Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

1Copper is indispensable for normal development and function of the central nervous system (CNS). The copper concentration of the human adult brain is significant, estimated at 7–10% of total body copper, similar to that of the liver, the major organ that regulates the copper status of the body. Regional variance in brain copper concentrations reflects differing metabolic requirements for copper, which change during development (reviewed in ). Copper is required as a cofactor for numerous critical enzymes that are involved in vital CNS processes such as respiration, neurotransmitter synthesis, activation of neuropeptides and hormones, protection from oxidative damage, myelination, pigmentation, and iron metabolism among others. The redox cycling of copper between Cu2+ and Cu+ oxidation states is utilized by enzymes involved in these processes for catalytic reactions. However, this redox activity also can lead to elevated reactive oxygen species (ROS) and corruption of critical proteins by adventitious binding of copper ions. High oxygen consumption in the brain (20% of total body oxygen), coupled with low levels of antioxidants and antioxidant enzymes, and high levels of metal ions, mean the brain is particularly susceptible to ROS-induced oxidative stress. Hence, precise regulation of brain copper is essential to ensure appropriate levels and distribution for the maintenance of brain function, without risking inadvertent interactions with other cellular components.

2Much of what is currently known about brain copper regulation comes from studies of diseases where copper dysregulation is associated with neurodegeneration. Menkes disease (MD; OMIM 309400) is an X-linked inherited disorder with serious neurological symptoms and neurodegeneration resulting from severe copper deficiency. Occipital horn syndrome (OHS; OMIM 304150) is an allelic variant of MD with milder neurological symptoms and predominantly connective tissue defects. In Wilson disease (WD; OMIM 277900), an inherited, autosomal recessive copper toxicosis disorder, patients present with hepatic and neurological symptoms (reviewed in ). There is mounting evidence that copper dysregulation plays a key role in more common neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, motor neuron, and prion diseases (reviewed in ).

3The mechanisms of brain copper import, distribution, and export are now beginning to be elucidated. The exchange of copper between the periphery and the brain is highly regulated by the brain barriers. The copper concentration of cerebrospinal fluid (CSF; ~0.25 μM;; ) is up to 100-fold lower than that in the plasma (11–25 μM; ). In a rat brain perfusion study that compared copper uptake into brain capillaries, parenchyma, choroid plexus, and CSF, non-protein bound free copper ion was the predominant copper species that entered the brain via both the blood–brain barrier (BBB) and the blood–CSF barrier (BCB; ). The higher rate of copper transport into the brain parenchyma compared to the CSF suggests that the BBB is the main site through which copper enters the brain. Copper influx into the brain parenchyma and CSF is regulated by copper transporters CTR1, ATP7A, and ATP7B, which are highly expressed in the brain capillaries and choroid plexus.

4Significant insight into the mechanisms controlling brain copper homeostasis began two decades ago with the identification of the genes encoding the essential copper-transporting ATPases, ATP7A and ATP7B. Mutations in ATP7A and ATP7B underlie MD and WD, respectively. ATP7A is located on chromosome Xq13.2-13.3 and comprises 23 exons that span approximately 150 kb1. ATP7B is located on chromosome 13q14.3 and comprises 21 exons that span approximately 80 kb2. Transcripts of approximately 7.5–8.5 kb are produced from both genes and contain coding regions of 4.5 kb, which are translated to produce proteins of 180 and 165 kDa, respectively. ATP7A and ATP7B are members of the P1B-subfamily of the P-type ATPases. They undergo ATP-dependent cycles of phosphorylation and dephosphorylation to catalyze the translocation of copper across cellular membranes. Their structure and biochemistry was thoroughly reviewed by. They are highly related in structure and function with approximately 60% amino acid identity. They have eight transmembrane domains that form a path through cell membranes for copper translocation; and a large N-terminus with six metal-binding domains (MBDs), each comprising approximately 70 amino acids and the highly conserved metal-binding motif GMxCxxC (where x is any amino acid). Other highly conserved domains include the intramembrane CPC motif that is required for copper translocation through the membrane, the N-domain containing the ATP-binding site, the P-domain containing the conserved aspartic acid residue and the A-domain comprising the phosphatase domain. Copper-binding together with other N- and C-terminal signals regulate their activity, intracellular location, and copper-induced intracellular trafficking (see below and reviewed in;; Figure 1).

5ATP7A and ATP7B possess dual functions, delivering copper for incorporation into copper-dependent enzymes, and removal of excess copper from cells. These functions are largely regulated by their sub-cellular localization (see below). ATP7A is ubiquitously expressed in extrahepatic cells and tissues, which explains the systemic defects caused by its absence or inactivation in MD and points to a house-keeping role for ATP7A. ATP7B has a more limited expression pattern, with the highest expression level in the liver, and lower levels in the kidney, placenta, brain, heart, and lungs. This restricted expression suggests more specialized functions for ATP7B in regulating copper physiology, such as biliary copper excretion. ATP7B also has a biosynthetic role, supplying copper to cuproenzymes such as ceruloplasmin. In cells where ATP7B is co-expressed with ATP7A, it often has a specific and distinct role, for example in copper secretion into milk during lactation, and in fine-tuning the intracellular copper balance in the kidney. The expression of both copper-transporting P-type ATPases (copper-ATPases) in the brain and the severe neurological symptoms that arise from a deficiency of either transporter, suggest that they play key roles in regulating brain copper homeostasis. This review will summarize our current knowledge of the expression, localization, and contribution of ATP7A and ATP7B to maintaining and managing copper levels within the brain.

