Intracellular Copper Transport Research Articles

S3196 Wilson Disease in a U.S. Navy Pilot: Delayed Diagnosis With Drastic Career Implications

Introduction: Wilson disease is an autosomal recessive genetic disorder of the intracellular copper transport ATP7B leading to impaired copper excretion and subsequent accumulation in the liver, brain and cornea. Index symptoms vary widely and include neurologic and psychiatric abnormalities. The wide array of subtle symptoms often lead to significant delays in diagnosis and thus prognostic implications for patients diagnosed with this rare disease. In the Armed Forces, the diagnosis and clinical sequelae can have drastic career implications especially when neurologic sequelae are resistant to treatment. Case Description/Methods: A 28 year old US Navy pilot male presented to behavioral health clinic with complaint of depressed mood. He was diagnosed with major depressive disorder which was resistant to traditional treatment for over a year. He developed fasciculations of his neck and arms which was associated with initiation of an SSRI. When he had difficulty landing an aircraft due these progressive symptoms, he was deemed incapable of flight. He later required admission for suicidal ideations. He had several concerning incidents occur including driving over a median and through several red lights. Neurology was consulted and examination showed hyperkinetic movements of his extremities with head and voice tremors. Lab work revealed ALT 52 and AST 48 and later, a ceruloplasmin of 6.7. A slit lamp exam was performed revealing Kayser-Fleischer rings. MRI brain showed abnormal T2 signal within the basal ganglia and midbrain consistent with copper deposition. MRI liver was consistent with cirrhosis. A liver biopsy confirmed the diagnosis of Wilson disease with cirrhosis. He was started on trientene and zinc for chelation and his mood markedly improved. However, his neurologic abnormalities have persisted despite ongoing therapy for over 4 years, permanently ending his piloting career. Discussion: Our case highlights the importance of careful clinical consideration in patients presenting with psychiatric complaints. We aim to highlight this patient’s response to therapy, and lack thereof, as the range of patient response can be quite broad. The majority of patient’s have significant or even total resolution of symptoms. Our patient had significant improvement in depression after chelation therapy but his neurologic symptoms did not recover. Regrettably, the patient’s Navy pilot career was brought to an early close given the severity of his symptoms and lack of response and thus, inability to perform as an aviator.

S2948 Challenges in Recognizing and Diagnosing Wilson Disease

Introduction: Wilson disease is a rare autosomal recessive disease leading to impaired copper homeostasis and accumulation in the liver, brain, and other organs. It is manageable, and physicians must familiarize themselves with the diversity of its presentation. Our case sheds light on the challenges; health care providers face in establishing the diagnosis. Case Description/Methods: A fifty-eight-year-old female was admitted to the hospital for altered mental status who had a dramatic hospital course with acute liver failure complicated by shock and multiorgan failure. Her past medical history is significant for COPD, liver cirrhosis. Family history is positive for Alpha-1 Antitrypsin deficiency. She presented with acute psychosis and altered mental status. Patient was admitted with suspected hepatic encephalopathy. She developed acute liver failure followed by shock and multiorgan failure. Her work up came back as Hg 6.7, Plt 27, INR 2.1, Haptoglobin < 8 mg/dl, Coombs negative, Ceruoplasmn 5.9, copper serum 32, 24 hours urine copper level 42 and urine copper level 351. Previously, she complained of tremor and slurred speech. She was evaluated by neurology team for seizure like activity, but it was found that her movements were compelling choreiform movements (Figure). Liver cirrhosis evaluation started on 2019 when she was evaluated for new onset ascites. Her exam showed abdominal distension and lower limb edema, labs showed ALP 247, AST/ALT 51/16, total bilirubin 0.7, ANA >1: 1280, alpha 1 antitrypsin low 72 with MZ phenotype, Ceruplasmin level 13. Liver Biopsy Showed findings consistent with moderately active steatohepatitis with cirrhosis. It was presumed that her liver cirrhosis was related to A-1AT deficiency/NASH. The low ceruplasmin has slipped and the diagnosis of Liver Cirrhosis secondary to A-1AT deficiency carried on. Discussion: Wilson disease caused by mutations in the ATP7B gene responsible for intracellular copper transporter's function, led to impairment in biliary copper excretion leading to the deposit of copper in several organs. The diagnosis is based on the score developed at the 8th International Meeting on Wilson Disease in Leipzig (8). If the score is ≥4, Wilson disease is highly likely. In our patient, points are given as follows: Neuropsychiatric symptoms suggestive of Wilson disease (2), Coombs-negative hemolytic anemia with high serum copper (1), Serum ceruloplasmin < 10 mg/dL (2) with a total score of 5 points, which made her diagnosis with Wilson disease is highly likely.Figure 1.: Kayser-Fleisher ring.

