Copper in physiology and diseases

While copper is essential throughout mammalian life and is distributed throughout the whole body, specific developmental stages and organs strongly rely on copper bioavailability. Copper is extremely important for the central nervous system (CNS) function, and must also be distributed to the liver, which regulates systemic homeostasis, and the heart as well as the placenta and mammary glands during pregnancy and lactation (Figure 11A). When delivered to various organs, copper is used as a cofactor of cuproenzymes that are either required and thus synthetized in all cells, such as CCO or SOD1, or needed for specialized tissue-specific functions, such as tyrosinase to synthetize melanin in melanocytes, dopamine-ß-monooxygenase to convert dopamine to norepinephrine in noradrenergic neurons or lysyl oxidase that cross-links tropocollagen into strong collagen fibrils required in the extracellular matrix of tissues (Lutsenko et al., 2007).

Mammals find copper in their food, since it is naturally found in numerous food products, including chocolate, nuts, mushrooms, crustaceans, soy, and gelatin (Rodriguez-Castro et al., 2015). The journey of copper in the body starts at the level of the duodenum, where it is taken at the apical side of the enterocytes lining the intestinal lumen by copper import transporters, and then distributed to the organelles (Figure 11A,C) (Nevitt et al., 2012). Copper is pumped out of intestinal cells by ATP7A-containing vesicles that fuse with the basolateral plasma membrane for release into the lymphatic vessels and eventually the portal vein circulation, especially when copper bioavailability in the intestine is high (Figure 11A,C) (Nyasae et al., 2007; Ravia et al., 2005). The dietary intake varies with age, sex and copper levels in the diet and in the body. For instance, the uptake transporter CTR1 is increased at the apical plasma membrane of enterocytes in suckling mice, highlighting an age-dependent variation in copper demand (Kuo et al., 2006). In the portal vein, copper is associated with albumin, although the ferroxidase ceruloplasmin is the main copper-associated blood protein, representing 40-70%

of the copper pool in the bloodstream (Linder et al., 1998; Linder, 2016; Nevitt et al., 2012).

Ceruloplasmin delivers copper to the tissues, in addition to exerts its oxidase function in iron metabolism (Linder et al., 1998; Linder, 2016; Nevitt et al., 2012).

The liver is the key organ in copper physiology: (i) it stores this element; (ii) it mobilizes stored copper to peripheral tissues if required; (iii) it incorporates copper into ceruloplasmin in the secretory pathway of hepatocytes, and releases it into the blood; (iv) it excretes copper into the bile in case of excess (Figure 11A,C) (Nevitt et al., 2012). Copper enters from the blood into hepatocytes at the basolateral membrane, while ATP7B mediates apical copper extrusion to the bile (Figure 11C) (Kuo et al., 2006; Ralle et al., 2010; Linder et al., 1998; Roelofsen et al., 2000). ATP7A has been suggested to send back copper basolaterally into the blood in the case of systemic copper demand, such as during organism development or altered copper availability in the diet (Figure 11C) (Lenartowicz et al., 2010; Nevitt et al., 2012). It is currently unknown how the liver and intestine sense peripheral changes in copper availability, but it has been hypothesized that the peripheral organs facing an inadequate copper supply might secrete a signal which is sensed by the copper acquisition and storage organs, similarly to what occurs for iron sensing (Nevitt et al., 2012).

Kidney is one of the organs that display the highest levels of copper, especially in the tubules and glomeruli (Linder et al., 1998; Kirby et al., 1998; Kodama, 1993). Kidney cells need copper for their regular metabolism, but also to produce two copper-associated enzymes in the secretory pathway, ceruloplasmin and diamine oxidase (Figure 11C) (Elmore et al., 2002;

Shvartsman et al., 1990; Gaitskhoki et al., 1990). Since copper is associated with proteins in the blood serum, low levels are filtered through the glomeruli, confirming that urinary excretion plays a minor role in copper excretion (Brewer, 2002; Wu et al., 2003; Nevitt et al., 2012).

CTR1 is localized at the apical side of tubule cells and imports copper from the primary urine back into the cells (Figure 11C) (Kuo et al., 2006). ATP7A pumps copper back to the bloodstream for storage or excretion in the liver, through its translocation to the basolateral membrane, especially during high copper conditions to protect renal cells against copper toxicity (Figure 11C) (Linz et al., 2008; Lutsenko et al., 2007; Nose et al., 2006; Allen et al.,

2006; Nevitt et al., 2012). Thus, the copper content of the kidney is tightly regulated, and this organ is less affected than other organs by systemic copper alterations (Linz et al., 2008;

Lutsenko et al., 2007; Nose et al., 2006; Allen et al., 2006). ATP7B is also detected in the kidney, but how it contributes to renal copper homeostasis remains to be characterized (Moore and Cox, 2002; Linz et al., 2008; Lutsenko et al., 2007).