6The ATP7A gene is transcribed to produce an 8.5 kb transcript that is expressed in all tissues examined except for the liver. In the mouse brain, the Atp7a transcript is expressed in the cerebrovascular endothelial (CVE) cells that form the BBB, and is strongly expressed in the choroid plexus, a structure that forms the BCB and regulates the concentration of substances in the CSF (Figure 2A). further showed that Atp7a is more highly expressed in the brain barriers (BBB and BCB), the brain capillaries and choroid plexus, than in brain parenchyma. Comparing the two barriers, Atp7a mRNA expression is 3.4-fold higher in the choroid plexus than in the cerebral capillaries. This observation is consistent with the finding that the Atp7a protein levels in the developing and adult mouse brain are highest in the choroid plexus/ependymal cells of the lateral and third ventricles.

7Based on abnormal copper accumulation in cultured astrocytes from the macular mouse, a model of MD, proposed that astrocytes play an important role in copper transport from the blood and CSF toward neurons, and that this pathway is disturbed in MD and animal models (Figure 2B). later confirmed the expression of mouse and rat Atp7a in astrocytes from various brain regions (cerebral cortex, corpus striatum, and cerebellum). Therefore, ATP7A is postulated to play a key role in the copper distribution from astrocytes to neurons. Importantly, the successful treatment of MD mouse models by intravenous administration of copper earlier than postnatal day 7 (P7; ) was proposed to be a consequence of the immaturity of the BBB, which includes astrocytes. This immaturity allows the penetration of copper that is prevented in these ATP7A-deficient models once development of the BBB is complete.

8Early in situ hybridization studies in mouse brain showed significant levels of the Atp7a transcript in the hippocampal CA1 region, the dentate gyrus, the cerebellar granular layer and the olfactory bulb, and lower levels in the hippocampal CA3 region and Purkinje neurons. Atp7a mRNA was detected also by RT-PCR in mouse and rat cerebral cortex and cerebellum, and in isolated rat cerebellar granule neurons. Consistent with these observations, a detailed immunohistochemical study by demonstrated the presence of the Atp7a protein in most CNS cell types at P11 in the developing mouse brain and in the adult brain. In transgenic mice that overexpressed human ATP7A, the protein is primarily produced in the CA2 region of the hippocampus, the Purkinje neurons of the cerebellum, and in the choroid plexus. The overexpression of ATP7A resulted in an overall reduction of brain copper concentrations, which is consistent with ATP7A in the choroid plexus functioning to efflux copper back into the circulation. In more recent studies of human brain tissue, ATP7A protein levels are most prominent in the cerebellum and substantia nigra. The significance of ATP7A expression in these brain regions is poorly understood.

9ATP7A expression is developmentally regulated. The widespread expression of Atp7a mRNA in neurons and ependymal cells during embryonic and postnatal development in the mouse suggests a house-keeping role for ATP7A in the brain and CNS. Interestingly, Atp7a is not detectable by RT-PCR in embryonic day 20 rat astrocytes, but it is detectable in P3, P8, and adult astrocytes. The study by showed that Atp7a protein levels are most abundant in the early postnatal period, peaking in the neocortex and cerebellum at P4. From birth (P0) to adulthood, there is a decline in Atp7a levels in most brain regions, and this decline is more pronounced in the hippocampus and cerebellum than in the hypothalamus. During this postnatal period, despite a general decline in Atp7a levels, there is increased Atp7a expression in CA2 hippocampal pyramidal cells and cerebellar Purkinje neurons. The authors proposed that since the CA2 region is resistant to epileptogenesis, the increase in CA2 Atp7a levels may contribute to seizure resistance. The observed increase in Atp7a levels in Purkinje neurons is consistent with significant levels of ATP7A in human cerebellar Purkinje neurons, but it is inconsistent with other reports of a postnatal decline in mouse Atp7a mRNA and protein levels in these cells. Although reported a postnatal switch in the expression of Atp7a from Purkinje neurons to Bergmann glia in the rodent brain, in human brain tissue, ATP7A could not be detected in the Bergmann glia. This discrepancy may allude to the possibility that different mechanisms regulate human and rodent cerebellar copper homeostasis. The increased ATP7A expression in the cerebellum may explain the increased sensitivity of this region, and in particular Purkinje neurons, to copper deficiency as observed in MD.

10In the olfactory system, neuronal Atp7a localization correlates with neuronal maturation. Atp7a is initially concentrated in neuronal cell bodies at early embryonic stages, then shifts to the extending axons during the postnatal period. Atp7a levels peak prior to synaptogenesis, which occurs postnatally. Similarly, increased expression levels precede synapse formation following injury-stimulated neurogenesis. Together with a follow-up study of the mottled brindled mouse model of MD (MoBr/y), these data suggest a role for ATP7A in axon extension and synaptogenesis, the absence of which may contribute to the neurodegeneration evident in MD and its mouse models.

11The increase in ATP7A during the early postnatal period indicates a crucial role for copper during early development, and particularly in synaptogenesis. This may underlie the critical window during which some human MD patients and mouse models (the mottled mouse mutants) respond favorably to postnatal copper injections, especially when administered in the early postnatal period. reported that this successful copper treatment depends on the presence of a mutant ATP7A protein with some residual copper transport function. Hence, the therapeutic efficacy of copper injections may be a consequence of increased levels of mutant ATP7A, albeit with significantly reduced catalytic activity.

