Reflections of a cupromaniac

Perspectives

Mammals only contain about a dozen cuproenzymes (copper binding proteins that depend on metal for catalytic function).¹ These enzymes participate in a diverse set of redox-based reactions that span many biological processes. Mammals also possess specific copper binding proteins that control influx and efflux across membranes as well as support intracellular reactions that escort and store copper as metallothionein. Details of many of these copper binding proteins will be summarized by others. Copper research has progressed during my career, now spanning 46 years. My initial work was with brain. When I began my studies there was relatively little known about copper and brain biology; today this topic is much better understood as recently reviewed by my friend and colleague, Julian Mercer.² Further, there was little known about many of the now established cuproenzymes. For example, activity of superoxide dismutase was only reported one year prior to my graduate work, dopamine beta hydroxylase was reported only 5 years prior and peptidyl glycine alpha amidating monooxygenase was not yet discovered.

My first published manuscript on copper was 42 years ago, shortly after David Danks revealed the copper connection in Menkes disease.³ His discovery gave immediate credibility to the essential nature of copper for humans.⁴ I began my graduate work in 1970 and my first introduction to copper by testing the hypothesis that high levels of phenylalanine could interfere with copper availability. This proved to be wrong, but yielded an ethanol reward after a wager with my thesis advisor W. W. Wells who proposed the hypothesis.

This experience and copper essentiality intrigued me for my entire research career, especially the importance of copper in brain development, and was the major topic of my PhD thesis in biochemistry at Michigan State University, awarded in 1974. I continued this work for 35 years as a faculty member of the University of Minnesota Medical School, Duluth campus (UMD) following my postdoctoral training at the University of Wisconsin.

My studies of copper biology in mammals relied on the use of dietary copper restriction in rodents and the use of several genetic mutants, most notably brindled mice, characterized as copper mutants by David Hunt.⁵ These models were used to investigate the roles of copper in mammals and the causes of Menkes disease in humans, because when I began my studies no one knew of ATP7A and the copper connection with Menkes disease, but did know that brindled mice seemed a good model for Menkes steely hair disease. My work did not involve copper overload or Wilson disease models.

Early work characterized neonatal models of copper-deficient (CuD) rats and mice.⁶ This expanded to a series of studies comparing dietary CuD mice to brindled mice, hoping to unravel a factor responsible for Menkes disease.⁷ There were similarities between dietary and genetic copper deficiency. For example, the CuD mice generally had lower activities of key cuproenzymes like superoxide dismutase (SOD1) and cytochrome c oxidase (CCO) compared to brindled mice. However, the reduction in norepinephrine was more severe in young Mo^br/y mice than age-matched CuD mice deprived of copper throughout pregnancy and lactation.^7a Puzzling at that time, we now know this was due to the ATP7A dependence on copper metallation of dopamine-β-monooxygenase, the cuproenzyme that synthesizes norepinephrine. Other experiments were conducted with brindled mice but the majority of my career focused on dietary manipulation of rodents.

I focused on four major biological systems in regard to the impact of dietary copper deficiency, with emphasis on the central nervous system and studies on the cardiovascular system, the immune system and most recently the requirement of copper for iron homeostasis.

The devasting impact of Menkes disease on the infant brain, as well as the impact of CuD forage on the lamb brain, stimulated a career long effort to determine the biochemical basis of aberrant brain development in CuD mammals. Pregnancy and lactation are particularly sensitive to restriction of nutritional copper as most of the brain copper accretion occurs shortly after birth.³ In fact seminal work by others showed that adult CuD rat brain does not lose copper.⁸ The reduction in brain Cu in the developing rodents lowers the activity of a number of cuproenzymes.⁹ Noteworthy, the major impact of CuD on brain development is significant loss of cytochrome c oxidase.¹⁰ The activity and the protein abundance are greatly lower. This results in aberrant mitochondrial function including impaired electron transport, elevated citrate production, increased brain lactate, and lower fructose-2,6-bisphosphate; consistent with altered energy metabolism.^10,11 Interestingly though, our study of in vivo brain NMR of CuD rats failed to detect alterations of adenine nucleotides.¹² Further studies of the impact of CuD on brain development seem merited as neonatal restriction of copper leads to permanent behavior alterations in sensory motor function.¹³

I was fortunate to have many collaborators at UMD since we had a limited research and graduate program on campus. This was most evident in my work on CuD mice and the immune system with my friend Omelan Lukasewycz.¹⁴

We showed that CuD mice had impaired humoral and cellular immunity.¹⁵ Although we never discovered a specific mechanism, our work on IL-2 was in the right direction.¹⁶ Later work from Mark Failla extended our findings.¹⁷ This area of copper research has progressed little in recent years.