Contraction of cardiomyocytes in the heart requires significant mitochondrial ATP production through the CCO, which needs copper to function (Nevitt et al., 2012). Cardiomyocytes also necessitate high SOD1 activity, which uses copper as cofactor, to detoxify the reactive oxygen species (ROS) generated by respiration (Nevitt et al., 2012). The uptake transporter CTR1, localized at intercalated discs, is especially important in these cells to satisfy the high demand in copper (Figure 11C) (Kuo et al., 2006; Nevitt et al., 2012).

Copper concentration in the brain is one of the highest, just below the liver concentration, and its distribution in the CNS is not uniform spatially and throughout life, highlighting variable metabolic demands (Lutsenko et al., 2010; Xiao et al., 2018). Brain critically requires a tight regulation of copper homeostasis as its development and function rely on several cuproproteins, such as the dopamine-ß-monooxygenase that converts dopamine to the neurotransmitter norepinephrine, peptidylglycine-α-amidating monooxygenase that participates in the synthesis of amidated neuropeptides (oxytocin, vasopressin,…), or the amyloid precursors suggested to be involved in synaptogenesis (Lutsenko et al., 2010; Nevitt et al., 2012). Copper also regulates myelination, even if the underlying mechanisms are unclear, and is suggested to play a protective role in neuronal excitability and related excitotoxicity (Liu et al., 2005; Schlief et al., 2006; Lutsenko et al., 2010). In addition, mitochondrial respiration requires copper-loaded CCO and ROS detoxification by superoxide dismutases which use copper as cofactor, illustrating the capital importance of copper for the CNS (Lutsenko et al., 2010). Accordingly, several neurological pathologies are associated with disrupted copper homeostasis (Lutsenko et al., 2010).

The molecular mechanisms mediating copper distribution among the different cell types in the CNS are not clear, but copper transport into the brain is mainly achieved through the blood-brain barrier, while efflux from the cerebrospinal fluid to the blood occurs at the choroid plexus through the blood-cerebrospinal fluid barrier (Figure 11C) (Choi and Zheng, 2009; Fu et al., 2014; Lutsenko et al., 2010). The highest copper accumulation was observed in the choroid plexus, which mediates blood-cerebrospinal fluid barrier, and in the locus coeruleus (LC) (Choi and Zheng, 2009; Xiao et al., 2018; Schmidt et al., 2018; Lutsenko et al., 2010). The LC is the major hub of the cuproenzyme dopamine-ß-monooxygenase, which converts dopamine to norepinephrine to modulate sleep, arousal, learning, attention, mood, and fear responses (Choi and Zheng, 2009; Xiao et al., 2018; Schmidt et al., 2018; Aston-Jones and Waterhouse, 2016; Lutsenko et al., 2010). The knowledge about how the copper handling machinery varies between the different CNS cell types is quite rudimentary, and the adaptation of the cellular copper toolkit to copper variation in the CNS seems cell-specific (Lutsenko et al., 2010). CTR1 presents a uniform expression in the brain, but it is more expressed in the choroid plexus and in endothelial cells of small blood vessels (Kuo et al., 2006; Gybina and Prohaska, 2006).

Under systemic copper deficiency, it is upregulated in the choroid plexus (Kuo et al., 2006;

Gybina and Prohaska, 2006). CTR1 is localized at the apical membrane of choroidal epithelial cells, facing the cerebrospinal fluid, and its increase upon copper deficiency is believed to enhance copper uptake into brain by restricting copper in cerebrospinal fluid (Figure 11C) (Zheng and Monnot, 2012; Zheng et al., 2012; Kuo et al., 2006; Nevitt et al., 2012; Lutsenko et al., 2010). CTR1 is also significantly detected in LC neurons, which express dopamine-ß-monooxygenase and produce norepinephrine (Figure 12) (Xiao et al., 2018). The expression of ATP7A and ATP7B in the brain evolves with the course of development, to adapt to the evolution of copper demand (Lutsenko et al., 2010; Nevitt et al., 2012). ATP7A is detected from embryonic to adult stage, suggesting its housekeeping role in the CNS (Lutsenko et al., 2010;