Antioxidant Capacity Is Decreased in Wilson's Disease and Correlates to Liver Function.

The metabolic disorder Wilson's disease (WD) is caused by copper accumulation in the tissues due to a biallelic pathogenic mutation of the gene ATP7B, encoding intracellular copper transporter ATPase-7B. As copper is a redox active metal; aberrations in its homeostasis may create favourable conditions for superoxide-yielding redox cycling and oxidative damage to the cells. We tried to characterise antioxidant defence in WD patients and to evaluate whether it is related to liver function.The blood glutathione concentration, the activity of manganese-SOD (MnSOD), catalase (Cat), glutathione peroxidase, and glutathione S-transferase glutathione (GST), and serum antioxidant potential (AOP-450) were measured in WD treatment-naive patients and healthy controls and correlated with clinical data.The blood glutathione concentration, the activity of MnSOD, Cat, glutathione peroxidase, and GST and AOP-450 are significantly decreased in WD patients. There was a positive correlation of AOP-450 with AST. Moreover, the Cat and GST activity as well as AOP-450 strongly correlated with parameters of synthetic liver function. MnSOD activity correlated positively with ALT and AST.The blood glutathione concentration, the activity of MnSOD, Cat, glutathione peroxidase, and GST and AOP-450 are significantly decreased in WD patients. There was a positive correlation of AOP-450 with AST. Moreover, the Cat and GST activity as well as AOP-450 strongly correlated with parameters of synthetic liver function. MnSOD activity correlated positively with ALT and AST.Liver injury in course of WD is linked with decreased antioxidant capacity.

Maladie de Wilson

AbstractWilson’s disease is a autosomal recessive inherited disorder that causes progressive copper overload. The ATP7B gene carried on chromosome 13 encodes an intracellular copper transporter ATP7B protein that allows cellular excretion of excess copper. In Wilson’s disease, the functional deficiency of ATP7B causes a failure in the elimination of copper from the bile, which then accumulates in the liver and is then released in free form into the bloodstream. Wilson’s disease is therefore an initially hepatic disorder which can progress to multisystem involvement with an accumulation of copper in the brain, eye, kidney, etc. if it is not diagnosed early. The first symptoms are most often hepatic in children and neurological in adults. The diagnosis is made on a bundle of clinical, biological and radiological arguments. Interpretation of the cupric balance, typically associating a decrease in ceruloplasminemia and total cupremia with an increase in 24-hour urinary copper excretion, can be difficult, justifying the determination of the free or exchangeable copper. The ratio of exchangeable copper/total copper or REC (relative exchangeable copper) is an excellent diagnostic biomarker. Molecular biology can confirm the diagnosis in 98 % of cases. The treatments combine a copper low diet, copper chelators or zinc salts. Liver transplantation is the treatment for fulminant liver forms and decompensated cirrhosis. This rare genetic disease has a good prognosis if treatment is started early and continued all lifelong. It is therefore important to know the clinical manifestations of the disease and the diagnostic tests in order to evoke it quickly and to then ensure regular clinical and laboratory monitoring of patients.

Interactions between knockout of schizophrenia risk factor Dysbindin-1 and copper metabolism in mice

