Copper Handling Research Articles

Decreased spinal cord motor neuron numbers in mice depleted of central nervous system copper.

Disrupted copper availability in the central nervous system (CNS) is implicated as a significant feature of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Solute carrier family 31 member 1 (Slc31a1; Ctr1) governs copper uptake in mammalian cells and mutations affecting Slc31a1 are associated with severe neurological abnormalities. Here, we examined the impact of decreased CNS copper caused by ubiquitous heterozygosity for functional Slc31a1 on spinal cord motor neurons in Slc31a1+/- mice. Congruent with the CNS being relatively susceptible to disrupted copper availability, brain and spinal cord tissue from Slc31a1+/- mice contained significantly less copper than wild-type littermates, even though copper levels in other tissues were unaffected. Slc31a1+/- mice had less spinal cord α-motor neurons compared to wild-type littermates, but they did not develop any overt physical signs of motor impairment. By contrast, ALS model SOD1G37R mice had fewer α-motor neurons than control mice and exhibited clear signs of motor function impairment. With the expression of Slc31a1 notwithstanding, spinal cord expression of genes related to copper handling revealed only minor differences between Slc31a1+/- and wild-type mice. This contrasted with SOD1G37R mice where changes in the expression of copper handling genes were pronounced. Similarly, the expression of genes related to toxic glial activation was unchanged in spinal cords from Slc31a1+/- mice but highly upregulated in SOD1G37R mice. Together, results from the Slc31a1+/- mice and SOD1G37R mice indicate that although depleted CNS copper has a significant impact on spinal cord motor neuron numbers, the manifestation of overt ALS-like motor impairment requires additional factors.

Integrated elemental analysis supports targeting copper perturbations as a therapeutic strategy in multiple sclerosis

Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound CuII(atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with CuII(atsm) showing initial promise.

The physiological and pathophysiological roles of copper in the nervous system.

Copper is a critical trace element in biological systems due the vast number of essential enzymes that require the metal as a cofactor, including cytochrome c oxidase, superoxide dismutase and dopamine-β-hydroxylase. Due its key role in oxidative metabolism, antioxidant defence and neurotransmitter synthesis, copper is particularly important for neuronal development and proper neuronal function. Moreover, increasing evidence suggests that copper also serves important functions in synaptic and network activity, the regulation of circadian rhythms, and arousal. However, it is important to note that because of copper's ability to redox cycle and generate reactive species, cellular levels of the metal must be tightly regulated to meet cellular needs while avoiding copper-induced oxidative stress. Therefore, it is essential that the intricate system of copper transporters, exporters, copper chaperones and copper trafficking proteins function properly and in coordinate fashion. Indeed, disorders of copper metabolism such as Menkes disease and Wilson disease, as well as diseases linked to dysfunction of copper-requiring enzymes, such as SOD1-linked amyotrophic lateral sclerosis, demonstrate the dramatic neurological consequences of altered copper homeostasis. In this review, we explore the physiological importance of copper in the nervous system as well as pathologies related to improper copper handling.

Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Applying a nanocomposite hydrogel electrode to mitigate electrochemical polarization and focusing effect in electrokinetic remediation of a Cu- and Pb-contaminated loess

Inappropriate handling of copper (Cu) and lead (Pb)-containing wastewater resulting from metallurgical and smelting industries in Northwest China encourages their migration to surrounding environments. Their accumulation causes damage to liver and kidney function. The electrokinetic (EK) technology is considered to be an alternative to traditional remediation technologies because of its great maneuverability. The EK remediation is accompanied by the electrode polarization and the focusing effect toward affecting removal efficiency. In this study, a nanocomposite hydrogel (NCH) electrode was proposed and applied to the EK remediation of Cu- and Pb-contaminated loess. The mechanical, adsorption capacity, adsorption kinetics, and electrochemical properties of the NCH electrode were investigated in detail, followed by microscopic analyses of Scanning Electron Microscope (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Raman spectrometer. Results showed that the enhancement of the mechanical properties of the NCH electrode was attributed to the crosslinks of graphene nanoparticles, calcium alginate, and hydrogen bonds, while the Cu or Pb adsorption by the NCH electrode was in a chemisorption manner. The second layer formation might address the increase in adsorption capacity with increasing temperature. These results highlight the relative merits of the NCH electrode and verify the potential of applying the NCH electrode to the EK remediation of Cu- and Pb-contamianted loess.

