综述:金属离子代谢平衡失调与阿尔茨海默病早期发病机制

研究表明,脑内金属离子代谢失衡与阿尔茨海默病(AD)有关,但其机理尚需深入探讨．结合本实验室研究结果,作者对金属离子代谢紊乱与氧化应激,金属离子代谢紊乱与β-淀粉样蛋白、转铁蛋白和转铁蛋白受体、铁调节蛋白、二价金属离子转运体以及天然抗氧化剂通过调节金属离子代谢平衡缓解β-淀粉样蛋白的毒性和保护细胞的作用进行探讨．提出：铁、铜等金属离子缺乏可能主要与AD早期关系密切,而铁、铜等金属离子过载可能主要与AD后期损伤关系密切的学术观点．     

Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.     

Nanoparticle and other metal chelation therapeutics in Alzheimer disease

Current therapies for Alzheimer disease (AD) such as the anticholinesterase inhibitors and the latest NMDA receptor inhibitor, Namenda, provide moderate symptomatic delay at various stages of disease, but do not arrest disease progression or supply meaningful remission. As such, new approaches to disease management are urgently needed. Although the etiology of AD is largely unknown, oxidative damage mediated by metals is likely a significant contributor since metals such as iron, aluminum, zinc, and copper are dysregulated and/or increased in AD brain tissue and create a pro-oxidative environment. This role of metal ion-induced free radical formation in AD makes chelation therapy an attractive means of dampening the oxidative stress burden in neurons. The chelator desferioxamine, FDA approved for iron overload, has shown some benefit in AD, but like many chelators, it has a host of adverse effects and substantial obstacles for tissue-specific targeting. Other chelators are under development and have shown various strengths and weaknesses. In this review, we propose a novel system of chelation therapy through the use of nanoparticles. Nanoparticles conjugated to chelators show a unique ability to cross the blood-brain barrier (BBB), chelate metals, and exit through the BBB with their corresponding complexed metal ions. This method may prove to be a safe and effective means of reducing the metal load in neural tissue thus staving off the harmful effects of oxidative damage and its sequelae.     

Copper in the brain and Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. The brain is particularly vulnerable to oxidative damage induced by unregulated redox-active metals such as copper and iron, and the brains of AD patients display evidence of metal dyshomeostasis and increased oxidative stress. The colocalisation of copper and amyloid β (Aβ) in the glutamatergic synapse during NMDA-receptor-mediated neurotransmission provides a microenvironment favouring the abnormal interaction of redox-potent Aβ with copper under conditions of copper dysregulation thought to prevail in the AD brain, resulting in the formation of neurotoxic soluble Aβ oligomers. Interactions between Aβ oligomers and copper can further promote the aggregation of Aβ, which is the core component of extracellular amyloid plaques, a central pathological hallmark of AD. Copper dysregulation is also implicated in the hyperphosphorylation and aggregation of tau, the main component of neurofibrillary tangles, which is also a defining pathological hallmark of AD. Therefore, tight regulation of neuronal copper homeostasis is essential to the integrity of normal brain functions. Therapeutic strategies targeting interactions between Aβ, tau and metals to restore copper and metal homeostasis are discussed.     

Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

Metallostasis in Alzheimer's disease

2012 has been another year in which multiple large-scale clinical trials for Alzheimer's disease (AD) have failed to meet their clinical endpoints. With the social and financial burden of this disease increasing every year, the onus is now on the field of AD researchers to investigate alternative ideas to deliver outcomes for patients. Although several major clinical trials targeting Aβ have failed, three smaller clinical trials targeting metal interactions with Aβ have all shown benefit for patients. Here we review the genetic, pathological, biochemical, and pharmacological evidence that underlies the metal hypothesis of AD. The AD-affected brain suffers from metallostasis, or fatigue of metal trafficking, resulting in redistribution of metals into inappropriate compartments. The metal hypothesis is built upon a triad of transition elements: iron, copper, and zinc. The hypothesis has matured from early investigations showing amyloidogenic and oxidative stress consequences of these metals; recently, disease-related proteins, APP, tau, and presenilin, have been shown to have major roles in metal regulation, which provides insight into the pathway of neurodegeneration in AD and illuminates potential new therapeutic avenues.     