Background and PurposeDTNBP1 gene variation and lower dysbindin-1 protein are associated with schizophrenia. Previous evidence suggests that downregulated dysbindin-1 expression results in lower expression of copper transporters ATP7A (intracellular copper transporter) and SLC31A1 (CTR1; extracellular copper transporter), which are required for copper transport across the blood brain barrier. However, whether antipsychotic medications used for schizophrenia treatment may modulate these systems is unclear. Experimental ApproachThe current study measured behavioral indices of neurological function in dysbindin-1 functional knockout (KO) mice and their wild-type (WT) littermates with or without quetiapine treatment. We assessed serum and brain copper levels, ATP7A and CTR1 mRNA, and copper transporter-expressing cellular population transcripts: TTR (transthyretin; choroid plexus epithelial cells), MBP (myelin basic protein; oligodendrocytes), and GJA1 (gap-junction protein alpha-1; astrocytes) in cortex and hippocampus. Key ResultsRegardless of genotype, quetiapine significantly reduced TTR, MBP, CTR1 mRNA, and serum copper levels. Neurological function of untreated KO mice was abnormal, and ledge instability was rescued with quetiapine. KO mice were hyperactive after 10 min in the open-field assay, which was not affected by treatment. Conclusions and ImplicationsDysbindin-1 KO results in hyperactivity, altered serum copper, and neurological impairment, the last of which is selectively rescued with quetiapine. Antipsychotic treatment modulates specific cellular populations, affecting myelin, the choroid plexus, and copper transport across the blood brain barrier. Together these results indicate the widespread impact of antipsychotic treatment, and that alteration of dysbindin-1 may be sufficient, but not necessary, for specific schizophrenia pathology.

Highlights of This Issue

Treatment of adult glioma has not changed for over a decade, and it remains unclear how to best translate insights from genomic studies to bedside. Zheng and colleagues tested the utility of panel sequencing for assisting in glioma diagnosis, prognosis prediction, and informing targeted therapy using a prospective cohort of 237 gliomas. The study showed that the platform can identify IDH mutations that align with disease histopathology and outcome, and had high specificity and modest sensitivity in predicting 1p/19q codeletion. It also enabled stratification of hypermutators. Three out of ten patients whose treatments were tailored based on this panel achieved exceptional responses.Treatment of aggressive breast cancer remains challenging. Finding novel anti-cancer targets is critical for progress. In this study, Karginova and colleagues evaluate the efficacy and biological activity of the small molecule DCAC50, designed to inhibit intracellular copper transport through chaperones ATOX1 and CCS. DCAC50 triggers apoptosis in breast cancer cells and suppresses tumor angiogenesis in xenograft models. Co-treatment with paclitaxel enhances cytotoxicity in breast cancer cells. Mechanistically, DCAC50 disrupts copper homeostasis and generates oxidative stress. The ability of DCAC50 to induce apoptosis in cancer cells and suppress angiogenesis supports intracellular copper transport as a promising target for breast cancer therapy.This study by Shatsky and colleagues compares and contrasts solid tumor tissue NGS and ctDNA NGS technology to assess for potentially actionable genomic alterations in advanced breast malignancies. Both technologies were found to be useful and potentially complementary in the discovery of actionable alterations. In addition, ctDNA was found to have prognostic significance, as patients with greater than or equal to 5% ctDNA had a poorer overall survival. Both types of NGS have potential value that may add to targeted treatment options and further our understanding of advanced breast cancer.Peptide receptor radiotherapies with β-emitters are currently used to treat neuroendocrine tumors in patients. However, not all patients respond to treatment and most patients experience hematological toxicity. α-emitters, however, have a shorter pathlength and higher linear energy transfer allowing them to be more potent agents. This study by Stallons and colleagues examined 212Pb-DOTAMTATE in a preclinical neuroendocrine model and was more effective at controlling tumor growth in a single dose efficacy than previously published data with a beta-emitting somatostatin analog with little hematological toxicity. These studies support the therapeutic potential and possible advantage of 212Pb-DOTAMTATE and have led to the initiation of a phase I clinical trial.

Inhibition of Copper Transport Induces Apoptosis in Triple-Negative Breast Cancer Cells and Suppresses Tumor Angiogenesis.

Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor.

Copper is an essential element in plants. When scarce, copper is acquired from extracellular environment or remobilized from intracellular sites, through members of the high affinity copper transporters family COPT located at the plasma membrane and internal membrane, respectively. Here, we show that COPT3 is an intracellular copper transporter, located at a compartment of the secretory pathway, that is mainly expressed in pollen grains and vascular bundles. Contrary to the COPT1 plasma membrane member, the expression of the internal COPT3 membrane transporter was higher at 12 h than at 0 h of a neutral photoperiod day under copper deficiency. The screening of a library of conditionally overexpressed transcription factors implicated members of the TCP family in the COPT3 differential temporal expression pattern. Particularly, in vitro, TCP16 was found to bind to the COPT3 promoter and down-regulated its expression. Accordingly, TCP16 was mainly expressed at 0 h under copper deficiency and induced at 12 h by copper excess. Moreover, TCP16 overexpression resulted in increased sensitivity to copper deficiency, whereas the tcp16 mutant was sensitive to copper excess. Both copper content and the expression of particular copper status markers were altered in plants with modified levels of TCP16. Consistent with TCP16 affecting pollen development, the lack of COPT3 function led to altered pollen morphology. Furthermore, analysis of copt3 and COPT3 overexpressing plants revealed that COPT3 function exerted a negative effect on TCP16 expression. Taken together, these results suggest a differential daily regulation of copper uptake depending on the external and internal copper pools, in which TCP16 inhibits copper remobilization at dawn through repression of intracellular transporters.