Copper Homeostatic Mechanisms and Their Role in the Virulence of Escherichia coli and Salmonella enterica.

Copper is an essential micronutrient that also exerts toxic effects at high concentrations. This review summarizes the current state of knowledge on copper handling and homeostasis systems in Escherichia coli and Salmonella enterica. We describe the mechanisms by which transcriptional regulators, efflux pumps, detoxification enzymes, metallochaperones, and ancillary copper response systems orchestrate cellular response to copper stress. E. coli and S. enterica are important pathogens of humans and animals. We discuss the critical role of copper during killing of these pathogens by macrophages and in nutritional immunity at the bacterial-pathogen-host interface. In closing, we identify opportunities to advance our understanding of the biological roles of copper in these model enteric bacterial pathogens.

Copper Handling in the Salmonella Cell Envelope and Its Impact on Virulence.

Copper (Cu) plays a key role at the host-pathogen interface as both an essential element and a toxic element. Intracellular strains of pathogenic Salmonella have acquired the periplasmic Cu chaperone, CueP, and the thiol oxidoreductases complex Scs, while losing the ancestral Cu-detoxification Cus system. Coregulation of these species-specific factors link Cu with redox stress and allows Salmonella to counteract Cu toxicity during infection.

Wilson disease.

Wilson disease (WD) is an inherited disorder of copper metabolism. The resultant defective handling of copper results in toxic effects on the hepatocytes and increased copper in the circulation. Copper accumulates in other organ sites especially the central nervous system. WD occurs worldwide, usually between 5 and 35 years; a wider age range is now recognized. Clinical presentations are diverse and include combinations of hepatic, neurological, ophthalmic and psychiatric manifestations. Biochemical abnormalities such as serum ceruloplasmin and 24-h urinary copper excretion are important for the diagnosis but are not always abnormal in WD. They can overlap with non-WD causes. Patients may present with hepatic or neurological disease or combinations thereof. Approx. 50% of WD patients present with liver disease. Liver presentation is variable: asymptomatic abnormal liver tests, chronic hepatitis picture, cirrhosis, and acute liver failure. Similarly, the histology has several different patterns: mild nonspecific changes, steatosis or steatohepatitis, chronic hepatitis, and acute hepatitis with submassive or massive necrosis. None of these are specific for WD. Aids to the histologic diagnosis include special stains for copper and copper associated protein, and copper concentration in liver tissue. The biopsy is performed in the context of the clinical algorithms for the diagnosis of WD put forth by the clinical hepatology organizations such as the European Association for the Study of Liver Disease and the American Association for the Study of Liver Disease. The discovery of the responsible gene ATP7B has made the molecular diagnosis feasible through genetic testing and sequencing of the gene.

Handling of nutrient copper in the bacterial envelope

In bacteria, copper (Cu) is often recognised for its potential toxicity and its antibacterial activity is now considered a key component of the mammalian innate immune system. Cu ions bound in weak sites can catalyse harmful redox reactions while Cu ions in strong but adventitious sites can disrupt protein or enzyme function. For these reasons, the outward transport of Cu from bacteria has received significant attention. Yet, Cu is also a bacterial nutrient, required as a cofactor by enzymes that catalyse electron transfer processes, for instance in aerobic and anaerobic respiration. To date, the inward flow of this metal ion as a nutrient and its insertion into target cuproenzymes remain poorly defined. Here we revisit the available evidence related to bacterial nutrient Cu trafficking and identify gaps in knowledge. Particularly intriguing is the evidence that bacterial cuproenzymes do not always require auxiliary metallochaperones to insert nutrient Cu into their active sites. This review outlines our effort to consolidate the available experimental data using an established energy-driven model for metalation.

