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. This article outlines some of this history.

A commonly used Drosophila model of Alzheimer's disease generates an aberrant species of amyloid-ß with an additional N-terminal glutamine residue.

Expression of human amyloid-ß (Aß) in Drosophila is frequently used to investigate its toxicity in vivo. We expressed Aß1-42 in the fly using a secretion signal derived from the Drosophila necrotic gene, as described in several previous publications. Surface-enhanced laser desorption/ionization TOF MS analysis revealed that the Aß produced contained an additional glutamine residue at the N-terminus. AßQ+1-42 was found to have increased protein abundance and to cause more severe neurodegenerative effects than wild type Aß1-42 as assessed by locomotor activity and lifespan assays. These data reveal that a commonly used model of Alzheimer's disease generates incorrect Aß peptide.

Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-ß (GSK3ß) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3ß kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3ß-dependent phosphorylation in SH-SY5Y cells.

Neuroprotective copper bis(thiosemicarbazonato) complexes promote neurite elongation.

Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, Cu(II)(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that Cu(II)(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, Cu(II)(atsm), but at a higher concentration. Induction of neurite elongation by Cu(II)(gtsm) was restricted to neurites within the length range of 75-99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM Cu(II)(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that Cu(II)(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM Cu(II)(gtsm), suggesting a potential link between Cu(II)(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes.

What can flies tell us about copper homeostasis?

Copper (Cu) is an essential redox active metal that is potentially toxic in excess. Multicellular organisms acquire Cu from the diet and must regulate uptake, storage, distribution and export of Cu at both the cellular and organismal levels. Systemic Cu deficiency can be fatal, as seen in Menkes disease patients. Conversely Cu toxicity occurs in patients with Wilson disease. Cu dyshomeostasis has also been implicated in neurodegenerative disorders such as Alzheimer's disease. Over the last decade, the fly Drosophila melanogaster has become an important model organism for the elucidation of eukaryotic Cu regulatory mechanisms. Gene discovery approaches with Drosophila have identified novel genes with conserved protein functions relevant to Cu homeostasis in humans. This review focuses on our current understanding of Cu uptake, distribution and export in Drosophila and the implications for mammals.

Copper modulates the large dense core vesicle secretory pathway in PC12 cells.

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

Presenilin promotes dietary copper uptake.

Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Metal dyshomeostasis and oxidative stress in Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia in the elderly and is defined by two pathological hallmarks; the accumulation of aggregated amyloid beta and excessively phosphorylated Tau proteins. The etiology of Alzheimer's disease progression is still debated, however, increased oxidative stress is an early and sustained event that underlies much of the neurotoxicity and consequent neuronal loss. Amyloid beta is a metal binding protein and copper, zinc and iron promote amyloid beta oligomer formation. Additionally, copper and iron are redox active and can generate reactive oxygen species via Fenton (and Fenton-like chemistry) and the Haber-Weiss reaction. Copper, zinc and iron are naturally abundant in the brain but Alzheimer's disease brain contains elevated concentrations of these metals in areas of amyloid plaque pathology. Amyloid beta can become pro-oxidant and when complexed to copper or iron it can generate hydrogen peroxide. Accumulating evidence suggests that copper, zinc, and iron homeostasis may become perturbed in Alzheimer's disease and could underlie an increased oxidative stress burden. In this review we discuss oxidative/nitrosative stress in Alzheimer's disease with a focus on the role that metals play in this process. Recent studies have started to elucidate molecular links with oxidative/nitrosative stress and Alzheimer's disease. Finally, we discuss metal binding compounds that are designed to cross the blood brain barrier and restore metal homeostasis as potential Alzheimer's disease therapeutics.

Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity.

Dyshomeostasis of extracellular zinc and copper has been implicated in ß-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the ß-amyloid precursor protein to release ß-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-month-old presenilin 1 heterozygous knock-out (PS1(+/-)) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1(+/-) brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact ß-amyloid aggregation through metal ion clearance.

The ADP-ribosylation factor 1 (Arf1) is involved in regulating copper uptake.

Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.

Copper promotes the trafficking of the amyloid precursor protein.

Accumulation of the amyloid ß peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid ß peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.