The Involvement of Copper Transport in Von Willebrand Factor Multimer Assembly

In the event of vascular injury, the underlying prothrombotic extracellular matrix becomes exposed to circulating blood. Endothelial cells at the site of injury secrete multimeric von Willebrand factor (VWF) that will bind to the exposed extracellular matrix and create a scaffold to which platelets can adhere, forming a platelet plug. These critical steps after vascular injury are essential for creating a stable clot. Intracellular VWF multimer assembly is required for its extracellular function, and defects in VWF assembly cause the bleeding diathesis, von Willebrand disease. In a cell-free in vitro system, we and others find that EDTA or EGTA inhibits multimer formation, and calcium ions have been proposed to be required for this process. However, we find that the binding affinity of calcium ions for VWF D domains is very weak (Kdof 300-400 μM) and that buffers containing less than 2 μM calcium ions support multimer assembly even though few calcium binding sites could be occupied. These results suggest a role for cations other than calcium in VWF multimer assembly. Here we show that copper ions support VWF multimer assembly in a cell-free system and that the intracellular copper transporter, ATP7A, is required for VWF multimer assembly in cultured cells.Some calcium binding proteins undergo a conformational change upon binding calcium ions and exhibit a decrease in the apparent molecular weight (an electrophoretic mobility shift) as assessed by SDS-PAGE and Western blot. Although the D domains of VWF bind calcium ions, they do not exhibit this behavior. We tested a panel of other divalent cations including: iron, magnesium, manganese, zinc, and copper. We found that the VWF N-terminal D’D3 dimer bound copper ions as demonstrated by a shift in electrophoretic mobility. We therefore assessed the effect of copper ions on VWF multimer assembly in vitro. Briefly, addition of monensin (1.5 μM) to human umbilical vein endothelial cells (HUVEC) caused secretion of C-terminal dimers of proVWF into the media. Pretreatment of the conditioned media with the divalent cation chelator, EGTA, or the copper chelator, tetraethylenepentamine (TEPA), preceded immunopurification of C-terminal dimers of proVWF using a rabbit polyclonal anti-hVWF antibody (DAKO A0082) bound to protein A magnetic beads. Beads were incubated at pH 7.4 or pH 5.8, and in the presence or absence of divalent cations for 3 hours at 37°C before analyzing the VWF by gel electrophoresis. Addition of calcium ions (100 μM) to proVWF dimers pretreated with only EGTA allowed some VWF multimer assembly; however, copper ions (100 μM) induced the assembly of higher molecular weight multimers from proVWF dimers pretreated with either EGTA or TEPA. With either divalent cation, multimers assembled at low pH, but not at neutral pH. These findings support a role for copper ions in VWF synthesis.In vivo, free copper ions are toxic; therefore, they are sequestered by a system of chaperones and transporters. We examined the role of the intracellular copper transporter, ATP7A, in VWF multimerization because it is expressed in the trans Golgi network of endothelial cells, the site of VWF multimer assembly. Mick Petris (University of Missouri-Columbia) provided both wildtype and ATP7A knockout mouse embryonic fibroblasts (MEFs). We stably expressed full length human VWF and analyzed the secreted multimers by agarose gel electrophoresis. Wildtype MEFs produced multimeric VWF; however, culturing the MEFs in the presence of the copper chelator, TEPA, markedly impaired VWF multimer assembly. Moreover, ATP7A-null MEFs secreted only VWF dimers. These results indicate that copper ions and ATP7A contribute to VWF multimer assembly in cells.The dependence of VWF assembly on ATP7A in cultured cells suggests that humans with congenital ATP7A deficiency (Menkes disease) could have detectable abnormalities in VWF multimer structure, although dietary copper and parenteral copper replacement therapy could affect this phenotype. Our preliminary analyses have identified a Menkes disease patient with low plasma levels of VWF antigen, multimers and activity. Further study will be required to determine the prevalence and significance of altered VWF multimer assembly in Menkes disease. DisclosuresSadler:BioMarin: Consultancy; 23andMe: Consultancy; Ablynx: Consultancy.