Using a similar reach out strategy when I wished to further characterize the well known impact of CuD diet on cardiac hypertrophy, I collaborated with my colleague Lois Heller.¹⁸ We characterized the impaired cardiac function of the hypertrophied hearts of CuD rats. The precise cause of the hypertrophy of CuD was challenging as we tested theories related to catecholamines and angiotensin,¹⁹ but ultimately must be due to an endogenous cardiac factor as nicely shown by Dennis Thiele's group with cardiac specific Ctr1null mice.²⁰

The essential need for copper was first demonstrated in rats by showing that copper was needed to reverse anemia.²¹ It was likely known in the 19th century that this was true for humans but never widely appreciated.²² I was interested in the copper–iron interaction issue of copper biology for a long time, ever since reporting that the brains of CuD rats have low iron.¹⁰ This was confirmed again by the careful work of my PhD student Anya Gybina with perfused brain samples.²³ It is still not certain if any of the brain and behavior deficits observed in CuD rats are due to secondary Fe deficiency. This question also seems worthy of future research endeavors.

The mechanism responsible for the severe anemia following CuD treatment of rodents is still not known. Clearly, there is more to the hypothesis than simple reduction of ferroxidase activity limiting ceruloplasmin (Cp) and hephaestin and thus iron mobilization.²⁴ Although CuD rodents have a major loss of Cp and GPI-Cp activity and protein, only in rats is there a reduction in serum iron, suggesting an alternate mechanism to explain the anemia. In fact Cp null mice are only mildly anemic and often have no change in serum iron.²⁵ Importantly, there must be some factor specific to pregnancy that impacts iron biology and hepcidin, a major signaling peptide in iron homeostatsis, as CuD dams respond differently than FeD dams during anemia.²⁶ Clearly this is an important area for further investigation.

As a biochemist I always hypothesized that the pathophysiology of Cu deficiency could be explained by reductions in activity of cuproenzymes with subsequent changes in metabolism when those changes became rate limiting.

This was the theory. Proving this was a career challenge. Here are a few examples.

SOD1 activity was lower following CuD treatment as first reported for rat brain.³ However no evidence of enhanced lipid peroxidation was found. Despite a major loss of CCO activity in the rodent brain, energy charge was not impacted.^10,27 It is still not certain if the reduction in brain norepinephrine due to limitation in DBM activity, first reported in rats, is sufficient to explain the neuropathology of Cu deficiency.³ Does the impact of CuD diet on levels of GPI-anchored Cp impact Fe mobilization?²⁸

One of the challenges of future mammalian Cu research will be the discovery of mechanisms responsible for the deleterious affect of Cu limitation. One factor limiting the assessment of Cu status in humans has been the availability of a suitable biomarker besides serum Cu (mainly ceruloplasmin). Because Cp is an acute phase protein, Cu assessment during inflammation becomes problematic. We hope our work on the impact of CuD diet on a potential biomarker for a Cu, namely the copper chaperone for superoxide dismutase (CCS), will continue.²⁹ This protein can be easily detected by immunoblot technology and is not impacted by inflammation. Detection of CuD humans is important as failure to do so can lead to permanent neurological damage.¹

Mammalian copper biology owes a great debt to the pioneer research in budding yeast conducted by Andrew Dancis, Dave Eide, Dennis Thiele, Dennis Winge, Val Culotta and others who discovered some of the fundamentals of copper transporters, copper chaperones, and accessory proteins. Also many others who use unique model systems, such as my friends Jim Camakaris with Drosophilia and Jonathan Gitlin with zebrafish, that lead the way for future discovery. At the 9th International copper meeting in 2014 I was asked to give a historical perspective on my career. I chose “Consequences of Copper Deficiency: What I Did and What Yet To Do”. There is much yet to do.

Acknowledgements

My career accomplishments would not have been possible without the outstanding scientific support of many technicians and students; as well as the generous financial aid of the US Public Health Service and Department of Agriculture.

References

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