The role of metals in modulating metalloprotease activity in the AD brain

Biometals such as copper and zinc have an important role in Alzheimer’s disease (AD). Accumulating evidence indicates that copper homeostasis is altered in AD brain with elevated extracellular and low intracellular copper levels. Studies in animals and cell cultures have suggested that increasing intracellular copper can ameliorate AD-like pathology including amyloid deposition and tau phosphorylation. Modulating copper homeostasis can also improve cognitive function in animal models of AD. Treatments are now being developed that may result in redistribution of copper within the brain. Metal ligands such as clioquinol (CQ), DP-109 or pyrrolidine dithiocarbamate (PDTC) have shown promising results in animal models of AD, however, the actual mode of action in vivo has not been fully determined. We previously reported that CQ-metal complexes were able to increase intracellular copper levels in vitro. This resulted in stimulation of phosphoinositol-3-kinase activity and mitogen activated protein kinases (MAPK). Increased kinase activity resulted in up-regulated matrix metalloprotease (MMP2 and MMP3) activity resulting in enhanced degradation of secreted Aβ. These findings are consistent with previous studies reporting metal-mediated activation of MAPKs and MMPs. How this activation occurs is unknown but evidence suggests that copper may be able to activate membrane receptors such as the epidermal growth factor receptor (EGFR) and result in downstream activation of MAPK pathways. This has been supported by studies showing metal-mediated activation of EGFR through ligand-independent processes in a number of cell-types. Our initial studies reveal that copper complexes can in fact activate EGFR. However, further studies are necessary to determine if metal complexes such as CQ-copper induce up-regulation of Aβ-degrading MMP activity through this mechanism. Elucidation of this pathway may have important implications for the development of metal ligand based therapeutics for treatment of AD and other neurodegenerative disorders. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience, held in Melbourne on 11 July 2007.     

Copper transport and Alzheimer’s disease

This brief review discusses copper transport in humans, with an emphasis on knowledge learned from one of the simplest model organisms, yeast. There is a further focus on copper transport in Alzheimer’s Disease (AD). Copper homeostasis is essential for the well-being of all organisms, from bacteria to yeast to humans: survival depends on maintaining the required supply of copper for the many enzymes, dependent on copper for activity, while ensuring that there is no excess free copper, which would cause toxicity. A virtual orchestra of proteins are required to achieve copper homeostasis. For copper uptake, Cu(II) is first reduced to Cu(I) via a membrane-bound reductase. The reduced copper can then be internalised by a copper transporter where it is transferred to copper chaperones for transport and specific delivery to various organelles. Of significance are internal copper transporters, ATP7A and ATP7B, notable for their role in disorders of copper deficiency and toxicity, Menkes and Wilson’s disease, respectively. Metallothioneins and Cu/Zn superoxide dismutase can protect against excess copper in cells. It is clear too, increasing age, environmental and lifestyle factors impact on brain copper. Studies on AD suggest an important role for copper in the brain, with some AD therapies focusing on mobilising copper in AD brains. The transport of copper into the brain is complex and involves numerous players, including amyloid precursor protein, Aβ peptide and cholesterol.     

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．     

Role of metal dyshomeostasis in Alzheimer's disease

Despite serving a crucial purpose in neurobiological function, transition metals play a sinister part in the aging brain, where the abnormal accumulation and distribution of reactive iron, copper, and zinc elicit oxidative stress and macromolecular damage that impedes cellular function. Alzheimer's disease (AD), an age-related neurodegenerative condition, presents marked accumulations of oxidative stress-induced damage, and increasing evidence points to aberrant transition metal homeostasis as a critical factor in its pathogenesis. Amyloid-β oligomerization and fibrillation, considered by many to be the precipitating factor underlying AD onset and development, is also induced by abnormal transition metal activity. We here elaborate on the roles of iron, copper, and zinc in AD and describe the therapeutic implications they present.     

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.     

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer’s disease patients

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer’s disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.     

Ceruloplasmin fragmentation is implicated in 'free' copper deregulation of Alzheimer disease

A dysfunction in copper homeostasis seems to occur in Alzheimer’s disease (AD). We recently demonstrated that an excess of non-ceruloplasmin-copper (i.e. ‘free’ copper) correlates with the main functional and anatomical deficits as well as the cerebrospinal markers of the disease, thus suggesting that copper contributes to AD neurodegeneration. Aim of this study was to investigate the profile of serum ceruloplasmin isoforms immunoreactive protein in relation to copper dysfunction in AD. Twenty-five AD patients and 25 controls were included in the study. All subjects underwent individual measurements of serum ceruloplasmin and copper concentrations, and the amount of ‘free’ copper was computed for each copper and ceruloplasmin pair. Serum samples were also pooled and analyzed by two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blot analysis. The mean concentration of ’free’ copper resulted higher in AD patients than in controls. Ceruloplasmin 2-D PAGE western blot analysis of pooled sera showed in the AD samples low-molecular-weight spots in the <50 kDa range that were not detected in controls’ pooled sera (p < 0.029). Our data indicate a ceruloplasmin fragmentation in the serum of AD patients, possibly related to ‘free’ copper deregulation in this disease.     

Ceruloplasmin fragmentation is implicated in ��free�� copper deregulation of Alzheimer's disease

A dysfunction in copper homeostasis seems to occur in Alzheimer''s disease (AD). We recently demonstrated that an excess of non-ceruloplasmin-copper (i.e., ‘free’ copper) correlates with the main functional and anatomical deficits as well as the cerebrospinal markers of the disease, thus suggesting that copper contributes to AD neurodegeneration. Aim of this study was to investigate the profile of serum ceruloplasmin isoforms immunoreactive protein in relation to copper dysfunction in AD. Twenty-five AD patients and 25 controls were included in the study. All subjects underwent individual measurements of serum ceruloplasmin and copper concentrations, and the amount of ‘free’ copper was computed for each copper and ceruloplasmin pair. Serum samples were also pooled and analyzed by two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blot analysis. The mean concentration of ‘free’ copper resulted higher in AD patients than in controls. Ceruloplasmin 2-D PAGE western blot analysis of pooled sera showed in the AD samples low-molecular-weight spots in the <50 kDa range that were not detected in controls'' pooled sera (p < 0.029).Our data indicate a ceruloplasmin fragmentation in the serum of AD patients, possibly related to ‘free’ copper deregulation in this disease.Key words: Alzheimer''s disease, copper, ceruloplasmin, serum, SDS-page     

Copper toxicity in Alzheimer's disease: cognitive loss from ingestion of inorganic copper

In this review I present the hypothesis that a toxic substance, inorganic copper, ingested from drinking water and vitamin/mineral supplements containing inorganic copper, is at least partially causal of the epidemic of Alzheimer's disease (AD) we are seeing in developed countries. I set the stage for this hypothesis by pointing out that the epidemic is a new disease phenomenon coinciding temporally with the use of copper plumbing in developed countries. The evidence is good that AD was nonexistent or rare in the 1800 s and early 1900 s, and the arguments that elderly people did not exist in those times, or that AD was simply attributed to senility, are refuted. The web of evidence tying ingestion of inorganic copper as a causal factor in AD is strong, and includes AD animal model data where trace amounts of inorganic copper in the drinking water markedly worsened AD, human studies where ingestion of copper supplements, along with a high fat diet, is associated with a marked loss of cognition, human studies showing a markedly higher mortality in elderly women ingesting copper supplements, as well as other data. It is likely that a high fat diet works in conjunction with ingestion of inorganic copper to increase the risk of AD. It is clear that some factor toxic to the brain is present in the environment in developed countries, but not undeveloped countries, and is a major risk factor for AD. I believe that that toxic factor is ingestion of inorganic copper.     

Copper chelator induced efficient episodic memory recovery in a non-transgenic Alzheimer's mouse model

Alzheimer's disease (AD) is a neurodegenerative syndrom involving many different biological parameters, including the accumulation of copper metal ions in Aβ amyloid peptides due to a perturbation of copper circulation and homeostasis within the brain. Copper-containing amyloids activated by endogenous reductants are able to generate an oxidative stress that is involved in the toxicity of abnormal amyloids and contribute to the progressive loss of neurons in AD. Since only few drugs are currently available for the treatment of AD, we decided to design small molecules able to interact with copper and we evaluated these drug-candidates with non-transgenic mice, since AD is mainly an aging disease, not related to genetic disorders. We created a memory deficit mouse model by a single icv injection of Aβ(1-42) peptide, in order to mimic the early stage of the disease and the key role of amyloid oligomers in AD. No memory deficit was observed in the control mice with the antisense Aβ(42-1) peptide. Here we report the capacity of a new copper-specific chelating agent, a bis-8-aminoquinoline PA1637, to fully reverse the deficit of episodic memory after three weeks of treatment by oral route on non-transgenic amyloid-impaired mice. Clioquinol and memantine have been used as comparators to validate this fast and efficient mouse model.     

Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking.     

