed intuitively in the literature [42, 44]. The concept is that Non-Cp-Cu is a causative risk factor for AD. However, we believe this concept pertains only to a certain proportion of AD cases, i.e., those who have a mild metabolic dysfunction of copper, which can be revealed by measuring Non-Cp-Cu in general circulation. This percentage of AD cases can be considered a subtype of AD, as described below.

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undoubtedly this mouse model of AD exhibited vulnerability to trace amounts of copper in drinking water even in the absence of cholesterol in the diet. This observation highlights the fact that both cholesterol and copper are causative factors that interact together to further enhance formation of A␤ plaques. They also have significant clinical insinuations, since they provide the rationale for the critical need to reevaluate safe drinking water copper levels. Another study confirmed the findings of Sparks and colleagues [40]. The authors demonstrated that Kunming strain mice fed with high-cholesterol diet and distilled water containing 0.21 ppm copper exhibited significantly increased APP mRNA level coupled with activation of caspase-3 in the brain, suggesting apoptosis mediated neurotoxicity. Strikingly, copper also increased cholesterol-induced learning and memory impairment in mice [40]. Moreover, it has been reported that, in Sprague-Dawley rats, which underwent bilateral common carotid artery occlusion (2VO) and were administered with 250 ppm copper containing water for 3 months, chronic copper toxicity exacerbated memory impairment induced by 2VO coupled with an augmented expression of brain A␤PP and ␤-site A␤PP-cleaving enzyme 1 (BACE1) at both mRNA and protein levels. However, these copper-aggravated changes were ameliorated after copper was withdrawn from the drinking water [41]. As a whole, these experimental animal models demonstrated the toxicity mediated by copper in the AD cascade, showing that increased level of copper ingested with drinking water, or more generally through the diet, affects AD neuropathology. TOWARD A UNIFIED VISION OF COPPER INVOLVEMENT IN ALZHEIMER’S DISEASE

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In humans it is well established that copper ingested through the diet reaches the liver through the portal circulation [1]. Grossly simplifying the metabolic fate of copper absorption, retention, and excretion (detailed in sophisticated studies carried out in metabolic research units using isotopic tracers [1]), it can be said that, after the liver absorption of the quantity necessary for biological needs, copper comes back to the general circulation in the form of Cp or of a Non-Cp-Cu. Some authors [42] argue that the copper taken in by drinking water as inorganic copper comes directly into the bloodstream without passing through the liver, becoming immediately part of the Non-Cp-Cu pool. Regardless of the exact path of the inorganic copper

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ASSOCIATION OF COPPER AND ALZHEIMER’S DISEASE: HUMAN STUDIES As we have described above, A␤PP and A␤ itself have been shown to react with copper producing oxidative stress [45–47], eventually damaging biomolecules

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decline and abnormal levels of the exchangeable fraction of low molecular weight Non-Cp-Cu in serum along with its connection with copper intake (reviewed above). This low molecular weight copper has very low concentrations in human physiology: lower than 1.6 ␮mol/L (i.e., 5–15% of total serum copper, which instead ranges between 11–24.4 ␮mol/L [59, 60]). In a series of investigations, it was demonstrated that the average of serum Non-Cp-Cu concentrations are increased in AD [61]. Mini-Mental State Examination (MMSE), as well as other neuropsychological tests of AD subjects, shows correlation between Non-CpCu levels and cognitive performance (the higher the serum Non-Cp-Cu levels, the lower the MMSE score) [25, 61, 62]. It has been estimated that it is filterable and in humans 3% of Non-Cp-Cu crosses the BBB [25]. Moreover, Non-Cp-Cu levels were predictive of MMSE score worsening in AD patients in a year study follow-up [26]; and that Non-Cp-Cu increases in mild cognitive impairment (MCI) [63]—the status which is considered a precursor of AD, due to the high statistical rate of progression from MCI to AD—predicting the rate of progression from MCI to AD [64]. As a whole, these results indicate that the levels of Non-Cp-Cu are higher in AD patients than in healthy individuals, and that this pool of copper is filterable (as exemplified by Wilson’s disease and by the experimental model of AD toxicity) and has effects on cognition (Fig. 1). These three facts were confirmed by the study of James et al. [53].