12A zebrafish mutant, calamity, with an embryonic-recessive lethal mutation in the ATP7A ortholog, shows impaired copper homeostasis with absent melanin pigmentation, defective notochord formation, and neurodegeneration in the hindbrain region of the developing brain. These authors proposed a developmental hierarchy of copper metabolism, where notochord formation is preferentially preserved during copper limitation, potentially explaining some of the vascular and neurologic abnormalities observed in MD. Drosophila has a sole ortholog of ATP7A, DmATP7, which appears to play an important role in the developing Drosophila brain. It is strongly expressed in the larval brain at different developmental stages. Strong DmATP7A expression is observed in the ventral ganglion but it is absent from most of the optic lobes.

13Although ATP7B is expressed in the brain, its expression patterns and contribution to brain copper homeostasis are less well characterized than that of ATP7A. In the brain of the developing mouse embryo, Atp7b mRNA cannot be detected, suggesting that there may be no prenatal expression or that expression is below the detection limits. Alternatively, consistent with the possibility that significant expression begins postnatally, brain copper levels in an Atp7b null mouse continues to increase slightly throughout adult life. In a study of Atp7b protein and mRNA distribution in the rat brain, Atp7b is detected in the hippocampus, the granular cells of the dentate gyrus and pyramidal cells of the CA1 to CA4 layers, the glomerular cell layer of the olfactory bulbs, Purkinje neurons of the cerebellum, pyramidal neurons of the cerebral cortex, and cores of several nuclei (e.g., pontine nuclei and lateral reticular nuclei) in the brainstem. In these brain regions, both Atp7b mRNA and protein correlate with copper distribution as determined by staining with the copper chelator bathocuproine disulfonic acid (BCS). Based on similar distribution patterns of cuproenzymes such as dopamine-β-hydroxylase (DBH) and Cu–Zn superoxide dismutase (Cu–Zn SOD), as well as abnormal catecholamine synthesis in the Long-Evans Cinnamon (LEC) rat model of WD, these authors speculated that ATP7B-mediated control of copper homeostasis in these brain regions is important in regulating DBH activity.

14In contrast to mouse Atp7a, there is continuous Atp7b expression in the adult mouse cerebellar Purkinje neurons, and an age-dependent down-regulation. Based on kinetic studies and experiments in mice lacking Atp7b, these authors proposed a homeostatic role for ATP7A in maintaining intracellular copper at a certain level, and a biosynthetic role for ATP7B mediating the synthesis of copper-dependent enzymes such as ceruloplasmin.

15In the human brain, immunohistochemical staining reveals expression of the ATP7B protein in the visual cortex, anterior cingulate cortex, body of caudate, putamen, substantia nigra, and cerebellum, with the most significant levels of ATP7B detected in the cerebellum, anterior cingulated cortex, and caudate putamen. Strong staining of ATP7B in Purkinje neurons and not the Bergmann glia is consistent with the findings in the adult rat and mouse brains. However, there is no correlation between ATP7A and ATP7B protein levels and copper levels in the brain regions investigated.

16Much of what is currently known about the copper-ATPases derives from studies in peripheral cells and tissues. Emerging studies of copper transport and the copper-ATPases in the brain and certain CNS cell types both support our current understanding of their mechanism of action and regulation, but also provide new insights into the complexity of copper’s role in the CNS.

17ATP7A and ATP7B have a dual role in cells; a biosynthetic role, delivering copper to the secretory pathway for metallation of cuproenzymes, and a homeostatic role, exporting excess copper from the cell. Under normal, physiological conditions, ATP7A and ATP7B are localized at the trans-Golgi network (TGN) where they provide copper to copper-dependent enzymes synthesized in the secretory pathway. ATP7A has a role in the metallation of enzymes such as peptidylglycine α-amidating monooxygenase (PAM;; ), tyrosinase, extracellular SOD3, DBH, and lysyl oxidase, all of which are expressed in the CNS. ATP7A-mediated copper delivery to lysyl oxidase is proposed based on similar temporal expression of these two proteins in the developing rat brain and the sensitivity of lysyl oxidase-dependent processes to ATP7A inactivation as evident in MD and OHS. Copper delivery to apo-ceruloplasmin in hepatocytes and mouse cerebellum is mediated by Atp7b, and by Atp7a in macrophages in response to hypoxia-mediated increased copper uptake.

18The key mechanism by which the copper-ATPases regulate cellular copper levels involves alteration of their cellular localization in response to changes in cytoplasmic copper concentration. When intracellular copper levels are elevated, ATP7A and ATP7B traffic from the TGN to the cell periphery to export excess copper from cells. The copper-induced trafficking of ATP7A was first described in Chinese hamster ovary (CHO-K1) cells, and since then, it has been reported in a wide range of non-polarized (e.g., human fibroblasts, HeLa, and mammary carcinoma cells;;;;;,; ) and polarized cell types (Madin–Darby canine kidney (MDCK), mouse enterocytes, Caco-2;;; ). Trafficking of ATP7B also has been observed in non-polarized hepatoma, human bladder carcinoma, and CHO-K1 cells, and in polarized hepatic cells (HepG2 and WIF-B;;; ).

19In polarized cells, ATP7A traffics from the TGN to a rapidly recycling vesicular pool located near the basolateral membrane, whereas ATP7B traffics to sub-apical vesicles. At these locations they sequester and mediate the export of excess copper. When copper levels return to normal, ATP7A and ATP7B recycle back to the TGN. The trafficking and recycling of the copper-ATPases requires specific signal sequences (Figure 1). For both ATP7A and ATP7B, only MBD 5 or 6 is necessary and sufficient to mediate copper-stimulated trafficking from the TGN to the cell periphery. C-terminal di- and tri-leucine motifs in ATP7A and ATP7B, respectively, are required for retrograde trafficking of the proteins to the TGN when copper levels return to normal, and for basolateral targeting of ATP7A in polarized MDCK cells. TGN retention of ATP7A is mediated by a 38 amino acid sequence contained within transmembrane domain three whereas ATP7A retention at the basolateral surface may require the PDZ-binding motif (D1497TAL) within the C-terminus. In ATP7B, a nine amino acid sequence (F37AFDNVGYE)in the N-terminus is essential for TGN retention under low copper conditions and for apical targeting in polarized hepatocytes when copper levels are elevated. Although details of the trafficking machinery involved in these processes remain to be fully elucidated, interacting partners that may play a role include AIPP1, a PDZ domain-containing protein that may bind to the PDZ motif in ATP7A, and dynactin subunit p62 that may be involved in ATP7B trafficking.

20In addition to the N- and C-terminal signals that control the copper-ATPase response to elevated copper, other processes also contribute to regulating copper-ATPase activity. They include, posttranslational modifications such as transient auto-phosphorylation at the invariant aspartate residue (D1044KTGT; Figure 1) to form an acyl-phosphate intermediate, a process that is characteristic of the P-type ATPase catalytic cycle, copper-stimulated phosphorylation by kinases, and protein interactions. ATOX1 was the first protein shown to interact in a copper-dependent manner with the N-terminal domain of both proteins for delivery of copper to the secretory pathway. MBD 1–4 is most important for this interaction, in particular MBD 2 and 4. This is supported by studies in the Atox1 null mice, which demonstrated a critical requirement for Atox1 in Atp7a function and copper homeostasis. A 45 kDa isoform of the promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor of Hox genes, interacts with the C-terminus of ATP7B. Both proteins co-localized within the TGN, and based on experiments in HepG2 cells and Drosophila, this interaction is proposed to have a role in regulating ERK signal transduction. However, the functional consequences of these associations remain to be elucidated. The glutaredoxin1 (GRX1)/glutathione (GSH) system may play a key role in regulating the trafficking and activity of the copper-ATPases by regulating their redox state through glutathionylation and deglutathionylation by GRX1. GRX1 protects proteins from oxidative damage, and it is expressed throughout the brain with highest activity in the hippocampus, cortex and midbrain. Hence, GRX1/GSH may preserve the integrity and function of ATP7A and ATP7B during oxidative stress, which accompanies neuropathological processes. Clusterin (ApoJ) and COMMD1 regulate the degradation of misfolded and mutant copper-ATPases via the lysosomal and proteasomal pathways, respectively.

21Information on the activity of the copper-ATPases in specific CNS cell types is limited and somewhat fragmentary, but emerging evidence is revealing specific roles for ATP7A and ATP7B in brain and CNS copper homeostasis. Peripherally, ATP7A and ATP7B mostly have cell type-specific expression patterns and where co-expressed in certain cells and tissues (e.g., kidney, placenta, and mammary gland), they have distinct roles (reviewed in and ). Similarly in the CNS, ATP7A and ATP7B may be uniquely expressed in certain cell types and co-expressed in others.

22The BBB and BCB regulate the movement of copper between the brain and peripheral circulation. In both choroid plexus epithelial cells and capillary endothelial cells of the brain, ATP7A predominantly localizes to the basolateral membrane, which is consistent with its location in other polarized epithelial cell types such as intestinal enterocytes. In contrast, ATP7B is concentrated at the apical membrane, again consistent with its localization in polarized hepatocytes. This distinct membrane localization of ATP7A and ATP7B combined with their discrete enzyme kinetics may be a mechanism to ensure strict control over copper transport across the BCB and BBB. The kinetically slower ATP7B at the CSF-facing apical membrane of choroid plexus epithelial cells may explain the slower rate of copper transport into the CSF relative to the choroid plexus copper uptake rate, and may contribute to copper sequestration in the choroid plexus. In contrast, the basolateral location of the kinetically faster ATP7A facilitates the removal of excess copper from the brain into the blood. This arrangement of the copper-ATPases is similar to that in other tissues such as the mammary epithelium or the kidney where ATP7A serves to protect from copper excess while ATP7B serves a biosynthetic role. Therefore, the BCB serves as the main pathway for eliminating excess brain copper (Figure 2). In a further study that utilized cultured choroidal epithelial Z310 cells, siRNA-mediated knockdown of ATOX1 and ATP7A resulted in increased cellular copper retention, confirming the involvement of ATP7A in copper transport across the choroid plexus and hence in regulating copper homeostasis of the CSF. The BBB, which regulates copper influx into the brain, has lower levels of ATP7A at the basolateral surface of brain capillary endothelial cells to enable rapid but limited transport of copper into the brain to accommodate sudden changes in brain copper concentration.

23Astrocytes play a pivotal role in brain copper homeostasis. They are strategically sandwiched between endothelial cells of the BBB and neurons, hence, they are the first brain parenchyma cells to encounter metal ions that cross the BBB. Astrocytes are able to efficiently take up and store copper, and with greater resistance to copper-induced toxicity, they protect neurons from copper toxicity (reviewed in; ). Atp7a has been detected in rodent astrocytes in culture and in brain tissue. These studies implicate Atp7a in copper export from astrocytes, which is consistent with the early observations by of copper accumulation in astrocytes of the macular mouse (Figure 2B). In astrocytes, similar to peripheral cell types, Atp7a localizes to a perinuclear region and undergoes copper-responsive trafficking between the TGN and the cell periphery. Therefore, it was proposed that Atp7a mediates copper export from astrocytes, which is time-, concentration- and temperature-dependent. However, in contrast to previous studies, copper export from cultured astrocytes is non-saturable and does not follow the established Michaelis–Menten kinetics for ATP7A-dependent copper transport. Therefore, additional ATP7A-independent mechanisms may be involved in copper export from astrocytes. The rate of copper export is proportional to the increase in copper content after copper exposure, which led to propose that the copper export rate is determined by the trafficking or fusion of Atp7a-containing vesicles with the plasma membrane, rather than by ATP7A-mediated transport of copper into vesicles.

24Cerebellar Purkinje neurons are highly sensitive to copper deficiency during development. In the human brain, ATP7A and ATP7B are co-expressed in Purkinje neurons, and in the mouse brain, for a postnatal period of up to 2 weeks. Co-expression of both copper-ATPases may be a mechanism to achieve a delicate balance of copper that is critical to cerebellar development and function. Based on the faster kinetics of copper transport by Atp7a relative to Atp7b, a homeostatic role is proposed for Atp7a in maintaining the intracellular copper level, and a biosynthetic role for Atp7b to mediate the synthesis of copper-dependent enzymes such as ceruloplasmin.

25Hippocampal ATP7A plays a pivotal role in learning and memory. In mature rat primary hippocampal neurons, Atp7a undergoes both copper-dependent and -independent redistribution from the TGN to dendrites and axons. The study by provides the first evidence of copper-independent and reversible trafficking of Atp7a that (i) is stimulated by N-methyl-D-aspartate (NMDA) receptor activation, (ii) requires Ca2+ influx through the NMDA receptor, and (iii) is associated with the rapid release of copper from hippocampal neurons (Figure 2A). The inability to detect Atp7a at the cell surface prompted the suggestion that copper may be released by exocytosis from vesicles once Atp7a has recycled back to the TGN. This possibility is consistent with the rapid efflux of copper following NMDA receptor activation. The authors suggest that NMDA receptor activation-mediated trafficking of copper-loaded, ATP7A-associated vesicles provides a readily available and releasable pool of copper following Ca2+ influx. In the hippocampus, following neuronal depolarization, about 15 μM of copper is released into the glutamatergic synaptic cleft from synaptic vesicles. While the physiological significance of synaptic copper release is not yet clear, it is proposed that copper down-regulates NMDA activity through redox processes that involve S-nitrosylation of specific cysteine residues within NMDA subunits NR1 and NR2A, thus protecting hippocampal neurons from excitotoxicity. These findings link ATP7A and copper with modulating memory and learning processes that depend on precise regulation of NMDA receptor activity. In MD, deficiencies in both brain copper and ATP7A activity will prevent copper release, thus impairing NMDA receptor modulation. The subsequent NMDA-mediated excitotoxicity may contribute to the pathological features, seizures and neuronal degeneration, that are characteristic of MD. In Alzheimer’s disease (AD), the combination of increased copper and Aβ [the amyloidogenic cleavage product of the amyloid precursor protein (APP)] in the synaptic cleft may promote the formation of neurotoxic Aβ-copper oligomers contributing to the neuropathology of AD.

26The recent identification of a new ATP7A-related distal hereditary motor neuropathy (dHMN), caused by mutations in ATP7A (see below), has drawn attention to the role of ATP7A in motor neurons. The cell bodies of motor neurons are located within the anterior horn of the spinal cord, and their axons extend for long distances to distal limb muscles. Hence, both the CNS and peripheral nervous system are affected in motor neuron disorders. In undifferentiated NCS-34 cells (a motor neuron-enriched cell line), Atp7a is localized at the TGN and traffics to the cell periphery with elevated copper, as it does in most other cell types. In differentiated NSC-34 cells, Atp7a can be detected along the full length of Tau-1-positive neuritic projections, with localization to the axonal membrane when copper levels are elevated. Based on the data presented, ATP7A was postulated to traffic along axons, and to mediate copper release from the axonal membrane of motor neurons.

27Retinal degeneration is observed in both MD and WD. The essential requirement for copper and ATP7B for retinal structure integrity is underscored by reduced thickness of total macula as well as ganglion cell and inner plexiform layer in WD patients. Up to 50% of WD patients have abnormal visual evoked potentials (VEPs; ). Immunohistochemical studies of mouse and human retina identified the presence of ATP7A in the retinal pigment epithelium (RPE), outer plexiform layer (OPL), and ganglion cell layer (GCL) of the BALB/c mouse retina. In contrast, ATP7B expression is restricted to the RPE. Within the RPE, both copper-ATPases localize to a perinuclear region that overlap with TGN and Golgi markers, consistent with their biosynthetic role in delivering copper to tyrosinase for melanogenesis, and to the iron homeostatic proteins ceruloplasmin and hephaestin. Increased copper levels trigger a redistribution of ATP7B to a diffuse cytoplasmic compartment in an immortalized human RPE cell line.

28Activated microglial cells concentrate at sites of neuronal damage and inflammation. Atp7a expression is elevated in activated microglia surrounding Aβ plaques in the TgCRND8 transgenic mouse model of AD. Data from cultured mouse BV-2 microglial cells suggest Atp7a expression and trafficking is responsive to stimulation by the pro-inflammatory cytokine interferon-gamma (IFN-γ). Concomitant copper accumulation due to upregulation of the copper import protein, Ctr1 and copper uptake, suggests a role for copper in the pro-inflammatory pathway. Furthermore, the IFN-γ-stimulated ATP7A redistribution differs from that induced by copper, and appears to mediate copper sequestration in cytoplasmic vesicles rather than copper export. This mechanism is postulated to represent a protective mechanism by activated microglia at sites of amyloidogenesis to reduce aberrant copper-Aβ interactions in the extracellular milieu.

29Pineal night-specific ATPase (PINA) is a novel splice variant of ATP7B, lacking the N-terminal MBDs and the first four transmembrane domains. It has a predominant nocturnal expression in the pineal gland and retina, under the control of the retina-specific protein, cone rod homeobox (CRX; ). Despite a large N-terminal truncation, PINA retains a weak copper transport function in a Saccharomyces cerevisiae ccc2Δ complementation assay. This finding implicates a yet-to-be-defined role for PINA-mediated copper metabolism in pineal and retinal circadian function.

30Neuroblastoma cells have a high demand for copper for proliferation. High concentrations of copper in neuroblastoma cells are likely achieved via down-regulation of ATP7A expression and thus, reduced copper efflux. Retinoid treatment of neuroblastoma cells causes transcriptional upregulation of ATP7A and an increase in ATP7A protein levels. Ectopic expression of the retinoic acid receptor β2 subtype (RARβ2) also induces ATP7A expression, which is associated a growth inhibitory effect. Conversely, knockdown of ATP7A is associated with reduced copper efflux and increased viability of retinoid-treated cells. These data support a model where malignant neuroblastoma cells have a high copper-dependency for viability and proliferation, and copper depletion by retinoid/RARβ2-induced ATP7A upregulation offers therapeutic benefit.

31An appreciation of the role of ATP7A and ATP7B in brain copper management derives largely from the functional consequences of their absence or inactivation in the genetically inherited diseases, MD, OHS, WD, and the recently identified ATP7A-related motor neuropathy. The importance of their correct function is also emerging in the context of other neurological diseases where there is perturbed copper metabolism, such as the prion diseases and AD. Our current understanding of the copper-ATPase contribution to brain copper homeostasis and the consequences of their absence or impaired function is summarized below.

32The widespread expression of ATP7A accounts for the systemic defects that arise from mutation of this gene in MD. This X-linked disease presents in males within the first few months of life, and in severe cases, it is fatal within 2–3 years. The severity of the clinical presentation can vary, but commonly includes abnormal neurodevelopment, seizures associated with cerebral atrophy and dysmyelination, a range of connective tissue and vascular abnormalities, fragile bones, an unusual kinky hair structure (pili torti), hair and skin pigmentation defects and failure to thrive (reviewed in; ). These symptoms arise from impaired intestinal copper absorption leading to systemic copper deficiency and consequently, reduced activity of critical copper-dependent enzymes. Paradoxically, copper retention is evident in certain tissues exposed to the limited copper that reaches them and in MD patient skin fibroblast cells. Histochemical analysis of brain tissue from affected MD patients revealed neurodegeneration in the cerebral cortex, cerebellum, and hippocampus where ATP7A is enriched (see above).

33Occipital horn syndrome is also caused by mutations in ATP7A, but it is a milder disease with primarily connective tissue defects and moderate neurological symptoms. Causative mutations are often splice site mutations that result in reduced levels of normal ATP7A mRNA (reviewed in ). The milder phenotype suggests that sufficient residual functional ATP7A is produced, but the prominent connective tissue defects indicate that copper delivery to lysyl oxidase is severely disrupted.

34The neurological symptoms of MD and OHS have been attributed to impaired ATP7A-mediated copper transport across the BBB (Figure 2B), leading to deficiencies of enzymes such as cytochrome c oxidase, SOD1, BDH, PAM, lysyl oxidase, and tyrosinase, some of which require ATP7A for metallation in the TGN. The bioactivity of many neuropeptides is dependent on their amidation by PAM, while DBH is important for the production of the neurotransmitter norepinephrine from dopamine (reviewed in; ). However, impairment of other ATP7A-mediated functions also is likely to contribute to the neurodegeneration in MD. For example, excitotoxicity due to impaired synaptic copper release, which down-regulates NMDA receptor activity, can contribute to seizures and neuronal degeneration (,; Figure 2). In MoBr/y mice, the absence of a functional ATP7A protein results in degeneration of Purkinje neurons, cytoskeletal abnormalities, and impaired synaptogenesis and axonal outgrowth.

35There are a number of MD mouse models. As noted in the preceding sections, these mouse models have been invaluable in revealing insights into the critical role of ATP7A in the brain. The mottled mice are a series of mouse mutants with mutations in the murine ortholog of ATP7A. They exhibit a range of phenotypes that recapitulate the variable clinical severity of the human disease. The MoBr/y mouse model resembles most closely classical MD with neurological deficits and early postnatal lethality. This mouse expresses close to normal levels of Atp7a that has severely reduced copper transport activity due to an in-frame deletion of six nucleotides, leading to the loss of highly conserved amino acids A799 and L800. Studies in this mouse elegantly demonstrated the importance of Atp7a in supplying copper to PAM for the amidation of neuropeptides, in axon extension and synaptogenesis, and in compensatory mechanisms to facilitate copper transport across the BBB. The latter includes upregulation of the mutant Atp7a in endothelial cells, as well as increased association of MoBr/y astrocytes and microglia with the BBB, and these mechanisms possibly contribute to the effectiveness of early copper treatment. The severe copper deficiency in the brain of MoBr/y mice can be rescued by transgenic expression of the human ATP7A. Expression of the ATP7A transgene was detected in the cerebellar Purkinje cell layer, the CA1 and CA2 regions of the hippocampus, the mitral layer of the olfactory bulb, the vascular endothelium, and the choroid plexus, but it could not be detected in astrocytes. The gene correction significantly improved the survival of the ATP7A-expressing transgenic MoBr/y mice compared with non-transgenic animals. Furthermore, a recent study demonstrated rescue of neonatal MoBr/y mice following lateral ventricle injections of adeno-associated virus serotype 5 (AAV5) harboring a truncated ATP7A cDNA, in combination with copper treatment. The authors proposed different effects of the two treatments: the copper injections increased the amount of brain copper available to levels that were 25–50% of the wild type, while the AAV5 gene delivery improved copper utilization. This was associated with enhanced activity of DBH and correction of brain pathology. These findings further illustrate the critical role of copper and ATP7A for brain function and highlight the potential for gene therapy in the treatment of MD patients.

