Role of copper in central nervous system physiology and pathology

Abstract

Copper is a transition metal and an essential element for the organism, as alterations in its homeostasis leading to metal accumulation or deficiency have pathological effects in several organs, including the central nervous system. Central copper dysregulations have been evidenced in two genetic disorders characterized by mutations in the copper-ATPases ATP7A and ATP7B, Menkes disease and Wilson’s disease, respectively, and also in multifactorial neurological disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. This review summarizes current knowledge about the role of copper in central nervous system physiology and pathology, reports about unbalances in copper levels and/or distribution under disease, describes relevant animal models for human disorders where copper metabolism genes are dysregulated, and discusses relevant therapeutic approaches modulating copper availability. Overall, alterations in copper metabolism may contribute to the etiology of central nervous system disorders and represent relevant therapeutic targets to restore tissue homeostasis.

Introduction

Copper (Cu) is a transition metal and an essential element for the organism, as alterations in its homeostasis leading to metal accumulation or deficiency have pathological effects in several organs, including the central nervous system (CNS) (Chen et al., 2020). Copper homeostasis is maintained at the cellular level by proteins involved in metal uptake (e.g. copper transport 1 [CTR1] and divalent metal transporter 1 [DMT1]), intracellular chaperones (e.g. glutathione [GSH], metallothioneins, Atox1, Cox, and superoxide dismutase 1 [SOD1]), cuproenzymes (e.g. monoamine oxidase, dopamine β-monooxygenase, peptidylglycine monooxygenase, ferroxidases, cytochrome-c oxidase, and superoxide dismutase) and Cu-ATPases involved in Cu export (ATP7A and ATP7B) (Chen et al., 2020).

Copper is indispensable for CNS physiology as it is a cofactor of enzymes involved in redox reactions, energy metabolism, and synthesis of neuropeptides and neurotransmitters (Scheiber et al., 2014). Copper stored in the secretory pathway is released in a Ca²⁺-dependent manner and can affect synaptic transmission as it can bind to and modulate the function of γ-aminobutyric acid receptors, N-methyl-D-aspartate (NMDA) receptors, voltage-gated Ca²⁺ channels, and purinoreceptors (Coddou et al., 2003; Gaier et al., 2013). Dysregulations in copper metabolism have been evidenced in genetic and multifactorial disorders, including multiple sclerosis (MS). This review summarizes current knowledge about the role of copper in CNS physiology and pathology, underlines recent advances in copper transport in multiple sclerosis and its animal models (Colombo et al., 2021; Morgan et al., 2022), and critically revises the commonly accepted view that copper deprivation due to copper chelation may be at the basis of CNS pathology at the light of relevant experimental evidence (Morgan et al., 2022). Overall, proper knowledge about copper metabolism is necessary to understand the etiology of CNS disorders and provide appropriate therapeutic targets to restore tissue homeostasis.

Copper is a transition metal with three oxidation states, Cu0, Cu(I)/Cu⁺ (cuprous ion), and Cu(II)/Cu²⁺ (cupric ion), which endows copper with the ability to donate or receive electrons in diverse biochemical reactions such as redox reactions (Chen et al., 2020; Møller and Aaseth, 2022). It is acquired from the diet, as it is present e.g. in shellfish, nuts, seeds, organ meats (e.g., liver), wheat-bran cereals, whole-grain products, and chocolate (Pennington et al., 1995). Dietary copper is absorbed at the level of the proximal small intestine, and from there it is transported to the liver, whose cells incorporate copper into proteins, such as cuproenzymes and ceruloplasmin (CP) (Collins, 2020). Most of the copper circulating in the blood is bound to plasma proteins like CP and serves as a supply for body tissues and organs (Collins, 2020). Human beings do not store considerable amounts of copper (total body copper in adults ranges from 50 to 120 mg (Collins, 2020), thus making necessary constant copper intake. Copper is present in several organs, and is mostly ligated in bones and skeletal muscle, but reaches the highest concentrations in the kidney, followed by the liver and brain (Focarelli et al., 2022).

Copper in excess is transported from hepatocytes into bile canaliculi, so that it can be eliminated via the bile (Collins, 2020). Our organism controls copper homeostasis by coupling intestinal absorption with hepatic copper excretion into the bile. For example, when both dietary copper intake and intestinal absorption are high, the body intensifies biliary copper excretion (Scheiber et al., 2014; Collins, 2020).

Search Strategy

In this narrative review, literature was searched in PubMed and Google Scholar using combinations of keywords including amyotrophic lateral sclerosis, Alzheimer’s disease, astrocytes, copper, copper homeostasis, Menkes disease, microglia, multiple sclerosis, neurodegenerative disorders, oligodendrocytes, Parkinson’s disease, Wilson’s disease. All years were chosen in the search. Papers with text language other than English were excluded.