Effects of dietary transition metals on oxidative DNA lesions in neonatal rats

Bulky endogenous oxidative lesions (type II I-compounds) reflect DNA damage associated with oxidative stress. As shown by 32P-postlabeling, their levels are enhanced by pro-oxidant genotoxins and also shortly after normal birth in several rat tissues as a function of time and the maternal diet. In order to elucidate which dietary components contribute to postnatal DNA damage, we have focused, herein, on the possible role of transition metals (iron, copper, and nickel). Pregnant Fischer 344 (F344) rats were fed AIN-93G purified diet containing different amounts of iron, copper, and nickel, or Purina-5001 natural-ingredient diet (which contains relatively high concentrations of these metals). Type II I-compounds were estimated by nuclease P1-enhanced 32P-postlabeling in liver and lung DNA of fetuses and at 24h and day 9 post-partum. Increased postnatal oxidative damage was detected in liver but not lung DNA of neonates exposed to higher amounts of dietary transition metals. There were significant positive linear correlations between maternal transition metal intake and neonatal, but not fetal and maternal type II I-compound levels. The results show that transition metals in the maternal diet affect perinatal oxidative DNA damage, presumably via a Fenton-type reaction. They also provide evidence for optimal levels in the maternal diet of transition metals, which on one hand, are essential for life, but on the other, can cause potentially deleterious DNA alterations in the offspring.     

Transition metal abnormalities in progressive dementias

Abnormal distributions of transition metals inside the brain are potential diagnostic markers for several central nervous system diseases, including Alzheimer’s disease (AD), Parkinson’s disease, dementia with Lewy bodies (DLB), bipolar disorders and depression. To further explore this possibility, the total concentrations of iron, zinc, copper, manganese, aluminum, chromium and cadmium were measured in post-mortem hippocampus and amygdala tissues taken from AD, DLB and Control patients. A statistically significant near fifty percent reduction in the total copper levels of AD patients was observed in both the hippocampus and amygdala. The statistical power of the hippocampus and amygdala copper analysis was found to be 86 and 74% respectively. No statistically significant deviations in the total metal concentrations were found for zinc, manganese, chromium or aluminum. Iron was found to be increased by 38% in AD amygdala tissues, but was unchanged in AD hippocampus tissues. Accounting for differences in tissue water content, as a function of both tissue type and disease state, revealed more consistencies with previous literature. To aid in the design of future experiments, the effect sizes for all tissue types and metals studied are also presented.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.     

The redox chemistry of the Alzheimer's disease amyloid beta peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid beta peptide (Abeta), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Abeta from the brain. Abeta can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Abeta will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Abeta oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Abeta levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Abeta species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