Nevitt et al., 2012). ATP7A is critical for CNS development, since it has a broad and high expression in the neonate brain, especially in the neocortex and cerebellum, whereas it is only enriched in blood vessels, choroid plexus, a subset of astrocytes and neurons in adult mice

(Niciu et al., 2006; Qian et al., 1998; Kuo et al., 1997; Choi and Zheng, 2009; Kodama et al., 1991; Iwase et al., 1996). In microvascular cells, ATP7A mediates copper efflux and crossing of the blood-brain barrier (Figure 11A) (Qian et al., 1998). During neurogenesis, ATP7A is initially present in neuronal cell bodies and moves to the extending axons prior to synaptogenesis (El Meskini et al., 2005). Additionally, ATP7A transcription or translocation from the TGN to the plasma membrane in neurons can be triggered by some receptors (Bohlken et al., 2009; Schlief and Gitlin, 2006; Schlief et al., 2006). The expression of ATP7B is comparable in many regions of the brain, except in Purkinje neurons where it is more important to mediate copper delivery to the ceruloplasmin produced by these cells (Barnes et al., 2005; Saito et al., 1999). Exposure of epithelial cells from the choroid plexus to excess copper triggers the relocation of ATP7A from the TGN to the apical microvilli facing the cerebrospinal fluid, while ATP7B moves to the basolateral membrane where it expels copper towards the blood (Figure 11A,C) (Fu et al., 2014). In LC neurons, both ATP7A and ATP7B regulate dopamine-ß-monooxygenase. ATP7A-mediated copper loading into the TGN supports norepinephrine synthesis, while ATP7B sequesters copper into vesicles, regulating the cytosolic copper available for ATP7A (Figure 12) (Xiao et al., 2018; Schmidt et al., 2018).

Furthermore, they oppositely modulate the constitutive export of soluble dopamine-ß-monooxygenase from resting neurons: ATP7A and copper promote its secretion, whereas ATP7B prevents it (Figure 12) (Schmidt et al., 2018).

Figure 12. Scheme illustrating the regulation of

dopamine-ß-monooxygenase by ATP7A and ATP7B in neurons of the locus coeruleus.

CTR1 imports copper (green) into the locus coeruleus (LC) neurons. ATP7A transports copper into the TGN lumen whereas ATP7B sequesters it in vesicles, regulating the amount of cytosolic copper available to ATP7A. Dopamine-ß-monooxygenase (DßM, grey circles) receives its copper cofactor through ATP7A in the secretory pathway. Holo-dopamine-ß-monooxygenase has then two fates: a fraction is sorted into vesicles and exported in the absence of neuronal stimulation (constitutive export);

another is sorted into secretory granules where it converts dopamine (Dop., brown hexagons) to norepinephrine (Norepi., yellow stars) and is released to the extracellular space in response to neuronal stimulation (regulated secretion). Based on Xiao et al. (2018) and Schmidt et al. (2018).

During pregnancy, the placenta must provide the increasing needs of copper to the fetus, and copper transport must be tightly regulated since a copper deficiency can cause abnormalities such as embryonic mortality, neonatal growth retardation or developmental defects (Nevitt et al., 2012; Lutsenko et al., 2007). CTR1 is present at the basal, fetal side of syncytiotrophoblasts to transport excess copper from the fetus to the mother and to prevent its accumulation since the fetal system might be not mature enough to cope with any copper excess (Figure 11C) (Hardman et al., 2006). Furthermore, both copper pumps are found in syncytiotrophoblasts.

ATP7A is localized at the basal side and mediates copper transport towards the fetus, whereas ATP7B is detected at the TGN, loading copper into the secretory pathway for the synthesis of cuproenzymes, and at the apical membrane to export copper to the mother (Figure 11C) (Hardman et al., 2004; Hardman et al., 2011; Hardman et al., 2007b; Hardman et al., 2007a).

Localization and protein levels of copper ATPases in the placenta are regulated by hormones such as insulin (Qian et al., 1996; Hardman et al., 2007b).