Phenotypic convergence of Menkes and Wilson disease.

Menkes disease is an X-linked multisystem disorder with epilepsy, kinky hair, and neurodegeneration caused by mutations in the copper transporter ATP7A. Other ATP7A mutations have been linked to juvenile occipital horn syndrome and adult-onset hereditary motor neuropathy.1,2 About 5%-10% of the patients present with "atypical Menkes disease" characterized by longer survival, cerebellar ataxia, and developmental delay.2 The intracellular copper transport is regulated by 2 P type ATPase copper transporters ATP7A and ATP7B. These proteins are expressed in the trans-Golgi network that guides copper to intracellular compartments, and in copper excess, it relocates copper to the plasma membrane to pump it out from the cells.3ATP7B mutations cause Wilson disease with dystonia, ataxia, tremor, and abnormal copper accumulation in the brain, liver, and other organs.4.

Transcriptomic response of yeast cells to ATX1 deletion under different copper levels

BackgroundIron and copper homeostatic pathways are tightly linked since copper is required as a cofactor for high affinity iron transport. Atx1p plays an important role in the intracellular copper transport as a copper chaperone transferring copper from the transporters to Ccc2p for its subsequent insertion into Fet3p, which is required for high affinity iron transport.ResultsIn this study, genome-wide transcriptional landscape of ATX1 deletants grown in media either lacking copper or having excess copper was investigated. ATX1 deletants were allowed to recover full respiratory capacity in the presence of excess copper in growth environment. The present study revealed that iron ion homeostasis was not significantly affected by the absence of ATX1 either at the transcriptional or metabolic levels, suggesting other possible roles for Atx1p in addition to its function as a chaperone in copper-dependent iron absorption. The analysis of the transcriptomic response of atx1∆/atx1∆ and its integration with the genetic interaction network highlighted for the first time, the possible role of ATX1 in cell cycle regulation, likewise its mammalian counterpart ATOX1, which was reported to play an important role in the copper-stimulated proliferation of non-small lung cancer cells.ConclusionsThe present finding revealed the dispensability of Atx1p for the transfer of copper ions to Ccc2p and highlighted its possible role in the cell cycle regulation. The results also showed the potential of Saccharomyces cerevisiae as a model organism in studying the capacity of ATOX1 as a therapeutic target for lung cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2771-6) contains supplementary material, which is available to authorized users.

Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional pesticides and metals was assayed, and eight agents that produced a significant decrease and five that produced a significant increase in activated E1 were identified. These studies suggest that E1 is a sensitive redox sensor that can be modulated by exposure to environmental agents and can regulate downstream cellular processes.

A case of Menkes disease caused by novel mutation in the ATP7A gene with infantile hypertrophic pyloric stenosis

Menkes disease is caused by mutations in the ATP7A gene that lead to intracellular copper transport defects and characterized by brownish twisted...

Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1

The Vitis vinifera copper transporter 1 is capable of self-interaction and mediates intracellular copper transport. An understanding of copper homeostasis in grapevine (Vitis vinifera L.) is particularly relevant to viticulture in which copper-based fungicides are intensively used. In the present study, the Vitis vinifera copper transporter 1 (VvCTr1), belonging to the Ctr family of copper transporters, was cloned and functionally characterized. Amino acid sequence analysis showed that VvCTr1 monomers are small peptides composed of 148 amino acids with 3 transmembrane domains and several amino acid residues typical of Ctr transporters. Bimolecular fluorescence complementation (BiFC) demonstrated that Ctr monomers are self-interacting and subcellular localization studies revealed that VvCTr1 is mobilized via the trans-Golgi network, through the pre-vacuolar compartment and located to the vacuolar membrane. The heterologous expression of VvCTr1 in a yeast strain lacking all Ctr transporters fully rescued the phenotype, while a deficient complementation was observed in a strain lacking only plasma membrane-bound Ctrs. Given the common subcellular localization of VvCTr1 and AtCOPT5 and the highest amino acid sequence similarity in comparison to the remaining AtCOPT proteins, Arabidopsis copt5 plants were stably transformed with VvCTr1. The impairment in root growth observed in copt5 seedlings in copper-deficient conditions was fully rescued by VvCTr1, further supporting its involvement in intracellular copper transport. Expression studies in V. vinifera showed that VvCTr1 is mostly expressed in the root system, but transcripts were also present in leaves and stems. The functional characterization of VvCTr-mediated copper transport provides the first step towards understanding the physiological and molecular responses of grapevines to copper-based fungicides.