Cellular Copper Homeostasis

Cellular copper homeostasis is regulated by transporters for the uptake and efflux of Cu and by a group of intracellular proteins (Figure 1; Kaplan and Maryon, 2016).

Figure 1.

Cellular copper homeostasis.

Cellular copper homeostasis is regulated by transporters for the uptake and efflux of Cu and by a group of intracellular proteins. Plasma membranes may contain metalloreductases, which reduce extracellular Cu²⁺ to Cu⁺. Copper import into eukaryotic cells is mediated by CTR1, CTR2, and/or DMT1. CTR2 is predominately located in intracellular compartments, with a small fraction present in the plasma membrane. Copper is a reactive metal, thus, once inside the cell, it is immediately coupled to intracellular proteins which deliver this metal to specific targets. It can be stored as complex with GSH or MTs. In addition, copper is transported by the copper chaperone CCS to SOD1 and by Cox17, Cox11, and Sco1/2 to CCO in mitochondria. Atox1 shuttles Cu⁺ to ATP7A or ATP7B which transport copper into the TGN or, in case of intracellular copper excess, move to the plasma membrane to release copper in its reduced form (Cu⁺) out of the cells. SP1 is a transcription factor recognizing the promoter of the human CTR1 gene and with a zinc finger domain sensing copper levels. Created with BioRender.com. ATP7A: ATPase copper transporting alpha; ATP7B: ATPase copper transporting beta; CCO: cytochrome-c oxidase; CCS: copper chaperone for superoxide dismutase; CTR: copper transporter; DMT1: divalent metal transporter 1; MT: metallothionein; SOD: superoxide dismutase; SP1: specificity protein 1; TGN: trans-Golgi network.

Copper import into eukaryotic cells is mediated by CTR1, which is a high-affinity integral membrane protein highly specific for the cuprous ion Cu⁺ (Figure 1; Zhou and Gitschier, 1997; Nevitt et al., 2008). Plasma membranes may contain metalloreductases like STEAP proteins, which reduce extracellular Cu²⁺ to Cu⁺ (Ohgami et al., 2006). The vital importance of CTR1 is elucidated by the fact that its complete genetic inactivation is embryonic lethal (Kuo et al., 2001). CTR1 is found mostly on the plasma membrane or intracellular vesicles of several cell types, thus resulting in Cu⁺ entry into the cells or its mobilization from endosomal compartments to the cytosol respectively (Nevitt et al., 2008). A second member of the CTR family called CTR2 has been described in human cells and is a low-affinity copper (Cu⁺) transporter, predominantly located in intracellular compartments (Figure 1) and with a small fraction present at the plasma membrane (van den Berghe et al., 2007; Bertinato et al., 2008). Copper uptake may also be mediated by CTR1-independent mechanisms relying on DMT1 (Figure 1), a transporter for various divalent metals (Gunshin et al., 1997; Arredondo et al., 2003).

Copper is a reactive metal, thus, once inside the cell, it is immediately coupled to intracellular proteins which deliver this metal to specific targets (Kaplan and Maryon, 2016). GSH, a key molecule for antioxidant responses, is an endogenous Cu-coordinating protein present in high abundance in eukaryotic cells; it is the initial Cu acceptor, which then delivers the metal to chaperones such as antioxidant 1 copper chaperone (Atox1) and copper chaperone for superoxide dismutase (CCS) (Kaplan and Maryon, 2016). While Atox1 is a cytoplasmic protein responsible for transporting Cu⁺ to the Cu-ATPases ATP7A and ATP7B residing in the trans-Golgi network (TGN) (Figure 1; Scheiber et al., 2014), CCS is a copper chaperone for Cu/Zn SOD1, whose activity is important for the defense against superoxide radicals (Møller and Aaseth, 2022). CCS is a sort of oxygen sensor and a factor regulating SOD1 compartmentalization: high oxygen concentration promotes CCS folding and, thereby, SOD1 levels in the cytosol; instead, physiological oxygen levels favor CCS import into the mitochondria and, consequently, SOD1 translocation into the intermembrane space (Kawamata and Manfredi, 2008). SLC25A3 transports copper into the mitochondrial matrix for its storage (Cobine et al., 2021). Moreover, in the mitochondria Cox17, Cox11, and Sco1 support copper insertion into cytochrome-c oxidase (Figure 1) and are so important for the correct function of the cell that mutations in one of them cause severe cytochrome-c oxidase deficits (Lutsenko, 2010; Scheiber et al., 2014). In addition to cytochrome-c oxidase and Cu/Zn SOD1, several other enzymes use copper as a cofactor (Scheiber et al., 2014). Amine oxidase plays the deamination of primary amines, while diamine oxidase inactivates the release of histamine during allergic reactions (Harris, 1997). The monoamine oxidases degrade serotonin and are important for the metabolism of catecholamines, like epinephrine, norepinephrine, and dopamine (Harris, 1997). Lysyl oxidase uses lysine and hydroxylysine found in collagen and elastin to produce cross-linkages for the development of connective tissue of bones, lungs, and circulatory system (Harris, 1997). Dopamine β-monooxygenase converts dopamine into norepinephrine; then there is peptidylglycine monooxygenase, which is involved in the α-amidation of neuropeptides (Harris, 1997). Other cuproenzymes are, for example, tyrosinase, entailed in melanin production (Harris, 1997), and the ferroxidase CP, the principal copper-bound protein in plasma (Harris, 1997).