After birth, copper is essential for the neonatal development and therefore must be present into the milk produced by the mammary glands. This is translated by a 20-fold increase uptake by mammary tissue upon lactation (Donley et al., 2002). Copper is taken from the blood basolaterally by CTR1. It is then loaded into the TGN by ATP7B for the production of substantial amounts of ceruloplasmin and secretion into the milk, or is directly excreted into the alveoli by ATP7A and ATP7B which relocalize from the TGN to the plasma membrane during lactation (Figure 11C) (Kelleher and Lonnerdal, 2006; Jaeger et al., 1991; Donley et al., 2002; Michalczyk et al., 2000; Kelleher and Lonnerdal, 2003). ATP7A is also detected at the basolateral, serosal side of the cells, to also export copper back to the maternal circulation (Figure 11C) (Kelleher and Lonnerdal, 2003; Kelleher and Lonnerdal, 2006). It is currently unknown whether the translocation of the copper pumps in the mammary epithelium is regulated by the increased intracellular copper levels during lactation and/or by signaling pathways induced by hormones (Lutsenko et al., 2007).

Copper homeostasis is therefore crucial in a wide array of physiological processes. As a consequence, its disruption is associated with severe pathological conditions, especially neurological.

Menkes disease, first described in 1962 by John Menkes, is a recessive X-linked multisystemic disorder due to mutations in the gene coding for the ATP7A copper pump (Menkes et al., 1962). Its severe and more common form (more than 90% of the patients) is associated with null ATP7A mutations and manifests soon after birth (Hartwig et al., 2019;

Tümer and Møller, 2010). It is characterized by neurodegeneration and multisystemic defects, including abnormalities in the connective tissues, laxity of the skin and joints, twisted hair, hypopigmentation and reduced bone density (Hartwig et al., 2019; Tümer and Møller, 2010).

Death typically occurs before the age of three (Hartwig et al., 2019; Tümer and Møller, 2010).

An intermediate, atypical form is characterized by longer survival and/or milder symptoms of the affected patients (Tümer and Møller, 2010). The mildest manifestation of the disease is the Occipital Horn Syndrome, mainly associated with connective tissue defects, with a variable but

substantially longer life expectancy (up to 50 years) (Tümer and Møller, 2010). Rare missense mutations in ATP7A gene have also been identified in the adult-onset spinal muscular atrophy type 3, which specifically impairs the spinal motor neuron (Kennerson et al., 2010; Hodgkinson et al., 2015). The clinical manifestations of the Menkes disease are due to a multisystemic deficiency in copper (Hartwig et al., 2019). Due to ATP7A inactivation, copper cannot be pumped across the basolateral membrane of enterocytes (Figure 11A,C) towards the bloodstream (Danks et al., 1972; Camakaris et al., 1980). As a result, copper accumulates in intestinal cells and less copper is delivered to the blood, resulting in deficient copper supply to the other tissues (Danks et al., 1972; Camakaris et al., 1980). The multisystemic clinical features are attributable to impaired copper loading of diverse cuproenzymes that remain inactive, with strong neurological disorders explained by the high needs of copper in the CNS for its development and function (Figure 11A). For instance, the hypopigmentation observed in Menkes patients can be explained by the need of copper for tyrosinase activity during melanogenesis (Petris et al., 2000). Similarly, a major biochemical hallmark used for Menkes diagnosis is the increased ratio of dopamine to norepinephrine. The explanation is that brain faces a marked copper deficiency, probably worsened by the disrupted ATP7A-mediated copper entry through the blood-brain barrier, and this prevents conversion of dopamine to norepinephrine in the LC (Figure 12) (Kollros et al., 1991; Liu et al., 2005; Qian et al., 1998;

Xiao et al., 2018; Schmidt et al., 2018; Lutsenko et al., 2010). Kidneys, and especially cortical tubular cells, of Menkes patients exhibit high levels of copper, on the opposite to the systemic deficiency, since copper is imported from the primary urine into the cells but cannot be pumped back to the bloodstream (Figure 11A,C) (Lutsenko et al., 2007). Menkes disease demonstrates the importance of adequate copper supply for human development and physiology. The different degrees of clinical severity may depend on the amount of residual ATP7A and on the intracellular copper-dependent TGN-to-plasma membrane trafficking ability of the variant (Mercer, 2001; Skjørringe et al., 2017). In some severe forms, the protein is completely or partially absent, due to aberrant RNA splicing or proteasomal degradation (Mercer, 2001;

Skjørringe et al., 2017). Mutations that lead to a permanent TGN localization of ATP7A confer

severe classical Menkes phenotype, whereas mutants that keep a copper-dependent trafficking or are in post-TGN cytoplasmic vesicles display a certain activity and are associated with mild Menkes phenotype and Occipital Horn Syndrome (Mercer, 2001; Skjørringe et al., 2017). Menkes syndrome cannot be cured, but injections of copper-histidine complex can attenuate some neurological symptoms (Tümer and Møller, 2010).

Wilson disease has been described for the first time in 1912 by Kinnear Wilson and is an autosomal recessive disorder due to mutations in the gene coding for the ATP7B copper pump (Compston, 2009; Bull et al., 1993). Clinical features are variable, but include cirrhosis and chronic hepatitis, a gold-brown halo around the cornea due to copper deposition (Kayser-Fleischer ring), and a large proportion of patients (around 40%) presents neurological and psychiatric symptoms (Mercer, 2001; Lorincz, 2010). Wilson disease represents a genetic copper toxicosis, since the inactivation of ATP7B prevents the extrusion of excess copper into the bile (Figure 11A,C). This leads to a systemic and especially hepatic accumulation of copper (Rodriguez-Castro et al., 2015). ATP7B-mediated copper loading into the TGN which is required for the synthesis of cuproenzymes in the secretory pathway is also disrupted, leading to impaired production of active ceruloplasmin in hepatocytes and thus very low levels in the serum (Figure 11A,C) (Lutsenko et al., 2007; Rodriguez-Castro et al., 2015). Kidney is also affected, showing increased copper levels and producing an urine enriched in copper (Brewer, 2002; Wu et al., 2003). Neurological symptoms of Wilson patients are due to copper overload in the brain, triggered by the systemic copper accumulation and probably worsened by the inaction of ATP7B-mediated extrusion from the cerebrospinal fluid to the blood at the choroid plexus (Figure 11A,C) (Lutsenko et al., 2007; Fu et al., 2014). The CNS can be affected in several regions, including the brainstem comprising the LC, and patients with advanced disease show atrophy, spongy degeneration, increased ventricular size, cavitation and demyelination (Lutsenko et al., 2010). Wilson patients also present an altered metabolism of dopamine and norepinephrine as well as an abnormal functioning of the dopaminergic system.

Indeed, higher copper availability and inactivation of ATP7B may trigger a dysregulation of

intracellular sorting and/or boost the constitutive secretion of soluble dopamine-ß-monooxygenase in the LC (Figure 12) (Schmidt et al., 2018). Thus, the copper phenotypes in Menkes and Wilson diseases result from the cell- and tissue-types specific functions of ATP7A and ATP7B and the polarity of their transport of copper across the plasma membrane (Figure 11A,C). The natural course of Wilson disease is characterized by progressive health deterioration leading to death due to liver or neurological disorders, but treatments to prevent appearance of symptoms or clinical deterioration exist and provide a normal lifespan to patients (Rodriguez-Castro et al., 2015). Their purpose is to normalize free plasmatic copper, by limiting dietary copper, blocking the intestinal copper absorption, and removing excess of copper with chelating agents (Rodriguez-Castro et al., 2015).

Other genetic neurodegenerative disorders are linked to copper homeostasis, such as amyotrophic lateral sclerosis (ALS), Huntington disease and transmissible spongiform encephalopathy (Ackerman and Chang, 2018). In ALS, SOD1, a superoxide dismutase that requires copper as cofactor, is mutated and forms aggregates, potentially due to a lack of copper loading triggering protein instability (Sheng et al., 2012; Karch et al., 2009). A defective SOD1 leads to increased oxidative stress and neuronal death (Sheng et al., 2012; Karch et al., 2009). Notably, the evolution of the disease can be modulated by changes in dietary copper (Ermilova et al., 2005). Huntingtin is mutated in Huntington patients, forming pathological aggregates. Copper levels are increased in the corpus striatum of patients and in brains of Huntington model mice (Dexter et al., 1991; Fox et al., 2007). Aggregation of huntingtin might be mediated by copper, since the mutation of potential copper-binding residues in huntingtin prevents its aggregation in response to neural copper changes (Fox et al., 2007; Xiao et al., 2013). This suggests that copper levels directly impact disease progression. Copper also interacts with the extracellular domain of the cellular prion protein (PrP), which serves as a copper-binding buffer in the synapses and influences neural copper levels (Brown et al., 1997;

Kretzschmar et al., 2000). Mutated PrP, which becomes protease resistant and does not bind to copper, forms fibrillar extracellular aggregates that lead to cell death and spongiform

encephalopathy (Brown, 2010). PrP and brain copper levels are tightly linked, as brain PrP expression is altered by copper changes, and altering PrP expression changes the amount of copper in cells in addition to modify the expression of many other copper-binding proteins,

encephalopathy (Brown, 2010). PrP and brain copper levels are tightly linked, as brain PrP expression is altered by copper changes, and altering PrP expression changes the amount of copper in cells in addition to modify the expression of many other copper-binding proteins,