36Recently, missense mutations in ATP7A were found to cause a form of dHMN, which represents the third clinical phenotype associated with mutations in ATP7A. dHMNs are a clinically and genetically heterogeneous group of diseases characterized by lower-motor neuron weakness and muscular atrophy. proposed that a common factor in the disease mechanism may be altered copper homeostasis. The phenotype of ATP7A-related motor neuropathy is clinically distinct from that of MD, with variable age of onset that ranges from the first to the sixth decade of life, no overt abnormalities of copper metabolism, and typically distal muscle weakness and atrophy of the lower extremities leading to hand and foot deformities. Nerve conduction studies suggest that the disease causes gradual degeneration of motor neuron axons in the limbs, beginning in the distal portion and progressing toward the cell body. The late-onset nature of the disease suggests that these mutations in ATP7A have subtle effects on ATP7A function that can take years to manifest clinically.

37The missense mutations within ATP7A that result in T994I and P1386S substitutions were identified in two families with multiple affected males. These mutations are outside of the conserved functional domains and they cause abnormal ATP7A trafficking, affecting specifically motor neuron function. An abnormal interaction between ATP7A (T994I) and valosin-containing protein (p97/VCP) was demonstrated, the latter, an ATPase with functions that include vesicular trafficking and protein degradation by the ubiquitin (Ub)-proteasome system (UPS;; ). Mutations in p97/VCP are also associated with other diseases that involve motor neuron degeneration. The significance of this interaction in mediating the disease phenotype awaits further investigation. Potential mechanisms that can lead to axonal degeneration as a result of these ATP7A mutations include copper deficiency, oxidative damage from mislocalized copper, and/or slowed synaptic copper release, resulting in inefficient down-regulation of the NMDA receptor and hence excitotoxicity leading to neuronal damage and axonal dieback.

38In a significant proportion of WD patients, neurodegeneration and neurological presentation reveals an important role for ATP7B in maintaining neuronal copper homeostasis. WD manifests primarily in the liver and brain. Mutations that inactivate ATP7B lead to impaired biliary copper excretion and consequently, hepatic copper overload, apoptotic cell death, liver damage, and spillage of copper into the plasma and CSF. Hence, copper also accumulates in extrahepatic tissues, notably the brain, kidneys, and cornea.

39Approximately 40–50% of WD cases present with neurological symptoms and these patients typically have a later onset than those with the liver disease, presenting in the second or third decade. Although ATP7B is expressed in several brain regions, brain copper accumulation in WD appears to be secondary to the liver disease, because it can be reversed by transplantation. The psychiatric symptoms also are reversible with chelation therapy. The main areas of the brain affected in WD patients include the thalamus, subthalamic nuclei, brainstem, cerebellum, and frontal cortex. The precise mechanisms mediating neuronal injury in WD are not clear, but potentially involve increased extracellular copper combined with impaired copper homeostasis in those regions of the brain suffering loss of ATP7B function. For example, impaired DBH synthesis may explain the predominant abnormalities of the basal ganglia that result in Parkinsonian symptoms such as rigidity and tremor.

40The toxic milk mouse (tx) and the LEC rat are mouse models of WD that harbor a point mutation or deletion, respectively, in Atp7b. Studies of the neurological symptoms and neurodegenerative processes in these models have been lacking until recently. One study found that despite an increase in brain copper levels of the tx mouse, there are no neurological or behavioral symptoms. In contrast, a more recent study reported copper deposition in the striatum and hippocampus of tx mice associated with an inflammatory response in these tissues, as well as motor and cognitive disturbances and impaired spatial memory. The reason for the discrepancy between these two studies is not clear but may relate to the genetic background of the tx mice used.

41Alzheimer’s disease is a progressive neurodegenerative disorder occurring late in life. Patients suffer memory loss and cognitive decline. Key pathological hallmarks include intra- and extracellular proteinaceous deposits (senile plaques comprising the Aβ peptides derived from the processing of APP and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau). The role of copper in AD is gaining prominence with the discoveries that: (i) it is increased and/or mis-localized in the AD brain; (ii) enriched in extracellular plaques; (iii) deficient in Aβ plaque neighboring brain regions; and (iv) disease-linked proteins, APP, Aβ, tau, and BACE1, are copper-binding proteins with key roles in brain metal regulation (reviewed in; ). These observations and findings of dysregulation of other metals such as iron and zinc have led to the “metal theory of AD” with the coining of the term, “metallostasis.” This represents the fatigue of brain metal regulation and distribution, leading to Aβ aggregation and deposition, intraneuronal iron accumulation, and consequently, oxidative injury and neurodegeneration.