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and membranes. The Lovell et al. study, which has been considered a milestone study in the field for many years (creating a bias in reference citation), after an accurate revision of its methods, has been judged with many flaws and shortcomings [48]. Detailed information is reported in a recent meta-analysis which reviewed all the data available through 2011, and demonstrated a significant decrease of copper in many areas and, generally, in the brain of AD patients [49]. Two subsequent studies recently confirmed this finding [50, 51]. In their study, Rembach and colleagues [51], found a decrease in the copper present in the soluble AD brain extract (but still bound to proteins), along with a lack of a copper increase in the formic acid fraction, which contained AD plaques that showed no increases of copper. Even though counterintuitive, the evidence of a decrease of copper in the brain is perfectly in agreement with an increase of systemic Non-Cp-Cu. This notion is well supported by animal models of Wilson’s disease. More precisely, the Long Evans Cinnamon (LEC) rats, which have natural mutations in the ATP7B gene, and are then an ecological model of Wilson’s disease, have decreased levels of total copper in their brain and increased systemic Non-Cp-Cu [52], growing the number of shared features between AD and Wilson’s disease. Moreover, the study by James et al. [53], examining in depth the copper distribution in the AD brain, reported either a decrease of total copper in some specific areas of the AD brain, or an increase of the labile (resembling systemic NonCp-Cu) in the same areas. This confirmed a systemic copper dysfunction in AD, in terms of deregulation of Non-Cp-Cu in diverse body districts. In this line, numerous clinical investigations in living patients carried out so far [53–56] have revealed that Cp and copper levels are disturbed in AD, indicating systemic homeostasis deregulation in AD (systemic reviews in [56, 57]). This knowledge has consistently increased in the last 3 years, strongly supporting the original hypothesis of copper toxicity in AD described earlier, based on the biochemical properties of the A␤ peptide and its interaction with metals. Various studies have been published about the link between copper and cognition loss/AD. In a milestone study by Morris et al. [58], it was observed that people who eat a diet rich in saturated and trans-fats and have a higher copper intake experienced a faster rate of cognitive decline. These persons were assumed to be taking copper supplements, by virtue of the fact that they were in the highest quintile of copper intake [58]. Of specific importance are a number of previous findings in human subjects, which provide direct relationships between cognitive

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COPPER SUBTYPE OF AD Literature on this specific topic has increased in recent years [2–4], even though evidence reported previously certainly sustains this new concept in AD. Generally speaking, to prove the existence of a subtype of a disease, four different strategies should be taken into account: i) identification of a bimodal distribution of the biomarker of interest in the patient population; ii) the response to a specific therapy; iii) cluster analysis; and iv) the identification of a specific biomarker that distinguishes the cases, classifying them in sub-groups that are then compared for demographic, clinical, genetic, or biological variables. When the two hypothetical peaks of the subpopulation are very close, a clear graphical bimodal distribution can be hard to be visualized, since the peaks appear overlapped. Moreover, even in case of clear graphical bimodal distribution, additional evaluations should be done to avoid bias related to sex and age or

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Fig. 1. The increase of systemic Non-Cp-Cu in AD causes an imbalance of copper in the brain. Once in the brain, Non-Cp-Cu can interact with physiological amyloid-␤ (A␤), forming clusters of metal-toxic soluble A␤ that evolve in diffuse amyloid and, finally, in toxic plaques. Non-Cp-Cu can also readily interact with reactive oxygen species (ROS), which are responsible for lipid peroxidation in membranes of neuron, protein oxidation, and cleavage of DNA and RNA molecules.

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additional potential confounders. In this case, the other approaches can be more informative. Regarding AD, the application of the procedure described in the fourth approach has recently been discussed. In the study by Murray et al. [65], the authors used the density and the distribution of neurofibrillary tangle counts in the brain at autopsy as a criterion to distinguish subgroups of AD, and then compared these subgroups for various neuropathological features. With regard to copper, we have applied a ‘copper criterion’ to distinguish AD patients in three recent studies, as follows.