Copper and oxidative stress in the pathogenesis of Alzheimer's disease

Copper is a redox-active metal with many important biological roles. Consequently, its distribution and oxidation state are subject to stringent regulation. A large body of clinicopathological, circumstantial, and epidemiological evidence suggests that the dysregulation of copper is intimately involved in the pathogenesis of Alzheimer's disease. Other light transition metals such as iron and zinc may affect copper regulation by competing for copper binding sites and transporters. Therapeutic interventions targeting the regulation of copper are promising, but large gaps in our understanding of copper biochemistry, amyloidogenesis, and the nature of oxidative stress in the brain must be addressed.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the misfolding and plaque-like accumulation of a naturally occurring peptide in the brain called amyloid beta (Abeta). Recently, this process has been associated with the binding of metal ions such as iron (Fe), copper (Cu), and zinc (Zn). It is thought that metal dyshomeostasis is involved in protein misfolding and may lead to oxidative stress and neuronal damage. However, the exact role of the misfolded proteins and metal ions in the degenerative process of AD is not yet clear. In this study, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to image the in situ secondary structure of the amyloid plaques in brain tissue of AD patients. These results were spatially correlated with metal ion accumulation in the same tissue sample using synchrotron X-ray fluorescence (SXRF) microprobe. For both techniques, a spatial resolution of 5-10 microm was achieved. FTIRM results showed that the amyloid plaques have elevated beta-sheet content, as demonstrated by a strong amide I absorbance at 1625cm(-1). Using SXRF microprobe, we find that AD tissue also contains "hot spots" of accumulated metal ions, specifically Cu and Zn, with a strong spatial correlation between these two ions. The "hot spots" of accumulated Zn and Cu were co-localized with beta-amyloid plaques. Thus for the first time, a strong spatial correlation has been observed between elevated beta-sheet content in Abeta plaques and accumulated Cu and Zn ions, emphasizing an association of metal ions with amyloid formation in AD.     

Metal dyshomeostasis and oxidative stress in Alzheimer’s disease

Alzheimer’s disease is the leading cause of dementia in the elderly and is defined by two pathological hallmarks; the accumulation of aggregated amyloid beta and excessively phosphorylated Tau proteins. The etiology of Alzheimer’s disease progression is still debated, however, increased oxidative stress is an early and sustained event that underlies much of the neurotoxicity and consequent neuronal loss. Amyloid beta is a metal binding protein and copper, zinc and iron promote amyloid beta oligomer formation. Additionally, copper and iron are redox active and can generate reactive oxygen species via Fenton (and Fenton-like chemistry) and the Haber–Weiss reaction. Copper, zinc and iron are naturally abundant in the brain but Alzheimer’s disease brain contains elevated concentrations of these metals in areas of amyloid plaque pathology. Amyloid beta can become pro-oxidant and when complexed to copper or iron it can generate hydrogen peroxide. Accumulating evidence suggests that copper, zinc, and iron homeostasis may become perturbed in Alzheimer’s disease and could underlie an increased oxidative stress burden. In this review we discuss oxidative/nitrosative stress in Alzheimer’s disease with a focus on the role that metals play in this process. Recent studies have started to elucidate molecular links with oxidative/nitrosative stress and Alzheimer’s disease. Finally, we discuss metal binding compounds that are designed to cross the blood brain barrier and restore metal homeostasis as potential Alzheimer’s disease therapeutics.     

Zinc, antioxidant systems and metallothionein in metal mediated-apoptosis: biochemical and cytochemical aspects

Copper, zinc and iron are essential metals for different physiological functions, even though their excess can lead to biological damage. This review provides a background of toxicity related to copper, iron and zinc excess, biological mechanisms of their homeostasis and their respective roles in the apoptotic process. The antioxidant action of metallothionein has been highlighted by summarizing the most important findings that confirm the role of zinc in cellular protection in relation to metallothionein expression and apoptotic processes. In particular, we show that a complex and efficient antioxidant system, the induction of metallothionein and the direct action of zinc have protective roles against oxidative damage and the resulting apoptosis induced by metals with redox proprieties. In addition, to emphasize the protective effects of Zn and Zn-MT in Cu and Fe-mediated oxidative stress-dependent apoptosis, some aspects of apoptotic cell death are shown. The most widely used cytochemical techniques also have been examined in order to critically evaluate the available data from a methodological point of view. The observations on the role of Zn and MT could potentially develop new applications for this metal and MT in biomedical research.     

