Copper comes of age in Melbourne.

When we were asked to produce articles for this volume, it seemed appropriate to us to co-author an article on the history and impact of copper research in Melbourne. It is appropriate because over many years, decades in fact, we worked closely together and with Professor David Danks to identify the molecular defect in Menkes disease. This work was always carried out with the intention of understanding the nature of the copper homeostatic mechanisms and a "copper pathway" in the cell, that David had the prescience to predict must exist despite scepticism from granting agencies! He indeed inspired us to pursue research careers in this field.

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX).

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency.

Copper dyshomoeostasis in Parkinson's disease: implications for pathogenesis and indications for novel therapeutics.

Copper is a biometal essential for normal brain development and function, thus copper deficiency or excess results in central nervous system disease. Well-characterized disorders of disrupted copper homoeostasis with neuronal degeneration include Menkes disease and Wilson's disease but a large body of evidence also implicates disrupted copper pathways in other neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Huntington's disease and prion diseases. In this short review we critically evaluate the data regarding changes in systemic and brain copper levels in Parkinson's disease, where alterations in brain copper are associated with regional neuronal cell death and disease pathology.

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry.

Effects of ATP7A overexpression in mice on copper transport and metabolism in lactation and gestation.

Placentae and mammary epithelial cells are unusual in robustly expressing two copper "pumps", ATP7A and B, raising the question of their individual roles in these tissues in pregnancy and lactation. Confocal microscopic evidence locates ATP7A to the fetal side of syncytiotrophoblasts, suggesting a role in pumping Cu towards the fetus; and to the basolateral (blood) side of lactating mammary epithelial cells, suggesting a role in recycling Cu to the blood. We tested these concepts in wild-type C57BL6 mice and their transgenic counterparts that expressed hATP7A at levels 10-20× those of endogenous mAtp7a.

Copper: effects of deficiency and overload.

Copper is an essential trace metal that is required for the catalysis of several important cellular enzymes. However, since an excess of copper can also harm cells due to its potential to catalyze the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. This chapter summarizes the current knowledge on the importance of copper for cellular processes and on the mechanisms involved in cellular copper uptake, storage and export.

Metabolism and functions of copper in brain.

Copper is an important trace element that is required for essential enzymes. However, due to its redox activity, copper can also lead to the generation of toxic reactive oxygen species. Therefore, cellular uptake, storage as well as export of copper have to be tightly regulated in order to guarantee sufficient copper supply for the synthesis of copper-containing enzymes but also to prevent copper-induced oxidative stress.

Copper pathology in vulnerable brain regions in Parkinson's disease.

Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss.

Copper transporter ATP7A protects against endothelial dysfunction in type 1 diabetic mice by regulating extracellular superoxide dismutase.

Oxidative stress and endothelial dysfunction contribute to vascular complication in diabetes. Extracellular superoxide dismutase (SOD3) is one of the key antioxidant enzymes that obtains copper via copper transporter ATP7A. SOD3 is secreted from vascular smooth muscles cells (VSMCs) and anchors at the endothelial surface.

Copper metabolism of astrocytes.

This short review will summarize the current knowledge on the uptake, storage, and export of copper ions by astrocytes and will address the potential roles of astrocytes in copper homeostasis in the normal and diseased brain. Astrocytes in culture efficiently accumulate copper by processes that include both the copper transporter Ctr1 and Ctr1-independent mechanisms. Exposure of astrocytes to copper induces an increase in cellular glutathione (GSH) content as well as synthesis of metallothioneins, suggesting that excess of copper is stored as complex with GSH and in metallothioneins.

Contributions of rat Ctr1 to the uptake and toxicity of copper and platinum anticancer drugs in dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons are affected by platinum-induced neurotoxicity and neurodegenerative processes associated with disturbed copper homeostasis and transport. This study aimed to understand the role of copper transporter 1 (Ctr1) in the uptake and toxicity of copper and platinum drugs in cultured rat DRG neurons, and the functional activities of rat Ctr1 (rCtr1) as a membrane transporter of copper and platinum drugs. Heterologous expression of rCtr1 in HEK293 cells (HEK/rCtr1 cells) increased the uptake and cytotoxicity of copper, oxaliplatin, cisplatin and carboplatin, in comparison to isogenic vector-transfected control cells.

Localization of copper and copper transporters in the human brain.

Disturbances in brain copper result in rare and severe neurological disorders and may play a role in the pathogenesis and progression of multiple neurodegenerative diseases. Our current understanding of mammalian brain copper transport is based on model systems outside the central nervous system and no data are available regarding copper transport systems in the human brain. To address this deficit, we quantified regional copper concentrations and examined the distribution and cellular localization of the copper transport proteins Copper transporter 1, Atox1, ATP7A, and ATP7B in multiple regions of the human brain using inductively coupled plasma-mass spectrometry, Western blot and immunohistochemistry.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy.

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking.

Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B.

ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases.

Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice.

Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p.

Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B.

The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively.

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue.

Background: ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.

Missense mutations in the copper transporter gene ATP7A cause X-linked distal hereditary motor neuropathy.

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families.

Copper accumulation by cultured astrocytes.

To study copper transport in brain astrocytes, we have used astrocyte-rich primary cultures as model system. Cells in these cultures contained a basal copper content of 1.1+/-0.

Mammalian copper-transporting P-type ATPases, ATP7A and ATP7B: emerging roles.

Copper (Cu) has a role in a diverse and increasing number of pathways, physiological and disease processes. These roles are testament to the fundamental importance of Cu in biology and the need to understand the mechanisms that regulate Cu homeostasis. The mammalian Cu-transporting P-type ATPases ATP7A and ATP7B are two key proteins that regulate the Cu status of the body.

Neuronal expression of copper transporter 1 in rat dorsal root ganglia: association with platinum neurotoxicity.

Purpose: We report the neuronal expression of copper transporter 1 (CTR1) in rat dorsal root ganglia (DRG) and its association with the neurotoxicity of platinum-based drugs.

Methods: CTR1 expression was studied by immunohistochemistry and RT-PCR. The toxicity of platinum drugs to CTR1-positive and CTR1-negative neurons was compared in DRG from animals treated with maximum tolerated doses of oxaliplatin (1.

Copper is taken up efficiently from albumin and alpha2-macroglobulin by cultured human cells by more than one mechanism.

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein.

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice.

ATP7A transgenic and nontransgenic mice are resistant to high copper exposure.

The protein affected in Menkes disease, ATP7A, is a copper (Cu)-transporting P-type ATPase that plays an important role in Cu homeostasis, but the full extent of this role has not been defined at a systemic level. Transgenic mice that overexpress the human ATP7A from the chicken beta-actin composite promoter (CAG) were used to further investigate the physiological function of ATP7A. Overexpression of ATP7A in the mice caused disturbances in Cu homeostasis, with depletion of Cu in some tissues, especially the heart.