Knockdown of copper chaperone antioxidant-1 by RNA interference inhibits copper-stimulated proliferation of non-small cell lung carcinoma cells

Copper is required for cell proliferation and tumor angiogenesis. Cellular copper metabolism is regulated by a network of copper transporters and chaperones. Antioxidant-1 (ATOX1) is a cytosolic copper chaperone important for intracellular copper transport, which plays a role in the regulation of cell proliferation by functioning as a transcription factor in cell growth signal-transduction pathways. The present study aimed to explore the role of ATOX1 in the copper-related regulation of lung cancer cell proliferation by immunohistochemical (IHC) analysis of ATOX1 expression in non-small cell lung cancer (NSCLC) tissue samples and by assessing the effects of RNA interference (RNAi)-mediated knockdown of ATOX1 on copper-stimulated proliferation of NSCLC cells. Overexpression of ATOX1 was detected in NSCLC by IHC analysis of the tissue samples from patients diagnosed with NSCLC when compared with expression of ATOX1 in non-malignant lung tissue samples. Knockdown of ATOX1 in the NSCLC cells transduced by a lentiviral vector encoding short hairpin RNA (shRNA) specific for ATOX1 was associated with reduction in copper-stimulated cell proliferation. These findings suggest that ATOX1 plays an important role in copper-stimulated proliferation of NSCLC cells and ATOX1 holds potential as a therapeutic target for lung cancer therapy targeting copper metabolism.

Selective Divalent Copper Chelation for the Treatment of Diabetes Mellitus

Oxidative stress and mitochondrial dysfunction have been identified by many workers as key pathogenic mechanisms in ageing-related metabolic, cardiovascular and neurodegenerative diseases (for example diabetes mellitus, heart failure and Alzheimer's disease). However, although numerous molecular mechanisms have been advanced to account for these processes, their precise nature remains obscure. This author has previously suggested that, in such diseases, these two mechanisms are likely to occur as manifestations of a single underlying disturbance of copper regulation. Copper is an essential but highly-toxic trace metal that is closely regulated in biological systems. Several rare genetic disorders of copper homeostasis are known in humans: these primarily affect various proteins that mediate intracellular copper transport processes, and can lead either to tissue copper deficiency or overload states. These examples illustrate how impaired regulation of copper transport pathways can cause organ damage and provide important insights into the impact of defects in specific molecular processes, including those catalyzed by the copper-transporting ATPases, ATP7A (mutated in Menkes disease), ATP7B (Wilson's disease), and the copper chaperones such as those for cytochrome c oxidase, SCO1 and SCO2. In diabetes, impaired copper regulation manifests as elevations in urinary CuII excretion, systemic chelatable-CuII and full copper balance, in increased pro-oxidant stress and defective antioxidant defenses, and in progressive damage to the blood vessels, heart, kidneys, retina and nerves. Linkages between dysregulated copper and organ damage can be demonstrated by CuII-selective chelation, which simultaneously prevents/reverses both copper dysregulation and organ damage. Pathogenic structures in blood vessels that contribute to binding and localization of catalytically-active CuII probably include advanced glycation end products (AGEs), as well as atherosclerotic plaque: the latter probably undergoes AGE-modification itself. Defective copper regulation mediates organ damage through two general processes that occur simultaneously in the same individual: elevation of CuII-mediated pro-oxidant stress and impairment of copper-catalyzed antioxidant defence mechanisms. This author has proposed that diabetes-evoked copper dysregulation is an important new target for therapeutic intervention to prevent/reverse organ damage in diabetes, heart failure, and neurodegenerative diseases, and that triethylenetetramine (TETA) is the first in a new class of anti-diabetic molecules, which function by targetting these copper-mediated pathogenic mechanisms. TETA prevents tissue damage and causes organ regeneration by acting as a highly-selective CuII chelator which suppresses copper-mediated oxidative stress and restores anti-oxidant defenses. My group has employed TETA in a comprehensive programme of nonclinical studies and proof-of-principle clinical trials, thereby characterizing copper dysregulation in diabetes and identifying numerous linked cellular and molecular mechanisms though which TETA exerts its therapeutic actions. Many of the results obtained in nonclinical models with respect to the molecular mechanisms of diabetic organ damage have not yet been replicated in patients' tissues so their applicability to the human disease must be considered as inferential until the results of informative clinical studies become available. Based on evidence from the studies reviewed herein, trientine is now proceeding into the later stages of pharmaceutical development for the treatment of heart failure and other diabetic complications.