Modulation of aconitase, metallothionein, and oxidative stress in zinc-deficient rat intestine during zinc and iron repletion

Potential interactions between zinc and iron during absorption and its functional consequences on intestinal oxidative damage and antioxidant status were studied using the zinc-deficient rat as a model. Zinc depletion produced mild-moderate iron deficiency in addition to zinc deficiency, which could be corrected by repletion with iron and zinc. The localization and intensity of both iron and zinc in the intestinal mucosa showed a pronounced decrease in the presence of the other metal, indicating negative interactions. Zinc-deficient intestine exposed to iron alone exhibited elevated peroxidative damage and compromised functional integrity, despite increased expression of ferritin. Inclusion of zinc significantly reduced the damage and improved the functional integrity, accompanied by decreased expression of ferritin. Decreased expression of ferritin in the presence of zinc was consistent with reduced aconitase activity, suggesting its modulation by zinc. Further, inclusion of iron along with zinc was associated with induction of ferritin and metallothionein in tune with the amount of iron and zinc localized in the intestinal mucosa, respectively. These results suggest that zinc and iron interact negatively with cytosolic aconitase, but prove beneficial in reducing the oxidative stress, apart from improving functional integrity and iron/zinc status.     

Metallobiology and therapeutic chelation of biometals (copper,zinc and iron) in Alzheimer’s disease: Limitations,and current and future perspectives

BackgroundAlzheimer's disease (AD) is the most prevalent cause of cognitive impairment and dementia worldwide. The pathobiology of the disease has been studied in the form of several hypotheses, ranging from oxidative stress, amyloid-beta (Aβ) aggregation, accumulation of tau forming neurofibrillary tangles (NFT) through metal dysregulation and homeostasis, dysfunction of the cholinergic system, and to inflammatory and autophagic mechanism. However, none of these hypotheses has led to confirmed diagnostics or approved cure for the disease.ObjectiveThis review is aimed as a basic and an encyclopedic short course into metals in AD and discusses the advances in chelation strategies and developments adopted in the treatment of the disease. Since there is accumulating evidence of the role of both biometal dyshomeostasis (iron (Fe), copper (Cu), and zinc (Zn)) and metal-amyloid interactions that lead to the pathogenesis of AD, this review focuses on unraveling therapeutic chelation strategies that have been considered in the treatment of the disease, aiming to sequester free and protein-bound metal ions and reducing cerebral metal burden. Promising compounds possessing chemically modified moieties evolving as multi-target ligands used as anti-AD drug candidates are also covered.Results and ConclusionSeveral multidirectional and multifaceted studies on metal chelation therapeutics show the need for improved synthesis, screening, and analysis of compounds to be able to effectively present chelating anti-AD drugs. Most drug candidates studied have limitations in their physicochemical properties; some enhance redistribution of metal ions, while others indirectly activate signaling pathways in AD. The metal chelation process in vivo still needs to be established and the design of potential anti-AD compounds that bi-functionally sequester metal ions as well as inhibit the Aβ aggregation by competing with the metal ions and reducing metal-induced oxidative damage and neurotoxicity may signal a bright end in chelation-based therapeutics of AD.     

In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals

There is a great deal of evidence to support a pathogenic role of oxidative stress in Alzheimer's disease (AD), but the sources of reactive oxygen species have not been directly demonstrated. In this study, using a novel in situ detection system, we show that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. Pretreatment with deferoxamine or diethylenetriaminepentaacetic acid abolishes the ability of the lesions to catalyze the H2O2-dependent oxidation of 3,3'-diaminobenzidine (DAB), strongly suggesting the involvement of associated transition metal ions. Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion-dependent catalytic redox reactivity. Although DAB oxidation can also detect peroxidase activity, this was inactivated by H2O2 pretreatment before use of DAB, as shown by a specific peroxidase detection method. Model studies confirmed the ability of certain copper and iron coordination complexes to catalyze the H2O2-dependent oxidation of DAB. Also, the microtubule-associated protein tau, as an in vitro model for proteins relevant to AD pathology, was found capable of adventitious binding of copper and iron in a redox-competent manner. Our findings suggest that neurofibrillary tangles and senile plaques contain redox-active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment.