Intracellular Copper Metabolism Research Articles

Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain

Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.

Changes in copper concentrations affect the protein levels but not the mRNA levels of copper chaperones in human umbilical vein endothelial cells

Copper chaperones are critical regulators of intracellular copper metabolism and distribution. The present study was undertaken to investigate the effects of changes in copper concentrations on the abundance of copper chaperones. Human umbilical vein endothelial cells (HUVECs) were treated with siRNA targeting copper transporter 1 (CTR1) or tetraethylenepentamine (TEPA) to decrease, or with copper sulfide to increase, intracellular copper concentrations, assayed using an atomic absorption spectrophotometer. Western blot analyses showed that CTR1 silencing or TEPA treatment increased the protein levels of copper chaperone ATOX1 and copper chaperone for superoxide dismutase 1 (CCS-1), but decreased copper chaperone for cytochrome c oxidase (COX17). In contrast, copper supplementation decreased the protein levels of ATOX1 and CCS-1 and increased COX17. Real-time RT-PCR analyses found no difference in the mRNA levels of the copper chaperones examined under the conditions defined above. This study thus demonstrated that changes in copper concentrations alter the protein levels, but not the mRNA levels, of copper chaperones, suggesting a role of copper in the post-translational modification of these proteins.

MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy

MEDNIK syndrome-acronym for mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratodermia-is caused by AP1S1 gene mutations, encoding σ1A, the small subunit of the adaptor protein 1 complex, which plays a crucial role in clathrin coat assembly and mediates trafficking between trans-Golgi network, endosomes and the plasma membrane. MEDNIK syndrome was first reported in a few French-Canadian families sharing common ancestors, presenting a complex neurocutaneous phenotype, but its pathogenesis is not completely understood. A Sephardic-Jewish patient, carrying a new AP1S1 homozygous mutation, showed severe perturbations of copper metabolism with hypocupremia, hypoceruloplasminemia and liver copper accumulation, along with intrahepatic cholestasis. Zinc acetate treatment strikingly improved clinical conditions, as well as liver copper and bile-acid overload. We evaluated copper-related metabolites and liver function retrospectively in the original French-Canadian patient series. Intracellular copper metabolism and subcellular localization and function of copper pump ATP7A were investigated in patient fibroblasts. Copper metabolism perturbation and hepatopathy were confirmed in all patients. Studies in mutant fibroblasts showed abnormal copper incorporation and retention, reduced expression of copper-dependent enzymes cytochrome-c-oxidase and Cu/Zn superoxide dismutase, and aberrant intracellular trafficking of Menkes protein ATP7A, which normalized after rescue experiments expressing wild-type AP1S1 gene. We solved the pathogenetic mechanism of MEDNIK syndrome, demonstrating that AP1S1 regulates intracellular copper machinery mediated by copper-pump proteins. This multisystem disease is characterized by a unique picture, combining clinical and biochemical signs of both Menkes and Wilson's diseases, in which liver copper overload is treatable by zinc acetate therapy, and can now be listed as a copper metabolism defect in humans. Our results may also contribute to understand the mechanism(s) of intracellular trafficking of copper pumps.

Experimental switching of copper status in laboratory rodents

There is an emerging link between copper metabolism, tumor growth and efficiency of antitumor treatment with platinum drugs or copper chelators. So there is an urgent need for well-defined and reproduced animal models with different states of copper metabolism. In the present study an animal model (rats and mice) with switching copper status in blood serum (copper concentration, oxidase activity and ceruloplasmin (Cp) protein content) is characterized. The drop of copper status is caused by addition of AgCl to fodder (Ag-animals). In rats and mice, the influence of silver ions on oxidase and ferroxidase activity of blood serum is similar, but copper concentration is reduced by 90% in rats, and by 60% in mice. The absorbed silver ions are transported to liver cells and included to Cp polypeptides, which are secreted to blood serum then. Cp, which circulates in bloodstream of Ag-animals contains silver atoms, and is misfolded, as judged by circular dichroism spectroscopy and differential scanning calorimetry. Single intraperitoneal or per oral injection of Cu(II) salt to Ag-animals causes recovery of oxidase and ferroxidase activity of blood serum within 4 hours in both rodent species, presumably by rapid metabolic insertion of copper into forming Cp in liver. The recovered copper status persists for 3 days under the continuing Ag-diet. The possibilities of use of Ag-rodents with switching copper status in investigation of influence of copper status on tissue-specific intracellular copper metabolism and role of copper in tumor genesis, bone metabolism and neurodegenerative diseases are discussed.

Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase

The metallochaperone Atox1 directly interacts with the copper-transporting ATPases and plays a critical role in perinatal copper homeostasis. To determine the cell biological mechanisms of Atox1 function, intracellular copper metabolism, and Menkes ATPase abundance, localization and trafficking were examined in immortalized fibroblast cell lines derived from Atox1(+/+) and Atox1(-/-) embryos. Consistent with the proposed role for Atox1 in copper delivery to the secretory pathway, a marked increase in intracellular copper content secondary to impaired copper efflux was observed in Atox1-deficient cells. Although the localization of the Menkes ATPase was identical in Atox1(+/+) and Atox1(-/-) cells under conditions of equivalent intracellular copper content, a significant impairment in copper-mediated Menkes ATPase trafficking was observed in the absence of Atox1. When quantitative confocal immunofluorescence was used, significant differences in the time and dose-dependent trafficking of the Menkes ATPase from the Golgi compartment in response to copper were observed between Atox1(+/+) and Atox1(-/-) cells. These data reveal an essential role for Atox1 in establishing the threshold for copper-dependent movement of the copper-transporting ATPases within the secretory compartment and that, in the absence of Atox1, this movement alone is not sufficient to restore normal copper efflux. Taken together, these findings provide a cell biological model for the role of this metallochaperone under the physiological conditions of copper limitation in mammalian cells.

A perspective on copper and liver disease in the dog.

Copper is a ubiquitous trace metal necessary for normal function of a variety of cellular proteins. Intracellular copper metabolism is complex, and only a few of the proteins/genes involved are known. Copper deficiency does not appear to be a clinical problem in dogs. Excess copper accumulation in the liver as a cause of hepatitis and cirrhosis was first demonstrated among Bedlington terriers. Subsequently, copper accumulation in the liver has been shown to occur in several other breeds of dogs. Excess hepatic copper has been found in dogs with normal liver histology, dogs with hepatitis, and dogs with end stage cirrhosis. Evidence is accumulating that copper is a cause of liver disease in breeds of dogs other than Bedlington terriers. Moreover, as more data are accumulated, the copper storage disease appears to have characteristics that are very similar among all of the affected breeds.

Intracellular localization of the Menkes and Wilson's disease proteins and their role in intracellular copper transport.

Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. In excess, copper is a very toxic ion and therefore efficient regulation of its metabolism is required. This is dramatically illustrated by the genetic disorders X-linked Menkes disease and autosomal recessive Wilson's disease. In 1993, both the Menkes and Wilson's genes were isolated and it was found that these genes encode homologous cation copper transporting P-type ATPase proteins. The Menkes protein (ATP7A) is expressed in most tissues, except liver. In contrast, the Wilson's protein (ATP7B) is abundantly expressed in liver. Intracellular localization of those proteins was investigated. Both ATP7A and ATP7B are localized in the trans-Golgi network and post-Golgi vesicular compartment (PGVC) in the cell. This intracellular localization was altered by the copper content present in the cell. This result may support the hypothesis that ATP7A and ATP7B are involved in cellular copper transport and those proteins could be suitable models for elucidating intracellular copper metabolism.

Identification and purification of a self-associating copper-binding protein from mouse hepatic cytosols.

The proteins which bind copper when it first enters cells and deliver that copper to the cellular sites where copper is incorporated into copper enzymes are unknown. When radiolabeled (64Cu(II)) cytosol proteins were fractionated on Superose columns, two large copper-binding protein fractions were detected which together bound 63% of the total 64Cu recovered from the column. A self-associating, 50-kDa copper-binding protein was identified whose tetramer and dimer appeared to elute in one of the major copper-binding fractions and the monomer eluted in the other. This protein was purified to homogeneity by successive Mono Q, chelating Superose, and phenyl-Superose columns. The concentrated, purified protein showed high amounts of tetramer and monomer plus some dimer, each of which bound copper. Rechromatography of the tetramer-dimer fraction on Superose gave rise to approximately equal amounts of tetramer, dimer, and monomer. Similarly, rechromatography of the monomer fraction gave rise to dimer and tetramer. Thus, the results with the purified protein confirmed that the 50-kDa protein exists as a tetramer-dimer-monomer equilibrium. The 50-kDa protein apparently makes a major contribution to the total copper-binding activity of the mouse hepatic cytosol and may play a significant role in intracellular copper metabolism.

Purification and properties of a self-associating, 50-kDa copper-binding protein from brindled mouse livers.

The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal 64Cu binding was detected in both the fraction which contained the tetramer plus dimer (approximately 26% less) and the fraction containing the monomer of CuBP (approximately 37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at approximately 20 min after starting the final water elution step, the brindled mouse protein eluted by approximately 5 min. This seemed to be due to abnormal self-association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consistent with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease.

Identification of proteins involved in intracellular copper metabolism: Low levels of a ≈ 48-kDa copper-binding protein in the brindled mouse model of Menkes disease

Abstract Little is known about copper metabolism at the cellular level. The brindled mouse is an animal model of Menkes disease which is an inborn error of copper metabolism. Control and brindled mice were used to identify copper-binding proteins with possible roles in normal copper metabolism that are affected by the defect in the brindled mice. When 64Cu-labeled hepatic or renal cytosols from control mice were applied to Mono Q or Superose columns, a approximately 48-kDa protein coeluted with the protein fractions which contained the radiolabeled copper. Large decreases in copper binding were detected in these fractions from the brindled mice. The same column fractions which showed decreased copper binding showed large decreases in the levels of the approximately 48-kDa protein. Decreased copper binding and approximately 48-kDa protein were not simply secondary to the abnormal hepatic and renal copper levels that are found in the brindled mice since although their liver copper levels are low, their kidney copper levels are high. Elevated levels of an approximately 80-kDa heat shock protein were also detected in the hepatic and renal cytosols from the brindled mice. Consistent with expression of the primary defect in both the liver and kidney, the levels of the approximately 48- and approximately 80-kDa proteins were affected similarly in both organs. Irrespective of how the low levels of the approximately 48-kDa protein may be related to the basic defect in the brindled mice, the data are consistent with an important role for the approximately 48-kDa protein in intracellular copper metabolism.

Cytosolic copper-binding proteins in rat and mouse hepatocytes incubated continuously with Cu(II)

The proteins that bind copper when it first enters cells are likely to play roles in its intracellular distribution and utilization. When hepatocytes were incubated with 64Cu(II), the time-dependence of the subcellular distribution of 64Cu was consistent with one or more cytosolic proteins distributing copper to the mitochondrial and nuclear fractions. Cytosolic copper was reproducibly distributed among four protein fractions from Sephadex G-150 columns at the earliest time (1 min) and at the lowest concentration used [2 microM-64Cu(II)] with both rat and mouse hepatocytes. Copper binding to proteins in these functions was sensitive to copper metabolic status. Hepatocytes from nutritionally copper-deficient rats or neonatal (9-30 days old) developing rats showed an inverse correlation between copper binding to metallothionein and copper binding to proteins in fraction I (approximately 88 kDa apparent) and fraction II (approximately 38 kDa apparent). The distribution of cytosolic 64Cu from the brindled-mouse model of Menkes disease indicated decreased binding by a protein in fraction I. Brindled-mouse hepatocytes also contain decreased levels of a approximately 55 kDa protein or subunit, which most likely represents a liver-specific secondary response to the primary defect. The results are consistent with one or more copper-binding proteins in fractions I and II having significant functions in intracellular copper metabolism.

Variations in the intralobular distribution of copper in the livers of copper-loaded rats in relation to the pathogenesis of copper storage diseases

There are differences in the hepatic intralobular distribution of copper in copper storage related diseases which may be of pathogenetic significance. Male rats fed a high copper diet (1500 ppm) for 16 weeks were killed at intervals in an attempt to compare copper distribution in their livers with those in human, canine and ovine copper toxicosis. Copper was found to accumulate almost exclusively in the periportal and mid-zones of the rat liver lobules and was associated with progressive pathological changes which included focal and periportal degeneration and necrosis. This pattern of copper distribution contrasts markedly with the centrilobular retention reported in familial canine copper toxicosis and chronic copper poisoning in sheep which suggests that, in these conditions, a secretory deficiency may be less important than a metabolic zonal defect of intracellular copper metabolism. The pathological changes observed in copper-loaded rats have a different micro-anatomical localization from those in dogs and sheep, but show similarities to the early changes reported in the latter species and indicate the possibility of a similar cellular lesion.