Copper dyshomeostasis in Wilson disease and Alzheimer's disease as shown by serum and urine copper indicators

Abnormal handling of copper is the cause of Wilson disease (WD), a rare disorder typified by increased levels in plasma copper not-bound to ceruloplasmin (nCp-Cu, also known as ‘free’ copper). In Alzheimer’s disease (AD), meta-analyses show that copper decreases in brain but increases in serum, due to the nCp Cu component increase. Despite the similarities, a direct comparison of copper biological status in the two diseases has never been carried out. To fill this gap, we evaluated serum copper, ceruloplasmin, nCp-Cu and Cu:Cp in 385 CE and 336 healthy controls previously investigated that were compared with 9 newly diagnosed WD patients. We then assessed 24h copper urinary excretion in 24 WD patients under D-penicillamine (D-pen) treatment and in 35 healthy controls, and compared results with those of AD patients participating to a D-pen phase II clinical trial previously published.After adjusting for sex and age, serum nCp-Cu and Cu:Cp resulted higher in AD and in WD than in healthy controls (both p<0.001). While nCp-Cu was similar between AD and WD, Cu:Cp was higher in WD (p<0.016). 24h urinary copper excretion in AD patients (12.05μg/day) was higher than in healthy controls (4.82μg/day; p<0.001). 77.8% of the AD patients under D-pen treatment had a 24h urinary excretion higher than 200μg/day, suggestive of a failure of copper control.This study provides new insight into the pathophysiology of copper homeostasis in AD, showing a failure of copper control and the Cu:Cp ratio as an eligible marker.

The mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve copper homeostasis in the murine heart.

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.

Thiol‐based copper handling by the copper chaperone Atox1

Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017.

The role of insufficient copper in lipid synthesis and fatty-liver disease.

The essential transition metal copper is important in lipid metabolism, redox balance, iron mobilization, and many other critical processes in eukaryotic organisms. Genetic diseases where copper homeostasis is disrupted, including Menkes disease and Wilson disease, indicate the importance of copper balance to human health. The severe consequences of insufficient copper supply are illustrated by Menkes disease, caused by mutation in the X-linked ATP7A gene encoding a protein that transports copper from intestinal epithelia into the bloodstream and across the blood-brain barrier. Inadequate copper supply to the body due to poor diet quality or malabsorption can disrupt several molecular level pathways and processes. Though much of the copper distribution machinery has been described and consequences of disrupted copper handling have been characterized in human disease as well as animal models, physiological consequences of sub-optimal copper due to poor nutrition or malabsorption have not been extensively studied. Recent work indicates that insufficient copper may be important in a number of common diseases including obesity, ischemic heart disease, and metabolic syndrome. Specifically, marginal copper deficiency (CuD) has been reported as a potential etiologic factor in diseases characterized by disrupted lipid metabolism such as non-alcoholic fatty-liver disease (NAFLD). In this review, we discuss the available data suggesting that a significant portion of the North American population may consume insufficient copper, the potential mechanisms by which CuD may promote lipid biosynthesis, and the interaction between CuD and dietary fructose in the etiology of NAFLD. © 2016 IUBMB Life, 69(4):263-270, 2017.

The mitochondrion: a central architect of copper homeostasis.

All known eukaryotes require copper for their development and survival. The essentiality of copper reflects its widespread use as a co-factor in conserved enzymes that catalyze biochemical reactions critical to energy production, free radical detoxification, collagen deposition, neurotransmitter biosynthesis and iron homeostasis. However, the prioritized use of copper poses an organism with a considerable challenge because, in its unbound form, copper can potentiate free radical production and displace iron-sulphur clusters to disrupt protein function. Protective mechanisms therefore evolved to mitigate this challenge and tightly regulate the acquisition, trafficking and storage of copper such that the metal ion is rarely found in its free form in the cell. Findings by a number of groups over the last ten years emphasize that this regulatory framework forms the foundation of a system that is capable of monitoring copper status and reprioritizing copper usage at both the cellular and systemic levels of organization. While the identification of relevant molecular mechanisms and signaling pathways has proven to be difficult and remains a barrier to our full understanding of the regulation of copper homeostasis, mounting evidence points to the mitochondrion as a pivotal hub in this regard in both healthy and diseased states. Here, we review our current understanding of copper handling pathways contained within the organelle and consider plausible mechanisms that may serve to functionally couple their activity to that of other cellular copper handling machinery to maintain copper homeostasis.

Suspected Wilson's Disease Presenting with Normal Serum Ceruloplasmin Levels

Wilson’s disease, also known as ‘Hepatolenticular degeneration’ is a disorder of copper handling. The clinical picture parallels the pathophysiology with symptoms of basal ganglia (10 to 100%), cerebellar dysfunction (18 to 73%) and liver dysfunction (18 to 84%). The patient profile usually describes a young male or female with signs of cerebellar dysfunction (Ataxia, Dysarthria and Nystagmus), basal ganglia dysfunction (Choreoathetosis), Kayser Fleisher (KF) rings in cornea and hepatic involvement (any form of acute or chronic liver disease). The basic pathophysiology relates to improper handling of copper by the liver owing to the dysfunctional ATP7B gene. The diagnostic tests include an increased urinary copper excretion (100 ug/dl), reduced serum ceruloplasmin levels ( 200 ug/gm of liver tissue). Although reduced serum ceruloplasmin levels are observed in most patients of Wilson disease, some proportion of patients particularly with acute hepatitis type of presentation can spuriously have raised ceruloplasmin levels thus posing the clinicians a diagnostic challenge especially in rural resource limited set ups. Presentations like these can be quite misleading as clinical suspicion index remains pretty low despite extensive laboratory work up. We report a case of such a patient of acute hepatitis with normal serum ceruloplasmin levels but significantly elevated urinary copper excretion. The patient showed wonderful response to oral zinc replacement evidenced with clinicobiocemical improvement.

Handling of Copper and Copper Oxide Nanoparticles by Astrocytes.

Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.

Effects of copper diffusion in gallium arsenide solar cells for space applications

High efficiency, thin-film Epitaxial Lift-Off (ELO) III–V solar cells offer excellent characteristics for implementation in flexible solar panels for space applications. However, the current thin-film ELO solar cell design generally includes a copper handling and support foil. Copper diffusion has a potentially detrimental effect on the device performance and the challenging environment provided by space (high temperatures, electron and proton irradiation) might induce diffusion. It is shown that heat treatments induce copper diffusion. The open-circuit voltage (Voc) is the most affected solar cell parameter. The decrease in Voc can be explained by enhanced non-radiative recombination via Cu trap levels in the middle of the band gap. The decrease in Voc is found to be dependent on junction depth. In all Cu cells annealed at T≥300°C signs of Cu diffusion are present, which implies that a barrier layer inhibiting Cu diffusion is necessary. Electron radiation damage was found to have no influence on Cu diffusion.

ATP7B activity is stimulated by PKCɛ in porcine liver

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)–ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10−8M), a PKC activator, increased Cu(I)–ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2μM Ca2+, whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

Involvement of CopZ in copper handling and delivery to cytochrome cbb3 oxidase

Involvement of CopZ in copper handling and delivery to cytochrome cbb3 oxidase

Quantification of copper binding to amyloid precursor protein domain 2 and its Caenorhabditis elegans ortholog. Implications for biological function

Aberrant regulation of transition metals and the resultant disregulation of neuronal reactive oxygen species (ROS) are considered significant in the etiology of Alzheimer's disease (AD). We determined the solution structure of the D2 domain of APL-1 (APL1-D2), the Caenorhabditis elegans ortholog of the amyloid precursor protein domain 2 (APP-D2). The copper binding affinities of APL1-D2 and APP-D2 were estimated and the ability of their copper complexes to promote formation of ROS was tested. The two protein domains are isostructural, consisting of a three-stranded β-sheet packed against a short α-helix in a βαββ fold. A six-residue insert in APL1-D2, absent in APP-D2, forms an extended loop. The putative copper binding ligands in APP-D2 are not conserved in APL1-D2 and this delineates a clear difference between them. APL1-D2 and APP-D2 bind one equivalent of Cu(I) weakly, with dissociation constants KD ∼10(-8.6) M and ~10(-10) M, respectively, and up to two equivalents of Cu(II) with KD values in the range 10(-6) -10(-8) M. The relative abilities of APL1-D2, APP-D2 and amyloid-β (Aβ) copper complexes to generate H2O2 correspond to their copper binding affinities. Copper affinities for Aβ (KD ~ 10(-10) M for both Cu(I) and Cu(II)) and APP-D2 are in a range allowing redox cycling to occur, albeit less efficiently for APP-D2. However, APL1-D2 binds Cu(I) and Cu(II) too weakly to sustain catalysis which further suggests that it plays no significant role in copper handling in C. elegans. Overall, the data are consistent with a possible role in copper homeostasis for APP-D2, but not APL1-D2.