Mammalian copper biology: hitting the pause button in celebration of three pioneers and four decades of discovery

This themed issue of Metallomics showcases recent progress in mammalian and bacterial copper biology made possible by the seminal discoveries of three pioneers in our field who recently announced their retirements. Here, we pause to celebrate the many significant contributions of Professors Julian Mercer, James Camakaris and Joseph Prohaska over the last four decades, which have paved the way to our sophisticated yet evolving understanding of the impact this double-edged (metal) sword on human health and disease.

The emergence of dioxygen in the Earth's atmosphere coincided with adaptive changes to copper and iron chemistry. In particular, the oxidation of soluble Fe(II) to insoluble Fe(III) led to loss of its bioavailability until the appearance of iron chelators (siderophores) and storage proteins (e.g. ferritin); whereas oxidation of the insoluble Cu(I) to the soluble Cu(II) rendered copper bioavailable and well-suited to exploit the oxidizing power of dioxygen. The bioavailability of copper led to new biological functions, some of which were perhaps previously assumed by iron, and coincided with the evolution of multicellular organisms with extracellular matrices that could resist attack by oxygen radicals.¹ Thus copper was used initially by extracellular enzymes and evolved to become essential for life as we know it.

While there are ancient references to the use of copper for human health, it was not until the 1800s that the occurrence of copper in plant and animal tissues was documented, and the early 1900s that a physiological function for copper was established.² In the century to follow, the linking of disrupted copper metabolism to certain animal and human diseases would reveal the essential, precious and toxic nature of copper to all aspects of mammalian development, and the highly complex and sophisticated systems that have evolved for the meticulous management of this metal.

Although the copper overload condition, Wilson disease, was first described in detail in 1912 by Samuel Alexander Kinnier Wilson, the significant involvement of copper in this disease was not established until several decades later in 1948. Subsequently, in 1952, ceruloplasmin deficiency in patients was reported by Scheinberg and Gitlin and remains the basis for the current diagnostic test. In 1985 the pattern of genetic inheritance of the disease was established. Almost in parallel, copper deficiency associated with demyelinating disease in ataxic lambs was reported in 1937 by Australian veterinary scientists, and in 1962 John Menkes described a new syndrome in male infants that was characterized by brain degeneration, unusual hair (pili torti), failure to thrive and X-linked inheritance. Twenty years later, in 1972, David Danks would make the connection between the similarity in texture between the unusual hair of Menkes disease infants and the brittle wool of sheep grazing on copper deficient soil in Australia, and identified copper deficiency as the basis for Menkes disease.

Intrigued by this discovery and on opposite sides of the world, Professor Joseph Prohaska (USA) and Professors James Camakaris and Julian Mercer (Australia), would soon become key participants in a period of significant and exciting developments in the history of mammalian copper metabolism. As documented in the ensuing editorials, Prof. J. Prohaska began his career by investigating the importance of copper in brain development and continued with a biochemical approach in a career-long pursuit to illuminate the mechanisms responsible for the deleterious effects of copper deficiency. Prof. J. Camakaris began working with Prof. David Danks to identify the defect in copper metabolism in cells from Wilson and Menkes disease patients and in the mottled mouse mutants, and to investigate treatment options. The assays he developed based on the use of the radioisotope copper-64 formed the basis of a pre-natal diagnostic test. He went on to develop various model systems for studying copper metabolism, including Chinese Hamster Ovary (CHO) cells, Escherichia coli (with Prof. Barry Lee) and Drosophila melanogaster (with Dr Richard Burke). Prof. J. Mercer began working with Prof. Camakaris and Prof. Danks in the late 1970s tasked with establishing gene cloning techniques to eventually clone the gene defective in Menkes disease. It would be 20 years after Prof. Danks' discovery linking copper with Menkes disease, that Prof. Mercer would lead one of the three international groups in the isolation and cloning of the ATP7A gene, reported in Nature Genetics in 1993. So began a new era in studying mammalian copper homeostasis.

The cloning of ATP7A enabled the subsequent cloning and characterization of the ATP7B gene defective in Wilson disease, and the discovery that both encoded copper-transporting P-type ATPases was pivotal. This latter revelation was aided by knowledge of bacterial P-type ATPases as recounted in this issue by M. Solioz (C6MT00111D). Indeed, structural and functional insights into the bacterial enzymes (Argüello and colleagues, C6MT00089D) continue to inform our understanding of their mammalian counterparts. In 2010, 17 years following the cloning of the ATP7A gene, M. Kennerson and colleagues described mutations in ATP7A that were the cause of a new disease phenotype, a late onset progressive distal motor neuropathy without overt signs of systemic copper deficiency. In this issue Pérez Siles and colleagues (C6MT00082G) describe the generation of a knock-in mouse model that will prove valuable in characterizing the molecular phenotype of this motor neuropathy and in ascertaining the temporal events leading to axonal degeneration.

The seminal finding that the copper-ATPases undergo trafficking in response to copper explained how these proteins could both deliver copper to secreted cuproenzymes, and remove excess copper, and this was reported for ATP7A by the Mercer–Camakaris team in ref. 3. This finding was the key to understanding cellular and physiological copper homeostasis, and ATP7A signals governing this process continue to emerge (Zhu et al., C6MT00093B). These discoveries in the mid-late 1990s paved the way for exponential growth in our understanding of cellular copper transport and the proteins, pathways and network of interactions involved, and indeed for gaining the appreciation of the far-reaching and ever-expanding role of copper in human physiology and pathology that we have today.