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Recent studies have revealed loci of susceptibility for AD in the master gene pertinent to the Non-Cp-Cu regulation pathway, namely ATP7B [66–70]. One specific study investigated the effect of these ATP7B gene variants on the variation of the biochemical levels of Non-Cp-Cu in AD [70]. In this latter study, after the subject samples were stratified into two classes on the

basis of the Non-Cp-Cu (≤than 1.6 ␮mol/L and > than 1.6 ␮mol/L), we have shown that AD patients with higher levels of Non-Cp-Cu have a higher frequency of some ATP7B variants than AD patients with lower levels of Non-Cp-Cu, thus distinguishing a copper subtype of AD. Prognosis of AD In a study exploring whether a deregulation of NonCp-Cu pool could predict AD clinical worsening, we stratified the AD patients on the basis of a cut-off of Non-Cp-Cu at baseline to better distinguish the two groups (2.1 ␮mol/L). Those patients with levels of Non-Cp-Cu higher than this cut-off had an increased probability to worsen after a year of follow-up than those patients who had lower Non-Cp-Cu values. NonCp-Cu can be then identified as a specific biomarker that distinguishes AD patients into two diverse subgroups that differ in terms of clinical worsening [26].

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Non-Cp-Cu levels in AD are not ascribable to any known recognizable condition; ii) ATP7B gene variants increase either the AD risk; iii) or serum Non-Cp-Cu levels in a percentage of AD [70], a primitive mild altered copper metabolism on a genetic basis can be demonstrated in AD. Nevertheless, hard work is still needed to identify the exact loci for AD susceptibility in the ATP7B gene or other genes pertinent to copper metabolism (work in progress). Regarding the exact value of the cut-off better distinguishing AD patients from healthy controls, we have obtained some preliminary data that define more accurately the normal reference values for Non-Cp-Cu, estimated in a cohort of 250 subjects by means of a new patented device to measure Non-Cp-Cu (C4D test, Colabufo, N. and R. Squitti, P.E. European Patent Office (EPO) (RO/EP), 2012). The upper reference limit, (95%) for the healthy population, has been set to 1.91 ␮mol/L (its 90% confidence interval is equal to 1.78–2.06; Code EDMA, 11 02 01 06 00 Copper Cu). Abnormal Non-Cp-Cu is a modifiable risk factor, so it can have relevant effects in contrasting the AD epidemic. In fact, we have calculated the estimated incidence of the copper subtype of AD, starting with the data estimating that there are 90 million inhabitants older than 60 in the region defined as Euro A [76]. In this population, the prevalence of AD is 5.4%, while the annual incidence, i.e., the new cases per year, is nine in a thousand of people. This incidence for those subjects with Non-Cp-Cu higher than normal values is 8% (estimated incidence), or ten times higher. For MCI, that has an incidence of conversion to AD of 15%, the estimated incidence in subjects with Non-Cp-Cu higher than normal values increases to 64%. We are now collecting data to run cluster analyses to substantiate these findings, and we are preparing to run a clinical trial with an anti-copper agent to definitely address the same issues.

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In a recent article [64], Non-Cp-Cu has been revealed as an index in the earliest clinical stages of AD in predicting an increase in the hazard conversion rate and a faster rate of progression from MCI to AD. As it has already been demonstrated for AD patients [26], after the stratification of the MCI subjects on the basis of a cut-off of Non-Cp-Cu at the baseline (1.6 ␮mol/L), the two subgroups differed in terms of their conversion to AD. Those patients with levels of Non-Cp-Cu higher than this cut-off had a three-fold increased hazard of the conversion rate to AD. As a whole, these data allow us to define a copper subtype of AD. If evaluated in the framework of the recent literature, this new concept also allows us to overcome the controversy fuelling a debate that was prevalent in 2006–2012, concerning the issue of whether copper is increased in AD living patients. In fact, meta-analyses, collecting scattered and heterogeneous results into one single piece of evidence, have repetitively and univocally demonstrated that copper and Non-Cp-Cu are increased in AD [56, 71–73]. However, it has to be considered that copper abnormalities are not a condition exhibited by all the AD patients. Taking this into consideration, we can account for the previously controversial results on the basis of the percentage of this copper AD subtype, which has been included in each study. The percentage also varies on the basis of the lifestyle, dietary habits, genetic make-up, ethnicity, and geographic area of the subject sample investigated. Of note is the finding that cognitive function is inversely correlated with Non-Cp-Cu levels, even in healthy subjects [74]. In our 15 years of experience in AD research, we have found that the percentage of AD patients with abnormal levels of Non-Cp-Cu is around 60%, and we have discussed this issue in a previous article published in this same Journal (information available at http://www.j-alz.com/node/182) [56]. In these subjects, the copper dysfunction exhibited cannot be ascribed to recognized conditions that increase NonCp-Cu, such as concomitant Wilson’s disease, acute liver failure of any etiology, chronic cholestasis, and copper intoxication, or supplement (we have also excluded additional diseases known to affect metal metabolism). Moreover, we have demonstrated in recent years the association of some genetic variants of ATP7B with AD [66–70], which have some effects on the concentrations of Non-Cp-Cu in general circulation [70]. Other authors have confirmed our findings [75]. On the basis of these three facts: i) increased