Syntaxin 5 is required for copper homeostasis in Drosophila and mammals.

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.

Conservation of copper-transporting P(IB)-type ATPase function.

Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.

Bis(thiosemicarbazonato) Cu-64 complexes for positron emission tomography imaging of Alzheimer's disease.

A bis (thiosemicarbazonato) complex radiolabeled with positron emitting Cu-64 can be used for a new and alternative method for the non-invasive diagnosis of Alzheimer's disease using positron emission tomography (PET). Most imaging agents being investigated for the diagnosis of Alzheimer's disease target amyloid-beta plaque burden but our new approach highlights altered copper homeostasis. This approach has the potential to offer complementary information to other diagnostic procedures that elucidate plaque burden.

Tissue-specific interplay between copper uptake and efflux in Drosophila.

The vinegar fly Drosophila melanogaster is proving to be an excellent system to study the in vivo regulation of the essential metal copper. The Ctr1A/B and DmATP7 copper transport proteins have well-established roles in Drosophila copper uptake and efflux, respectively. Both Ctr1A and DmATP7 are essential genes, whereas Ctr1B mutants are viable but die in excess or depleted copper conditions. Less is known about the tissue-specific requirements for these three genes and how they interact to maintain copper homeostasis in different cell types. Here, we use targeted overexpression and suppression of each gene to examine these questions in vivo. We find that in the epidermal cells that form the adult thoracic and abdominal cuticle, Ctr1A plays a major role in copper uptake, whereas Ctr1B plays only a minor supporting role and DmATP7, as previously shown, is essential for transfer of copper to the trans-Golgi network. We also find that the copper chaperone dSco1 appears necessary for supplying the mitochondria with copper in these tissues. In contrast, in the developing Drosophila eye, DmATP7 appears to be non-essential unless copper levels in these cells are artificially elevated. Again, Ctr1A is the main copper uptake gene in the eye, but when ectopically expressed, Ctr1B has greater phenotypic effects than Ctr1A. Furthermore, Ctr1A and Ctr1B show a dramatic synergistic interaction, indicating their relationship is more complicated than a simply additive one and that they may in fact act cooperatively for optimal copper import.

Control of Alzheimer's amyloid beta toxicity by the high molecular weight immunophilin FKBP52 and copper homeostasis in Drosophila.

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Abeta toxicity. Towards this goal, we generated Abeta transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Abeta and increased lifespan in Abeta flies, whereas loss of function of FKBP52 exacerbated these Abeta phenotypes. Interestingly, the Abeta pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (-/-) cells have increased intracellular copper and higher levels of Abeta. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Abeta peptides.

Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase.

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.

The multi-layered regulation of copper translocating P-type ATPases.

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.

Regulation of prion gene expression by transcription factors SP1 and metal transcription factor-1.

Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA "knockdown" of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease.

Investigating copper-regulated protein expression in Menkes fibroblasts using antibody microarrays.

Neurodegenerative illnesses are characterized by aberrant metabolism of biometals such as copper (Cu), zinc (Zn) and iron (Fe). However, little is known about the metabolic effects associated with altered metal homeostasis. In this study, we used an in vitro model of altered Cu homeostasis to investigate how Cu regulates cellular protein expression. Human fibroblasts containing a natural deletion mutation of the Menkes (MNK) ATP7A Cu transporter (MNK deleted) were compared to fibroblasts overexpressing ATP7A (MNK transfected). Cultures of MNK-transfected (Low-Cu) cells exhibited 95% less intracellular Cu than MNK-deleted (High-Cu) cells. Comparative proteomic analysis of the two cell-lines was performed using antibody microarrays, and significant differential protein expression was observed between Low-Cu and High-Cu cell-lines. Western blot analysis confirmed the altered protein expression of Ku80, nexilin, L-caldesmon, MAP4, Inhibitor 2 and DNA topoisomerase I. The top 50 altered proteins were analysed using the software program Pathway Studio (Ariadne Genomics) and revealed a significant over-representation of proteins involved in DNA repair and maintenance. Further analysis confirmed that expression of the DNA repair protein Ku80 was dependent on cellular Cu homeostasis and that Low-Cu levels in fibroblasts resulted in elevated susceptibility to DNA oxidation.