Intracellular copper storage is supported by metallothioneins (MT), a group of metal-binding proteins with low molecular weight and high cysteine content involved in protection against metal toxicity (Calvo et al., 2017). They take on the function of cytosolic copper storage under normal copper metabolism and serve as metal reservoirs in the event of copper deficit (Ogra et al., 2006). Copper release involves two Cu-ATPases, ATP7A and ATP7B, whose activity and intracellular localization are modulated by copper (Gupta and Lutsenko, 2009; Lutsenko, 2010). Normally located in TGN (Figure 1), these transporters accept copper from Atox1 and use the energy of adenosine 5’-triphosphate (ATP) hydrolysis to transfer copper into the secretory pathway, where copper is incorporated into copper-dependent enzymes (Gupta and Lutsenko, 2009; Lutsenko, 2010). However, in case of intracellular copper excess, they move to the plasma membrane, where they use the energy of ATP hydrolysis to transport copper in its reduced form (Cu⁺) out of the cells (Gupta and Lutsenko, 2009; Lutsenko, 2010). Mutations in ATP7A or ATP7B cause severe genetic disorders, Menkes disease (MD) and Wilson’s disease (WD) respectively (Chen et al., 2020).

Organ Copper Distribution as a Function of Copper-Binding Proteins

The intestinal copper pool is formed by dietary and supplemental copper, and endogenous copper is present in gastric, intestinal, and pancreatic secretions (Collins, 2020). Before transport across the brush-border membrane copper is reduced from the cupric state (Cu²⁺) to the cuprous state (Cu⁺), then it is transported into enterocytes by CTR1 or the non-CTR1-dependent mode via DMT1 (Arredondo et al., 2003). Once in enterocytes, copper interacts with chaperone proteins that deliver the metal to different intracellular locations (Collins, 2020). The binding between copper and these proteins is fundamental to preventing reactions with oxygen and the production of superoxide radicals (Collins, 2020). First, copper interacts with GSH and forms the copper-GSH complex which passes copper along its chaperones, Atox1, CCS, Cox17, Cox11, SOD1, and SOD2 (Kaplan and Maryon, 2016). The export of copper out of enterocytes is partly mediated by exocytosis, partly by ATP7A which moves from TGN to the basolateral membrane of enterocytes (Zimnicka et al., 2007). Once copper is out of the cells, the oxidating environment of the intestinal fluids converts Cu⁺ to Cu²⁺ which can bind diverse serum proteins (e.g., albumin or 2-macroglobulin) for delivery in the portal blood to the liver (Gulec and Collins, 2014). Copper derived from the intestine is taken up by the liver but before this event, copper must be reduced again, so that it can enter hepatocytes through CTR1. In liver cells, copper binds to the same chaperones as in enterocytes and is distributed into intracellular organelles to support cuproenzyme synthesis (Collins, 2020). If in enterocytes copper enters TGN by ATP7A, in hepatocytes it does via ATP7B; in addition, copper exits the liver as an integral component of the CP ferroxidase protein and as atomic copper (Linder, 2016). Thus, ATP7A and 7B play critical roles in intestinal copper transport (ATP7A) and its biliary excretion (ATP7B), and their dysfunction underlies MD and WD respectively. Some biliary copper may be reabsorbed but excess biliary, unabsorbed dietary, and endogenous copper is lost via feces (Collins, 2020). To protect the organism from copper excess or depletion adaptive mechanisms evolved so that about 10% of dietary copper is absorbed under physiological conditions (Harvey et al., 2005), but this percentage increases when intake levels are low (Danks, 1988). In addition, when intake is high, copper is sequestered by metallothioneins in enterocytes, and biliary excretion increases (Danks, 1988; Turnlund et al., 2005).