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DIETARY COPPER CONTENT: DRINKING WATER AND FOODS Copper enters the body exclusively through dietary intake (food, dietary supplements, drinking water, beverages). Drinking water copper concentrations range from a few ␮g/L to 4.8 mg/L. Taking into consideration the Singh et al. finding [43], it is intriguing to note that 1.3 ppm (1.3 mg/L) copper in drinking water is permissible by the US Environmental Protection Agency, in comparison to the 0.17 ppm (0.17 mg/L) permitted by the California

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Table 1 Dietary reference intake of copper in various age groups as defined by the Food and Nutrition Board of the Institute of Medicine at the National Academies of Sciences. RDAs (Recommended Dietary Allowances) are reported.

THE INTERPLAY BETWEEN GENETICS AND DIETARY COPPER INTAKE IN BODY COPPER TOXICOSIS OR ACCUMULATION IN HUMANS It is established that whole-body copper metabolism is regulated by a variable efficiency of copper absorption and through excretion of endogenous copper into

RDA (␮g/d) – 340 440 700 890 900 700 890 900 1000 1300

Table 2 List of top-10 food with the highest bioavailable copper content.

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Environmental Protection Agency. However, the World Health Organization (WHO) interim drinking water guidance level for copper remains at 2 mg/L. As reported by the WHO, the minimal acceptable intake of copper is about 0.9 mg/day, and the recommended dietary allowance (RDA) is 1–1.3 mg/day. The average daily intake per person has been estimated at around roughly 2–3 mg/day (WHO, 1996. Copper. Trace elements in human nutrition and health. World Health Organization, Geneva, pp. 123–43) [77]; WHO, 2004,WHO/SDE/WSH/03.04/88 [78]). The Chicago Health and Aging project evaluated more the 3,700 subjects during a 9-year longitudinal study, through four cognitive tests administered during in-home interviews at 3-year intervals for 6 years, and a dietary assessment was performed with a food frequency questionnaire. The article reports that persons with a median copper intake of 2.75 mg/day, which is associated with a diet rich in saturated and trans fats, had a faster rate of cognitive decline, estimated to be around the equivalent of 19 or more years of age [61]. The Iowa Women’s Health Study showed that the ingestion of inorganic copper in the form of copper supplements was associated with high AD mortality [79]. In fact, most multi-vitamin/mineral dietary supplements contain approximately 2 mg of inorganic copper/pill [10], which further escalates the copper pool in the body. Thus, the intake of copper with dietary supplements usually exceeds the RDA [77, 78]. Moreover, a recent study demonstrated a strong association between the annual mortality of AD and copper concentration in the soil of mainland China [80]. This suggests a relation between the soil and the quality of the water, as well the product of agriculture in those regions. Finally, the Tolerable Upper Intake Level of copper has been set at 10 mg/day, and some foods easily exceed this amount (Tables 1 and 2; Food and Nutrition Board of the Institute of Medicine, The National Academies, 2001; information available at http://www.dsld.nlm.nih.gov/dsld/docs/Dietary Refer ence Intakes Recommended Intakes for Individuals. pdf).