Copper dysfunction in Alzheimer's disease: from meta-analysis of biochemical studies to new insight into genetics

The involvement of body copper metabolism in the development of Alzheimer's disease (AD) - the most common form of dementia - is a deeply investigated issue in recent years. Copper is essential for life, but in excess it can be toxic. Recently, it has been hypothesized that copper toxicity may be a contributory factor in the etiology of the neurodegenerative disease AD. Studies on copper evaluation in AD vs. healthy controls collected in the latest 30 years and merged in a meta-analysis demonstrate that serum copper is slightly increased in AD. A specific form of copper, the copper non-bound to ceruloplasmin, or 'free' copper, seems to best characterize this increase in copper in AD patients. Clinical studies from us and other groups have demonstrated that free copper is associated with the typical deficits of AD, incipient AD and mild cognitive impairment, and specific cerebrospinal markers. Moreover, very recent data addressing molecular processes underlying copper dysfunction in AD have indicated that genetic variations of K832R and R952K Single Nucleotide Polymorphisms (SNPs) of the Wilson's disease gene ATP7B are associated also with sporadic AD. Specifically, ATP7B encodes for the protein ATPase 7B which controls free copper status in the body, and both R allele in K832R and K allele in R952K ATP7B SNPs are associated with an increased risk of having AD. Even though copper dysfunction cannot be assumed as a determinant of the disease, its causative, rather than associated, role in AD pathology as risk factor can be claimed.     

Possible mechanisms of APP-mediated oxidative stress in Alzheimer's disease

Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.     

Copper subtype of Alzheimer's disease (AD): Meta-analyses,genetic studies and predictive value of non-ceruloplasmim copper in mild cognitive impairment conversion to full AD

Alzheimer's disease (AD) is the most common form of dementia. A myriad of complex factors contribute to AD, promoting the deposition in plaques of amyloid-beta (Aβ), which is the main constituent of this pathognomonic sign of AD at autopsy brain inspection. Aβ toxicity is related to oxidative stress, which results in synaptic loss in specific brain areas, eventually leading to cognitive decline. Metal, and especially copper, dyshomeostasis is a key factor in these processes. Recent studies have demonstrated that the serum fraction of copper that is not bound to ceruloplasmin (Non-Cp copper, also known as ‘free’ or labile copper) increases in a percentage of AD patients and mild cognitive impairment (MCI) subjects; this is considered a precursor of AD. Non-Cp copper is the exchangeable fraction of low molecular weight copper in serum. It is distinguished from the copper structurally bound to the ceruloplasmin protein, a master protein of iron metabolism. Non-Cp copper levels are higher than normal reference values (range 0–1.6 μmol/L) in about 50% of amnestic MCI subjects and 60% of AD patients, typifying them in a subset of AD. Meta-analyses, genetic studies and a prognostic study evaluating the predictive value of Non-Cp copper in MCI conversion to full AD demonstrate the existence of this copper phenotype of AD.     

Apolipoprotein E and Alzheimer's disease: molecular mechanisms and therapeutic opportunities

Multiple genetic and environmental factors are likely to contribute to the development of Alzheimer's disease (AD). The most important known risk factor for AD is presence of the E4 isoform of apolipoprotein E (apoE). Epidemiological studies demonstrated that apoE4 carriers have a higher risk and develop the disease and an early onset. Moreover, apoE4 is the only molecule that has been associated with all the biochemical disturbances characteristic of the disease: amyloid-beta (Abeta) deposition, tangle formation, oxidative stress, lipid homeostasis deregulation, synaptic plasticity loss and cholinergic dysfunction. This large body of evidence suggest that apoE is a key player in the pathogenesis of AD. This short review examines the current facts and hypotheses of the association between apoE4 and AD, as well as the therapeutic possibilities that apoE might offer for the treatment of this disease.     

Amyloid-beta: an antioxidant that becomes a pro-oxidant and critically contributes to Alzheimer's disease.

Elevated production of amyloid-beta (A beta) as a preventive antioxidant for brain lipoproteins under the action of increased oxidative stress in aging is postulated to represent a major event in the development of Alzheimer's disease (AD). Increase in A beta production is followed by chelation of transition metal ions by A beta, accumulation of A beta-metal lipoprotein aggregates, production of reactive oxygen species and neurotoxicity. Chelation of copper by A beta is proposed to be a most important part of this pathway, because A beta binds copper stronger than other transition metals and because copper is a more efficient catalyst of oxidation than other metals. This amyloid-binds-copper (ABC) model does not remove A beta peptide from its central place in our current thinking of AD, but rather places additional factors in the center of discussion. Most importantly, they embrace pathological mechanisms known to develop in aging (which is the major risk factor for AD), such as increased production of reactive oxygen species by mitochondria, that are positioned upstream relative to the generation of A beta.     