Copper and Transcription

In vitro studies indicate that copper may influence the transcription of genes, including CTR1. In fact, CTR1 mRNA levels may vary in response to changes in copper concentration (Song et al., 2004), and the transcription factor SP1 is involved in the homeostatic control of human CTR1 mRNA (Song et al., 2008). SP1 is a zinc finger protein that may sense copper levels (Song et al., 2008), recognize the promoter of the human CTR1 (Yuan et al., 2017) and thereby regulate its expression (Figure 1). Further, copper may regulate the expression and/or stability of transcription factors, such as specificity protein 1 (SP1), hepatocyte nuclear factor 4 alpha (HNF4α), and c-myc (Song and Freedman, 2011; Liang et al., 2012; Xie et al., 2023). However, the impact of copper-regulated transcription on CNS function is a neglected topic in the literature and deserves future investigation.

In conclusion, copper homeostasis may influence all cellular compartments and copper dysregulation may lead to profound and long-lasting effects in cellular functions.

Copper Trafficking in the Central Nervous System

Copper reaches the CNS via blood circulation and is transported as free ion across the blood-brain barrier and blood-cerebrospinal fluid barrier, the first separating CNS parenchyma from blood circulation and the second separating the blood from the cerebrospinal fluid (Choi and Zheng, 2009). Copper transport proteins may be present in CNS tissues but their levels may change depending on the brain region (Davies et al., 2013; Scheiber et al., 2014). Pioneer histochemical studies indicate main copper content in rodent substantia nigra and glial cells (Szerdahelyi and Kasa, 1986); however, systematic description of copper handling proteins in diverse CNS regions and cells is still missing.

Astrocytes are in close contact with blood vessels and thus represent the first parenchymal cells that metal ions encounter after crossing endothelial cells (Scheiber and Dringen, 2013). Interestingly, X-ray fluorescence microscopy may detect Cu-rich aggregates in astrocytes located in the subventricular zone and hippocampus of rats but not mice (Sullivan et al., 2017a, b). Cu handling by astrocytes may be carried out by the previously cited network of Cu transporters, chaperones, and storage proteins (Dringen et al., 2013). In addition, cellular prion protein may regulate copper levels and distribution in vivo (Pushie et al., 2011) and has been suggested for copper uptake by astrocytes (Tiffany-Castiglioni et al., 2011), which also express a membrane-bound glycosylphosphatidylinositol-anchored form of ceruloplasmin (Linder, 2016). Neuropathological studies evidenced CTR1 protein in neurons (Davies et al., 2013), while no in vivo information is available for oligodendrocytes and microglia. A microglial cell line has been shown to upregulate CTR1 expression in response to in vitro stimulation with interferon-γ (Zheng et al., 2010), suggesting that microglia can enhance copper trafficking during neuroinflammation.

Copper can enter the storage pool as three MTs (MT1, MT2, and MT3) are expressed at barriers and in CNS-resident cells such as astrocytes and neurons, while absent in microglial cells and oligodendrocytes (Hidalgo et al., 1994 2001). Copper can also bind to intracellular GSH whose de novo synthesis takes place in astrocytes using its constituent amino acids glutamic acid, cysteine, and glycine and whose precursors are then provided by astrocytes to neurons (Dwivedi et al., 2020). Intracellular copper trafficking in CNS cells is mediated by Atox1, CCS, and the copper chaperones of cytochrome-c oxidase Cox17 and Sco1/2 (Rothstein et al., 1999; Scheiber et al., 2014).

Regarding copper export proteins, ATP7A and ATP7B have been detected in the developing CNS (Kuo et al., 1997). ATP7A is also known to be expressed in both neuronal and nonneuronal cells, peaks at early postnatal time, and decreases at adult age (Niciu et al., 2006).

Copper Contribution to Central Nervous System Function

The CNS uses copper for general metabolic functions and specific tissue functions (Dringen, 2000). The brain is one of the most energy-demanding tissues of the human body, with 95% of ATP generated by mitochondria (Rossi et al., 2004; Vergun et al., 2007), and is sensitive to oxidative stress (Dringen, 2000). Cells of the human brain consume approximately 20% of the oxygen utilized by the body but constitute only 2% of the body weight; consequently, reactive oxygen species, which are continuously generated during oxidative metabolism, are produced at high rates within this organ (Dringen, 2000). Copper metabolism, which is essential for the regulation of redox reactions and energy metabolism, is therefore at the basis of CNS physiology (Scheiber et al., 2014). Other biological processes supported by copper and cuproenzymes in the CNS are iron metabolism, synthesis of neurotransmitters and neuropeptides, and synaptic transmission (Scheiber et al., 2014).

Copper and iron metabolism are closely interrelated, starting from the sharing of the cellular uptake transporter DMT1 to the role of copper as a cofactor in proteins involved in iron homeostasis such as ceruloplasmin, or as a modulator of the iron regulatory hormone hepcidin (Skjørringe et al., 2012). Abnormal systemic copper levels may affect iron metabolism in the brain and, vice versa, iron levels influence CNS copper homeostasis (Skjørringe et al., 2012).