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Animal liver (veal) Oysters Sesame seeds Cocoa Nuts (cashew) Seafood (Calamari) Sunflower seeds Sun dried tomatoes Pumpkin Dried herbs (basil)

Copper content (mg)/100 g serving 15 mg 1–8 mg 4.1 mg 3.8 mg 2.2 mg 2.1 mg 1.8 mg 1.4 mg 1.4 mg 1.4 mg

the gastrointestinal tract. In healthy individuals, when dietary copper is high and more is absorbed, endogenous excretion increases, protecting against excess accumulation of copper in the body [1]. These mechanisms of protections can be disturbed on a genetic basis. It is well known that Wilson’s disease is caused by mutations in the ATP7B gene. However, genetic make-up on the individual, along with environmental factors (lifestyle), can have some effects on the disease presentation. In this regard, some studies in animal models of Wilson’s disease, along with case reports of this disease, show diverse presentation of the disease, especially within a family sharing the same mutation (better discussed in [5]). Accordingly, the data gleaned about the LEC rats may be called into question. LEC rats have an early and rapid onset of fulminant hepatitis on a high dietary copper regimen. But they survive long-term on a low-copper dietary regimen [81]. Comparing this basic evidence to the complexity and longevity of a human life, it can be

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appreciated the burden of the environmental factors (copper in the diet, comprehending the inorganic copper) in accelerating the onset and progression of a disease that occurs in old age.

sion, or in terms of possible interventions intended to prevent or improve conditions associated with AD risk.

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ACKNOWLEDGMENTS

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AP and RP are thankful to the Indian Council of Medical Research (ICMR, New Delhi) for the financial assistance as JRF/SRF to Dr. Amit Pal [3/1/3/JRF2009/HRD-13 (11279)]. RS thanks the National Research Council “Aging Program 2012-2014”; MIUR Cod. 1182 /Ric/V o prot. 2010SH7H3F ‘Functional connectivity and neuroplasticity in physiological and pathological aging [ConnAge]; FISM – Fondazione Italiana Sclerosi Multipla – Cod.2011/R/32 Fatigue Relief in Multiple Sclerosis by transcranial Direct Current Stimulation (tDCS): can we Differentiate stimulation Targets within the primary sensorimotor cortices? [FaReMuS DiCDiT]; Ministry of Health Cod. GR-2008-1138642 Promoting recovery from Stroke: Individually enriched therapeutic intervention in Acute phase [ProSIA]; Ricerca Corrente, Italian Ministry of Health; Canox4drug SpA 2013-2016 ‘Non-Ceruloplasmin copper in Alzheimer’s disease’ (Prot. 30/2013). Authors apologize to any colleagues whose work was not cited in this review because of length restrictions. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2528).

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In vitro and animal models demonstrated that copper acts as a causative factor in A␤ toxicity, and numerous human studies on living patients showed the association of copper with the clinical picture of AD, in terms of cognitive performances, cerebrospinal fluid markers of AD, and clinical worsening in a percentage of AD patients. Moreover, recent genetic data, along with meta-analyses sustain a systemic deregulation of copper in AD. This finds support in a local decrease of total copper in the brain, as is also exemplified in model of Wilson’s disease. This evidence demonstrates the existence of a subtype of AD, which can be typified by abnormal levels of serum Non-Cp-Cu. Higher levels of copper in the diet, primarily if they are taken in in the form of inorganic copper (vitamin/mineral supplements), together with the consumption of a high fat diet, in people with peculiar genetic variants (APOE4, APP, ATP7B) appear to be linked with an increased risk of AD. These concepts are dire, if one considers that, for example, 100 grams of liver contains 14 mg of copper (Table 2). Even though endogenous mechanisms of copper balance protect against excess accumulation of copper in the body [1], these mechanisms can be disturbed in AD and have a relevance in consideration of the fact that MCI subjects with abnormal serum NonCp-Cu levels had a threefold increased hazard of the conversion rate to AD [64]. The outlined findings discussed in this review suggest that modifications in drinking water/dietary supplements, as well as diet in those subjects with an ascertained copper dysfunction can be a safe and inexpensive strategy to prevent or delay the onset of neurodegeneration/AD [5, 82]. However, the research community is still in a dilemma over the minimum safe levels of copper in drinking water or vitamin/mineral supplements. Even though RDA data have been released, it is obvious that they are routinely overlooked by many when planning their diets [77]. More research in healthy/AD animal models is warranted for further insights into the pathophysiological mechanisms of copper toxicity [83]. Research on humans should also be conducted, either in terms of testing the efficacy of treatments that can reestablish normal copper physiology to halting or delaying AD progres-

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