Following the ATP7A/7B gene discoveries in mammalian cells, the baker's yeast, Saccharomyces cerevisiae was pivotal in further illuminating copper transport pathways and revealing additional protein components of these pathways with homologues in mammals. Other model organisms such as bacteria, zebrafish, Drosophila melanogaster and Caenorrhabditis elegans have and continue to play a part in providing important insight.

What we now know is that the versatility and diverse biological roles of copper are driven by its facile transition between its reduced (Cu⁺) and oxidized (Cu²⁺) forms. This property may also play a role in copper toxicity, and so tightly regulated homeostatic mechanisms operate in response to nutritional supply and demand. As a consequence, free, uncomplexed copper does not exist in cells (or is negligible) and at a cellular level copper handling operates at the level of uptake, storage, speciation, distribution and trafficking, and efflux. Distribution may be controlled thermodynamically (along affinity gradients), in combination with metal-mediated protein–protein interactions for copper transfer, and these will also be influenced by the conditions (e.g., pH, redox balance) within a given cellular compartment. Indeed in this issue, S. Lutsenko (C6MT00176A) provides a detailed update on recent developments in copper delivery to the secretory pathway, highlighting new concepts, proteins and pathways, and the complexities governing this process, while challenging current models of Atox1-mediated copper transfer to the copper-ATPases. Much is also known about copper physiology, with absorption of dietary copper through the intestinal mucosa regulated by copper intake, and the liver primarily responsible for regulating the copper status of the body by mobilizing copper stores and excretion of the excess.⁴ Systemic signaling mechanisms⁵ and hierarchical distribution of copper in response to copper deficiency also have been proposed.

Copper is required as a structural or enzymatic cofactor for numerous enzymes involved in vital processes such as respiration, neurotransmitter synthesis, activation of neuropeptides and hormones, antioxidant activity, tissue integrity, angiogenesis, myelination, pigmentation and iron metabolism among others. Copper is indispensable for development and function of the central nervous system (CNS), and in addition to functioning as a co-factor, copper has specific roles in the CNS in neurodevelopment, synaptogenesis and axon extension, modulation of neurotransmitter receptor activity and synaptic transmission. Copper also has a key regulatory role in a range of signaling cascades. New information on the role of copper and copper-dependent enzymes (LOX, PAM, SOD1, ceruloplasmin) in different systems (cardiac, anterior pituitary, CNS, mammary) and subcellular locations (lysosomes, endoplasmic reticulum) are also highlighted in this issue, and reveal the challenges associated with integrating our current understandings with the peculiarities of copper distribution/maldistribution under certain pathological conditions (Xiao et al., C6MT00037A; Bonnemaison et al., C6MT00079G; Hilton et al., C6MT00099A; Linder, C6MT00103C; Freestone et al., C6MT00086J; Polishchuk and Polishchuk, C6MT00058D; Concilli et al., C6MT00148C).

While fundamental advances continue to drive our understanding of copper's biological roles and transport mechanisms, research focus is also moving towards understanding the pathological role of copper and its dysregulation in neurodegenerative disease and cancer, as well as its therapeutic potential. In the last decade, copper dysregulation as a key pathological feature in motor neuropathies (e.g., amyotrophic lateral sclerosis (ALS)), prominent neurodegenerative disorders such as Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) and prion diseases, as well as cancer and diabetes has become accepted. In this issue, M. Greenough et al. (C6MT00095A) and R. Squitti et al. (C6MT00101G) provide an update on new connections between key copper transporters, copper dysregulation and pathological features of AD. Copper involvement in various pathologies has stimulated interest in the therapeutic development of lipophilic ionophoric copper compounds (hydroxyquinolines, dithiocarbamates and thiosemicarbazones) that can cross the brain barriers for restoration of neuronal function; and for targeting cancer through chelation or through exacerbating the toxic redox cycling of copper, as reviewed by D. Richardson (C6MT00105J) in this issue.

There is still much to be learned, but in the last four decades since establishing the link between the devastating Menkes disease and disrupted copper metabolism, significant progress has been achieved towards a holistic view of the necessity and danger of copper and how this essential nutrient metal is managed and mismanaged. This themed issue in copper biology spans the role of copper in health and disease, the chemistry and structural biology of key copper homeostasis proteins, the development of cutting edge copper-based imaging tools and therapies, and the model systems that have informed all facets of research on this intriguing and exciting metal in the context of mammalian copper homeostasis.

It is fitting that the release of this issue coincides with the 10th iteration of the International Copper Meeting (Copper 2016) that is supported by the Menkes Foundation, and which began in 1997 in Italy, co-founded and organised by Prof. Julian Mercer and the late Prof. Arturo Leone. This meeting has continued biennially in Italy and brings together researchers from around the world with a passion and enthusiasm for understanding all aspects of copper in biological systems.

References

1. R. R. Crichton and J. L. Pierre, BioMetals, 2001, 14, 99–112.
2. E. Underwood, Trace Elements in Human and Animal Nutrition, Elsevier, 4th edn, 2012.
3. M. J. Petris, J. F. Mercer, J. G. Culvenor, P. Lockhart, P. A. Gleeson and J. Camakaris, EMBO J., 1996, 15, 6084–6095.
4. J. R. Turnlund, Am. J. Clin. Nutr., 1998, 67, 960S–964S.
5. B. E. Kim, M. L. Turski, Y. Nose, M. Casad, H. A. Rockman and D. J. Thiele, Cell Metab., 2010, 11, 353–363.

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