Regarding the synthesis of neuropeptides and neurotransmitters, dopamine β-monooxygenase catalyzes the final step of noradrenaline synthesis, which is the principal sympathetic neurotransmitter and an important modulator of mood, attention, arousal, and cardiovascular functions (Klinman, 2006; Scheiber et al., 2014). Amidated neuropeptides are synthesized in neurons and are involved in several activities, like the proliferation of neural stem cells of the olfactory system, energy metabolism, and neuromodulation (Bousquet-Moore et al., 2010a). Peptidylglycine monooxygenase is the only enzyme known to catalyze the α-amidation of peptide precursors, and due to its physiological importance, the lack of functional peptidylglycine monooxygenase in mice is embryonic lethal (Klinman, 2006; Bousquet-Moore et al., 2010b).

Copper can bind to and modulate the function of γ-aminobutyric acid receptors, NMDA receptors, voltage-gated Ca²⁺ channels, and purinoreceptors (Coddou et al., 2003; Gaier et al., 2013). It is stored in synaptic vesicles from where it is released in a Ca²⁺-dependent manner (D’Ambrosi and Rossi, 2015). For example, calcium entry in hippocampal neurons after NMDA-receptor stimulation induces ATP7A translocation to synapses and copper release (Schlief et al., 2005). Copper plays a role in synaptic physiology, possibly affecting neuronal transmission, long-term potentiation, synaptic plasticity, and excitotoxic cell death (D’Ambrosi and Rossi, 2015).

Dysregulation of Copper Metabolism in Central Nervous System Disorders

Dysregulations in copper metabolism have been evidenced in genetic and multifactorial disorders.

Menkes’ disease

MD is a rare X-linked disease due to mutations (including deletion of a single exon to almost the entire gene, missense and splice-site mutations, exon duplication, and point mutations) in the ATP7A gene located on the long arm of chromosome X (Xq21) (Manto, 2014; Maung et al., 2021). The lack of a functional ATP7A gene product leads to the failure of release of copper from intestine epithelia into the bloodstream, with consequent copper accumulation in the intestine and copper deficiency in the rest of the organism, including the CNS (Chen et al., 2020). MD hallmarks are neurological degeneration, seizures, degeneration of connective tissues, abnormal lightly pigmented hair and skin, low muscle tone, bone fragility, and aortic aneurysms (Manto, 2014).

Neuropathology is characterized by cortical neuron loss, gliosis, subcortical myelin loss associated with axonal degeneration, and atrophy of grey and white matter (Menkes et al., 1962; Barnard et al., 1978). Mice with mutations in the ATP7A gene, called mottled mutants, are well-established models of MD as they recapitulate the Menkes phenotype (Lenartowicz et al., 2015).

Therapy for MD patients is based on copper supplementation via parenteral administration, but there is no treatment to reverse neurological damage associated with MD (Maung et al., 2021).

Wilson’s disease

WD is an autosomal recessive disorder caused by loss-of-function mutations in the copper transporter gene ATP7B (Maung et al., 2021). ATP7B dysfunction hampers copper excretion from the liver into the bile; consequently, Cu accumulates in the liver, enters the circulation unbound, and deposits in multiple organs, including the CNS (Gaier et al., 2013). Ophthalmological manifestations are one of the hallmarks of the disease. Corneal deposition of copper, called Kayser–Fleischer ring, is typical in WD and constitutes one of the diagnostic criteria for this disease (Chevalier et al., 2022). Neurological symptoms of WD typically occur at 20–40 years of age (Czlonkowska et al., 2012) and commonly consist of tremors, dystonia, parkinsonism, and ataxia, and are frequently associated with dysphagia, dysarthria, and drooling (Machado et al., 2006; Taly et al., 2007). Distinct rodent models exist for Wilson disease, display increased copper levels in the CNS, and may develop neurological impairment (Reed et al., 2018).

If diagnosed early, WD can be successfully treated by systemic chelation therapy with D-penicillamine or triethylenetetramine, which restore and maintain copper homeostasis in the body (Aggarwal and Bhatt, 2018).

Alzheimer’s disease

Alzheimer’s disease (AD) is a common complex neurodegenerative disease of aging causing loss of memory and psychiatric disturbances (Scheiber et al., 2014; Maung et al., 2021). Its pathological hallmarks are the accumulation of extracellular senile plaques and intracellular neurofibrillary tangles in the grey matter of the brain (Maung et al., 2021).