Wilson Disease: Pathogenesis and Clinical Considerations in Diagnosis and Treatment

Nearly a century after Dr. Samuel Alexander Kinnier Wilson composed his doctoral thesis on the pathologic findings of "lenticular degeneration" in the brain associated with cirrhosis of the liver we know that the underlying molecular basis for this autosomal recessive inherited disorder that now bears his name is mutation of a copper transporting ATPase, ATP7B, an intracellular copper transporter mainly expressed in hepatocytes. Loss of ATP7B function is the basis for reduced hepatic biliary copper excretion and reduced incorporation of copper into ceruloplasmin. During the intervening years, there was recognition of the clinical signs, histologic, biochemical features, and mutation analysis of ATP7B that characterize and enable diagnosis of this disorder. These include the presence of signs of liver or neurologic disease and detection of Kayser-Fleischer rings, low ceruloplasmin, elevated urine and hepatic copper, and associated histologic changes in the liver. Medical therapies and liver transplantation can effectively treat patients with this once uniformly fatal disorder. The earlier detection of the disease led to the initiation of treatment to prevent disease progression and reverse pathologic findings if present, and family screening to detect the disorder in first-degree relatives is warranted. Gene therapy and hepatocyte cell transplantation for Wilson disease has only been tested in animal models but represent future areas for study. Despite all the advances we still have to consider the diagnosis of Wilson disease to test patients for this disorder and properly establish the diagnosis before committing to life-long treatment.

Copper incorporation into ceruloplasmin is regulated by Niemann–Pick C1 protein

Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans-Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann-Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes. We induced the NPC phenotype using U18666A to modulate the vesicle traffic from the late endosome to TGN. Then, we examined the effect of NPC1 overexpression on the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. Overexpression of NPC1 increased holo-Cp secretion to culture medium of U18666A-treated cells, but did not affect the secretion of albumin. Manipulation of NPC1 function affected the localization of ATP7B and late endosome markers, but did not change the localization of a TGN marker. ATP7B co-localized with the late endosome markers, but not with the TGN marker. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via an NPC1-dependent pathway and incorporated into Cp.

Maladie de Menkes néonatale : intérêts diagnostiques de la cuprémie et de l'examen microscopique des cheveux

Background Menkes disease is a rare X-linked disorder due to a defect in intracellular copper transport. Clinical symptoms appear during the first months of life, with a progressive developmental delay leading to death within a few years. Diagnosis is confirmed by the demonstration of copper retention in fibroblasts and/or DNA testing. However, these investigations are complexes and time consuming. Case report We report 1 case of Menkes disease with neonatal onset, diagnosed on multiple organ failure, hypothermia, and major central nervous system damage, leading to death in a few weeks. The diagnosis, suggested by the clinical features, was rapidly supported by the microscopic examination of the hairs, showing pili torti, and the demonstration of severely decreased levels of plasma copper and ceruloplasmin. Diagnosis was further confirmed by the demonstration of an increased copper uptake and retention in fibroblasts. Conclusion This report highlights the clinical variability of Menkes disease with the possibility of a neonatal onset. Microscopic examination of the hairs and the determination of copper and ceruloplasmin plasma levels are simple and inexpensive investigations, which can provide rapidly valuable information to support this diagnosis.

An Anesthetic Experience in a Patient with Menkes Disease - A case report -

Menkes disease, socalled kinky hair disease or steely hair disease, is a rare Xlinked recessive disorder of intracellular copper transport protein ATP7A defect, due to mutation of ATP7A gene, resulting in copper deficiency. It is characterized by seizure, retarded neurological development, kinky hair, skeletal abnormality, recurrent infection and subnormal body temperature. In addition, gastroesophageal reflux with the risk of aspiration is another important feature. This article is the first report of anesthetic management in a patient with Menkes disease who underwent gastrostomy and bladder diverticulectomy in Korea.