Selenium homeostasis in human brain cells: Effects of copper (II) and Se species

BackgroundBoth essential trace elements selenium (Se) and copper (Cu) play an important role in maintaining brain function. Homeostasis of Cu, which is tightly regulated under physiological conditions, seems to be disturbed in Alzheimer´s (AD) and Parkinson´s disease (PD) patients. Excess Cu promotes the formation of oxidative stress, which is thought to be a major cause for development and progression of neurological diseases (NDs). Most selenoproteins exhibit antioxidative properties and may counteract oxidative stress. However, expression of selenoproteins is altered under conditions of Se deficiency. Serum Se levels are decreased in AD and PD patients suggesting Se as an important factor in the development and progression of NDs. The aim of this study was to elucidate the interactions between Cu and Se in human brain cells particularly with respect to Se homeostasis.MethodsFirstly, modulation of Se status by selenite or SeMet were assessed in human astrocytes and human differentiated neurons. Therefore, cellular total Se content, intra- and extracellular selenoprotein P (SELENOP) content, and glutathione peroxidase (GPX) activity were quantified. Secondly, to investigate the impact of Cu on these markers, cells were exposed to copper(II)sulphate (CuSO₄) for 48 h. In addition, putative protective effects of Se on Cu-induced toxicity, as measured by cell viability, DNA damage, and neurodegeneration were investigated.ResultsModulation of cellular Se status was strongly dependent on Se species. In detail, SeMet increased total cellular Se and SELENOP content, whereas selenite led to increased GPX activity and SELENOP excretion. Cu treatment resulted in 133-fold higher cellular Cu concentration with a concomitant decrease in Se content. Additionally, SELENOP excretion was suppressed in both cell lines, while GPX activity was diminished only in astrocytes. These effects of Cu could be partially prevented by the addition of Se depending on the cell line and Se species used. While Cu-induced oxidative DNA damage could not be prevented by addition of Se regardless of chemical species, SeMet protected against neurite network degeneration triggered by Cu.ConclusionCu appears to negatively affect Se status in astrocytes and neurons. Especially with regard to an altered homeostasis of those trace elements during aging, this interaction is of high physiological relevance. Increasing Cu concentrations associated with decreased selenoprotein expression or functionality might be a promoting factor for the development of NDs.     

The Amyloid Precursor Protein Copper Binding Domain Histidine Residues 149 and 151 Mediate APP Stability and Metabolism

One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of the APP-derived amyloid β peptide (Aβ) in the brain. Altered copper homeostasis has also been reported in AD patients and is thought to increase oxidative stress and to contribute to toxic Aβ accumulation and regulate APP metabolism. The potential involvement of the N-terminal APP copper binding domain (CuBD) in these events has not been investigated. Based on the tertiary structure of the APP CuBD, we examined the histidine residues of the copper binding site (His(147), His(149), and His(151)). We report that histidines 149 and 151 are crucial for CuBD stability and APP metabolism. Co-mutation of the APP CuBD His(149) and His(151) to asparagine decreased APP proteolytic processing, impaired APP endoplasmic reticulum-to-Golgi trafficking, and promoted aberrant APP oligomerization in HEK293 cells. Expression of the triple H147N/H149N/H151N-APP mutant led to up-regulation of the unfolded protein response. Using recombinant protein encompassing the APP CuBD, we found that insertion of asparagines at positions 149 and 151 altered the secondary structure of the domain. This study identifies two APP CuBD residues that are crucial for APP metabolism and suggests an additional role of this domain in APP folding and stability besides its previously identified copper binding activity. These findings are of major significance for the design of novel AD therapeutic drugs targeting this APP domain.     

Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease

In this special issue about biofactors causing cognitive impairment, we present evidence for and discuss two such biofactors. One is excess copper, causing neuronal toxicity. The other is zinc deficiency, causing neuronal damage. We present evidence that Alzheimer's disease (AD) has become an epidemic in developed, but not undeveloped, countries and that the epidemic is a new disease phenomenon, beginning in the early 1900s and exploding in the last 50 years. This leads to the conclusion that something in the developed environment is a major risk factor for AD. We hypothesize that the factor is inorganic copper, leached from the copper plumbing, the use of which coincides with the AD epidemic. We present a web of evidence supporting this hypothesis. Regarding zinc, we have shown that patients with AD are zinc deficient when compared with age-matched controls. Zinc has critical functions in the brain, and lack of zinc can cause neuronal death. A nonblinded study about 20 years ago showed considerable improvement in AD with zinc therapy, and a mouse AD model study also showed significant cognitive benefit from zinc supplementation. In a small blinded study we carried out, post hoc analysis revealed that 6 months of zinc therapy resulted in significant benefit relative to placebo controls in two cognitive measuring systems. These two factors may be linked in that zinc therapy significantly reduced free copper levels. Thus, zinc may act by lowering copper toxicity or by direct benefit on neuronal health, or both.