Contradictory evidence is available about copper levels in AD brain. For example, while whole tissue measurement does not detect alterations in the frontal cortex of AD cases compared to non-demented elderly controls (Szabo et al., 2016), polyvalent metal cations including copper have been found at high concentrations specifically in senile AD plaques (Lovell et al., 1998). Copper ions may bind to beta-amyloid peptides with high affinity (Miller et al., 2006), foster the formation and aggregation of amyloid fibrils (Hane et al., 2013; Crooks et al., 2020), and promote reactive oxygen species production (Parthasarathy et al., 2014). A recent study investigating metal concentrations in several human brain regions evidences copper reduction in AD for all investigated areas, including the cerebellum despite being this region relatively spared by the disease (Xu et al., 2017). Previous biochemical assessments also indicate lower total Cu content in AD tissues but underline higher redox-reactive exchangeable Cu, which correlates with increased oxidative damage in AD (James et al., 2012). Similar findings are reproduced in the APP/PS1 mouse model (James et al., 2012, 2017). PET imaging of copper trafficking indicates fast Cu accumulation and clearance in a mouse model of AD (Torres et al., 2016). Further, hippocampal copper content is augmented in a mouse model of non-insulin-dependent diabetes mellitus (Hackett et al., 2019), a condition predisposing to AD (Barbagallo and Dominguez, 2014).

The 8-hydroxy quinoline compound PBT2, which was developed to interfere with amyloid β association with metals including copper, decreases soluble amyloid β levels within hours and ameliorates cognitive functions in APP/PS1 mice (Adlard et al., 2008), however, its administration to AD subjects generated controversial results (Adlard and Bush, 2018).

Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease in humans (Desai and Kaler, 2008) and is characterized by tremors, stiffness, slowness, and imbalance (Armstrong and Okun, 2020).

The pathological hallmarks of PD are the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies, which are an aggregated form of α-synuclein protein (Desai and Kaler, 2008; Scheiber et al., 2014). As shown by in vitro studies, copper may bind to both soluble and membrane α-synuclein with high affinity (Dudzik et al., 2013) and accelerate the aggregation of α-synuclein (Davies et al., 2011). Further, copper ions accelerate prion-like propagation of α-synuclein fibrils by promoting cellular internalization of α-synuclein fibrils, and intracellular α-synuclein aggregation (Li et al., 2020).

Copper levels measured by inductively coupled plasma spectroscopy in frozen post-mortem PD and control tissues indicate metal reduction in several CNS regions of PD patients (Dexter et al., 1991; Davies et al., 2014; Genoud et al., 2020; Scholefield et al., 2021), suggesting that regional copper deficit may contribute to disease pathogenesis. Further, the lower Cu levels correlate with lower CTR1 expression in the substantia nigra of PD patients (Davies et al., 2014).

Interestingly, prolonged administration of copper to wild-type male mice alters motor functions along with aging and induces dopaminergic neuronal loss, gliosis, and α-synuclein accumulation and aggregation in the midbrain (Gonzalez-Alcocer et al., 2023), suggesting that chronic exposure to copper may impact CNS state and function with aging.

Alterations in copper homeostasis genes, including CTR1, may exist in PD (as recently reviewed in Montes et al., 2014). Importantly, in vitro studies with α-synuclein-overexpressing cells demonstrate that CTR1 expression supports intracellular Cu accumulation, which then triggers α-synuclein aggregation, as CTR1 silencing hampers this process (Gou et al., 2021). In accordance with these in vitro findings, mice with conditional deletion of CTR1 in dopaminergic cells display reduced levels of S129-phosphorylated α-synuclein and dopaminergic neuronal loss following unilateral injection of adeno-associated virus human α-synuclein in the substantia nigra (Gou et al., 2021). Notably, motor dysfunction induced by α-synuclein dependent pathology was also alleviated in transgenic mice (Gou et al., 2021). These data demonstrate that CTR1 is a key modulator of α-synuclein neurotoxicity and may represent an interesting therapeutic target in PD (Gou et al., 2021).

CuATSM is used as a PET-imaging agent for hypoxic tumors in humans (Dearling et al., 2002), has low toxicity, and penetrates the blood–brain barrier in the PD brain, where it detects striatal oxidative stress (Ikawa et al., 2011). Interestingly, this compound has been found to rescue PD phenotypes in relevant animal models (Hung et al., 2012). In fact, after oral administration, CuATSM may reach the CNS, support survival of dopaminergic neurons, and improve motor coordination and cognitive function in toxic and genetic PD models (Hung et al., 2012). These investigations prompted a Phase I clinical trial of CuATSM in idiopathic Parkinson’s disease (ClinicalTrials.gov Identifier: NCT03204929) providing initial promising results (Evans et al., 2019), but final results are not publicly available yet.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons, begins in the limbs but then impairs functions such as speaking, chewing, and breathing (Brown and Al-Chalabi, 2017). Oxidative stress and dysregulation of redox-active metals are common phenotypes associated with genetic and sporadic forms of ALS (Maung et al., 2021). Controversial data are available about Cu alterations in the cerebrospinal fluid of ALS subjects (Sauzéat et al., 2018; Chen et al., 2022), while spinal cord analyses indicate unaltered total copper levels in human ALS and G93A- SOD1 mice (Lelie et al., 2011; Hilton et al., 2024b). Still, lower copper content in ventral grey matter and higher levels in the white matter of sporadic ALS spinal cord have been described (Hilton et al., 2024b), indicating that copper unbalances may occur locally.