42Genetic analysis of AD patients and healthy controls uncovered ATP7B as a genetic risk factor for AD. A number of single nucleotide polymorphisms (SNPs) in ATP7B are associated with increased risk for AD. These changes occur in either a MBD, the ATP-binding N-domain or transmembrane domains, which may negatively affect ATP7B function in relation to metal-binding, ATP hydrolysis or copper translocation across the membrane. SNPs in the transmembrane domains present the strongest association for AD risk. These observations further support a crucial role for ATP7B in maintaining brain copper homeostasis and a potential role in AD pathogenesis. In contrast to these observations, transgenic APP mice (CRND8) that are homozygous for the txJ mutation, and therefore lacking a functional ATP7B protein, exhibit elevated brain copper levels, but a markedly reduced number of amyloid plaques and decreased plasma Aβ levels. The authors postulated that the mechanism of this beneficial effect of the txJ mutation involves increased clearance of peripheral Aβ pools. Alternatively, elevated intracellular copper due to the txJ mutation may correct the copper-deficient phenotype of the CRND8 mice. The copper-induced retention of APP at the cell surface, leading to reduced Aβ production and interaction with copper in lipid rafts may explain the decrease in Aβ and amyloid plaques.

43APOE and CLUSTERIN (APOJ) polymorphisms represent the strongest and third strongest genetic risk factors for AD, respectively. The encoded proteins have long associations with AD. ApoE and clusterin are well-known for their function as secreted extracellular chaperones with key roles in lipid transport. They have neuroprotective functions, cooperatively regulating the deposition and clearance of Aβ. In humans, there are three common APOE alleles, ε2, ε3, and ε4. APOE-ε4 confers increased risk for AD. The presence of APOE-ε4 in WD patients with the common H1069Q mutation in ATP7B is associated with an earlier onset of symptoms. In contrast, APOE-ε3 is associated with a significant delay in the onset of WD symptoms compared with APOE-ε4 carrying patients. This difference may be linked to the isoform-specific antioxidant activity of the ApoE isoforms. ApoE4 is less effective than ApoE2/ApoE3 as an antioxidant, which may explain the greater susceptibility of ApoE4 patients to copper toxicity. While the exact mechanism remains unclear, these studies suggest a role for ApoE in copper regulation and in influencing WD phenotypes.

44We recently demonstrated that clusterin interacts with ATP7A and ATP7B, and this interaction appears to facilitate the degradation of misfolded copper-ATPase molecules predominantly via the lysosome. Both MD and WD exhibit a high degree of clinical variability, with reports of identical mutations, even among siblings, conferring variable clinical expression. Hence, these observations implicate other factors in determining the clinical phenotype. Based on functional similarities between clusterin and ApoE, the association between ApoE isoforms and WD onset, and the role of clusterin in copper-ATPase degradation, these molecules potentially could play a role in modifying the expression of neurological disease such as AD, MD, and WD. Conceivably, genetic variations in the clusterin and ApoE alleles, together with environmental factors, could contribute to the variability in the clinical expression of MD and WD.

45Prion diseases are characterized by the continual conformational change of the normal prion protein (PrPC) to an infectious, protease-resistant, β-sheet-rich form of the protein (PrPSc; ). The resultant toxic PrPSc aggregates can disrupt axonal transport and synaptic transmission and/or trigger apoptosis, leading to the neurodegenerative pathologies that are collectively termed transmissible spongiform encephalopathies (TSE) (reviewed in ).

46Prion protein is a membrane glycoprotein with four N-terminal octameric repeats and a nearby site that binds copper. This protein is ubiquitously expressed but enriched in neurons and concentrated at the synapse. Its normal physiological role still remains to be fully elucidated but insight into the cell biology and biochemical properties of PrPC is revealing some clues regarding its function. Various studies have demonstrated that copper-binding induces PrPC internalization prompting the suggestion that PrPC may be involved as a receptor for copper uptake or efflux. More recently, confirmed previous findings that copper modulates NMDA receptor activity. They further demonstrated that this occurs through a copper-dependent interaction between PrPC and the GluN1 subunit, which reduces glycine affinity for the receptor, thus suppressing its activity. The role of ATP7A-mediated synaptic copper release may be important in this context but whether it plays a direct role in PrPC-copper interactions is not clear.

47In contrast to the beneficial role of copper in PrPC function noted above, other studies reported that copper binding to PrPC increases its conversion to PrPSc, and that copper chelation delays the onset of prion disease. In support of these studies, disruption of copper homeostasis due to a hypomorphic mutation in ATP7A delays the onset of prion disease in mice. Copper levels in the brain are reduced by 60% and the amount of the disease-causing PrPSc is significantly lower than that of the controls. The controversies over the role of copper in PrPC function and prion disease remain to be clarified.

48Copper plays a central role in a complex network of signaling pathways that regulate a host of physiological and pathophysiological processes. The crucial role of copper in brain and CNS development and function was highlighted three decades ago when the connection was made between infants with MD and copper deficiency. However, despite its importance, detailed knowledge of the mechanisms controlling brain copper homeostasis remains limited. Significant advances have been made with the subsequent discovery of key components of the copper regulatory network. The copper-ATPases, ATP7A and ATP7B are among these and since their discovery two decades ago, significant progress has been made toward understanding their pivotal role in normal copper homeostasis. Emerging data now reveal a complex and varied role for copper in the brain, and that the copper-ATPases are integral to the regulation and maintenance of copper-mediated processes within the brain. The consequences of their malfunction are clearly illustrated by the severe neurological deficits and neurodegeneration that accompany the disorders of copper transport, MD and WD. More subtle functional defects in the copper-ATPases or in factors that regulate their activity and/or stability are likely to contribute to other neurodegenerative diseases where copper is dysregulated, such as Alzheimer’s, motor neuron and prion diseases. Given the complexity of the CNS, much remains to be learned about the role of ATP7A and ATP7B in neurological development and neurodegenerative processes. Current ongoing research into the factors that affect the regulation of their expression, post-translational modification and activity will continue to provide new insights into their involvement and adaptive capacity during neuropathological processes associated with aging and disease.

49The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.