Observations obtained with ALS mice bearing alterations in diverse copper metabolism genes provide a complex picture about the role of copper in these models (Kiaei et al., 2004; Son et al., 2007; Roberts et al., 2014; Tokuda et al., 2014). For example, animal survival is enhanced by in vivo expression of human CTR1 in SOD1-G37R mice (Roberts et al., 2014) or by ATP7a deficiency in the SOD1-G86R background (Kiaei et al., 2004), but is associated with higher or lower spinal cord Cu compared with control mice respectively (Kiaei et al., 2004; Roberts et al., 2014). Overexpression of MT1 in the SOD1-G93A background also prolongs the life span and normalizes the exacerbated copper levels present in SOD1-G93A mice (Tokuda et al., 2014). On the contrary, mice overexpressing the copper chaperone CCS in the SOD1-G93A background display mitochondrial pathology and acceleration of neurological deficits leading rapidly to death (Son et al., 2007).

Cu-ATSM PET imaging shows increased oxidative stress primarily in the motor cortex correlating with clinical severity in ALS patients (Ikawa et al., 2015). As CuATSM can selectively release Cu⁺ in hypoxic tissues (Donnelly et al., 2012), it has been used as a copper delivery tool to treat SOD1-G37R mice and SOD1-G93A mice co-expressing CCS (McAllum et al., 2013; Roberts et al., 2014; Williams et al., 2016). Notably, CuATSM improves locomotor function and survival in SOD1-G37R mice (McAllum et al., 2013; Roberts et al., 2014), prevents the early mortality of CCSxSOD mice (Williams et al., 2016), and its discontinuation leads to disease exacerbation in CCSxSOD mice (Williams et al., 2016). Additionally, the therapeutic benefit can also be achieved by treatment of SOD1-G93A mice with the copper chelator ammonium tetrathiomolybdate, which reduces spinal cord Cu levels and suppresses SOD1 activity (Tokuda and Furukawa, 2016). In the light of the observation that the ALS spinal cord may be characterized by copper accumulation and deficiency in distinct areas (Hilton et al., 2024b), the efficacy of apparently contradictory therapies achieving copper delivery versus chelation may be interpreted as the regional partial rescue of the pathological phenotype. Further investigation is however needed to address this relevant issue.

Dysregulation of Copper Metabolism in Multiple Sclerosis and Its Animal Models

MS is a complex, chronic inflammatory disease of the central nervous system, with onset in young adulthood and major female prevalence (Cotsapas et al., 2018). Typical symptoms of MS include discrete episodes (“attacks” or “relapses”) of neurological dysfunction such as optic neuritis, sensory disturbance, gait impairment, and cognitive deficit (McGinley et al., 2021).

It is characterized by multifocal plaques in white and grey matter where immune cell infiltration, demyelination, and neuroaxonal damage are present (Cotsapas et al., 2018).

A study reports higher copper concentration in MS sera and cerebrospinal fluid than in control samples, however, the two groups were not balanced according to sex as the MS population had a major female component (De Riccardis et al., 2018). A more recent publication by our group describes higher serum copper in women than in men but no difference between healthy and MS subjects (Colombo et al., 2021), demonstrating an interesting sexual dimorphism in circulating total copper levels in the human population. Further, the analysis of transcriptomes of circulating immune cells in healthy and MS subjects indicates dysregulations in gene regulatory networks centered around the three copper-dependent transcription factors SP1, HNF4α, and c-myc (Song et al., 2008; Song and Freedman, 2011; Balsano et al., 2018), whose activity sustains disease expression in an animal model of disease (Menon et al., 2012; Colombo et al., 2023).

Different types of experimental models are available to reproduce distinct aspects of human MS pathology, including the experimental autoimmune encephalomyelitis (EAE) model and the cuprizone (CPZ) model (Zirngibl et al., 2022). EAE is the most frequently used experimental model for MS and reproduces immune cell-mediated processes that trigger neuropathological mechanisms leading to white matter inflammation, demyelination, axonal loss, and gliosis (Constantinescu et al., 2011). EAE is induced in genetically susceptible mouse strains by immunization with CNS tissue or myelin proteins or peptides. Disease onset typically occurs after 9–12 days and is followed by variable clinical courses depending on the genetic background of the host and immunizing antigen (Constantinescu et al., 2011). Cuprizone is typically administered with the diet at concentrations of about 0.2%–0.3% (w/w) for 4–6 weeks and is used to study non-immune cell-mediated white matter demyelination of the CNS (Zirngibl et al., 2022). This model reproduces the state of chronic MS white matter lesions, characterized by extensive demyelination, axonal damage, and strong gliosis in the absence of immune cell infiltration (Zirngibl et al., 2022).

While no information is available about copper levels in human MS tissues, total copper content is comparable in the spinal cord of perfused EAE and naïve mice (Colombo et al., 2021). Upregulation of copper transporters CTR1, ATP7A, and ATP7B occurs on reactive astrocytes in lesions of animal models and human MS, and CTR1 levels inversely correlate with myelin content, that is the higher CTR1 the higher demyelination (Colombo et al., 2021). In the cuprizone model astrocyte activation and proliferation are already evident after 1 week CPZ diet and precede demyelination, which requires at least 3 weeks of CPZ administration to develop (Tezuka et al., 2013; Colombo et al., 2021). In vitro experiments demonstrate that primary astrocytes may sense CPZ and respond to it with an increase in proliferation (Colombo et al., 2021). Astrocyte activation and proliferation depend in vivo and in vitro on the neurotrophin receptor TrkB, as conditional transgenic mice or primary astrocytes deficient for astrocyte TrkB do not display adaptive proliferation when exposed to CPZ (Colombo et al., 2021). Notably, CPZ, inflammatory mediators or free copper at the concentration present in human serum (100 ng/mL; McMillin et al., 2009) may enhance CTR1 protein levels in wild-type astrocytes and not in TrkB-deficient astrocytes (Colombo et al., 2021). Further, mice lacking astrocyte TrkB fail to upregulate CTR1 and are protected from demyelination under the CPZ diet in vivo, suggesting a link between copper trafficking in astrocytes and demyelination (Colombo et al., 2021). Indeed, wild-type but not TrkB-deficient astrocytes may release copper in vitro in such an amount that may induce myelin loss and oligodendrocyte death (Colombo et al., 2021), formally demonstrating that dysregulation of copper trafficking in astrocytes during neuroinflammation may impact myelin state.

The commonly accepted assumption about the action of the cuprizone diet on white matter pathology is that CPZ induces demyelination via its copper chelating properties which reduce local copper availability. The assessment of copper partitioned into soluble and insoluble fractions within distinct brain regions of cuprizone-fed and control mice indicates a copper decrease in the soluble fraction specifically in the corpus callosum (Hilton et al., 2024a), however causal relevance of this association also at the light of the enhanced copper trafficking in the same model (Colombo et al., 2021) remains to be established. Further, recent observations challenge the common view. First, if demyelination depends on diminished copper availability due to copper chelation, copper supplementation to mice during the cuprizone diet is expected to rescue from demyelination. This experiment shows that copper supplementation does not protect from cuprizone-induced demyelination, however, it does not report about efficient Cu uptake from the diet as a control (Morgan et al., 2022). Second, if demyelination depends on copper chelation, other Cu-chelating agents are expected to trigger central demyelination. Administration of D-penicillamine, a potent copper chelator used for the treatment of WD, does not induce any white matter injury despite a tenfold higher dose than cuprizone (Morgan et al., 2022). Third, if the mechanism of action of cuprizone-induced demyelination requires depletion of Cu, the addition of a second chelator to cuprizone in the chow is expected to worsen white matter injury (Morgan et al., 2022). When D-penicillamine is administered with the diet together with cuprizone, it prevents myelin loss in a dose-dependent manner, with complete protection in mice receiving the highest (0.25%) D-penicillamine dose (Morgan et al., 2022). However, it is unclear to what extent such findings depend on copper availability as this critical issue has not been investigated in the same in vivo settings.

Overall, these pieces of evidence indicate that cuprizone-induced demyelination is mediated by enhanced and not reduced copper levels, that enhancement of copper transport takes place in animal models of MS, and that copper chelation may be beneficial in experimental neuroinflammation.

Conclusions and Perspectives

Copper is an essential metal for our organism and its metabolism regulates proper cellular homeostasis also within the central nervous system. Observations in genetic and multifactorial neurological disorders indicate that copper unbalances may trigger and/or sustain neurodegeneration and glial pathology. However, limited information is available about alterations in copper metabolism genes in distinct CNS cell types in distinct disorders and about the impact of copper unbalances on glia-neuron and glia-glia interactions. Finally, it is also important to generate solid evidence that available therapeutic applications for CNS disorders may target copper metabolism and/or that targeting of copper trafficking is sufficient to ameliorate neurological conditions.

Data availability statement

Not applicable.