Mitochondrial Copper in Human Genetic Disorders

Abstract

Copper is an essential micronutrient that serves as a cofactor for enzymes involved in diverse physiological processes, including mitochondrial energy generation. Copper enters cells through a dedicated copper transporter and is distributed to intracellular cuproenzymes by copper chaperones. Mitochondria are critical copper utilizing organelle that harbors an essential cuproenzyme cytochrome c oxidase, which powers energy production. Mutations in copper transporters and chaperones that perturb mitochondrial copper homeostasis result in fatal genetic disorders. Recent studies have uncovered the therapeutic potential of elesclomol, a copper ionophore, for the treatment of copper deficiency disorders such as Menkes disease. Here we review the role of copper in mitochondrial energy metabolism in the context of human diseases and highlight the recent developments in copper therapeutics.

Copper as an ancient enzymatic cofactor

Copper is an essential trace element utilized by nearly all organisms from bacteria to humans. The evolution of copper-containing enzymes (cuproenzymes) likely occurred in response to the increasing need to utilize oxygen and oxygen-containing molecules in the primordial ocean [1]. This feature is still present today as many cuproenzymes use oxygen as a substrate to catalyze redox reactions. These enzymes participate in diverse physiological processes including mitochondrial energy generation and detoxification of free radicals [2]. Before describing different aspects of mitochondrial copper biology, we first present a brief overview of copper absorption, transport, and intracellular distribution in humans to introduce key players in copper homeostasis.

Copper absorption and transport in humans

Total body copper in an average adult human is approximately 100 mg. The recommended daily intake of copper for adults is ~1.5 mg, which is mostly acquired through diet [3]. Dietary copper is imported as cuprous (Cu¹⁺) ions via copper transport protein 1, CTR1 (Figure 1) [4]. Various cupric (Cu²⁺) reductases facilitate this process, including cytochrome b ferric/cupric reductase, STEAP metalloreductases, and cytochrome B reductase 1 [5, 6]. Copper could also be imported as cupric (Cu²⁺) ions via divalent metal transporter, DMT1 [7, 8]. Dietary copper is then exported from enterocytes into the portal blood circulation via ATP7A, a copper-transporting P-type ATPase, which translocate to the basolateral membrane from its trans-Golgi location in response to excess cellular copper in enterocytes (Figure 1) [9].

Figure 1.

Copper trafficking in a polarized mammalian cell.

Extracellular Cu²⁺ is reduced to Cu¹⁺ by plasma membrane-localized reductases that allows its import via the high-affinity plasma membrane copper transporter CTR1. Extracellular Cu²⁺ could also be imported by a non-specific divalent metal transporter DMT1. Once in the cytosol, copper is immediately bound to either a non-proteinaceous ligand (GSH or CuL) or a metallochaperone for subsequent trafficking to various organelles. CuL is proposed to transport copper to mitochondria, where it is stored within the mitochondrial matrix and acts as a feedstock for the metallation of mitochondrial cuproenzymes. In the cytosol, CCS, a metallochaperone, binds and transfers copper to Cu/Zn SOD1. Copper is delivered to the Golgi lumen, via the combined action of cytosolic metallochaperone ATOX1 and the Golgi membrane localized copper pumps ATP7A and ATP7B. Recently, histones H3–H4 tetramer have been shown to act as copper reductases that increase the bioavailable copper pool by catalyzing the reduction of Cu²⁺ to Cu¹⁺. Excess copper is either bound to metallothionein (MT) or pumped out of the cell via the action of ATP7A/B, which translocates to the plasma membrane via vesicular trafficking. The figure was created with Biorender.com.

Once in the blood, copper is bound to α2-macroglobulin, albumin, and other ligand(s) and is delivered to the liver [10]. As in enterocytes, copper transport into hepatocytes is mediated by cupric reductases and CTR1. In these cells, ATP7B, a homolog of ATP7A, is expressed which facilitates copper loading into secretory cuproenzymes in the trans-Golgi network. One critical secretory cuproenzyme specifically expressed in the liver is ceruloplasmin, which is released into the blood and facilitates cellular absorption of serum iron [10]. When liver copper storage exceeds body requirements, ATP7B translocates to the canalicular membrane, where it mediates cellular copper export into the bile which is eliminated through feces [11].

Copper abundance varies across organs with tissues such as the skeletal muscle, brain, and heart, containing approximately 23, 8, and 1% of total body copper, respectively [12]. Copper entry into the brain through the blood-brain barrier requires the combined action of CTR1 and ATP7A [13]. In the brain, copper functions as an essential cofactor for several cuproenzymes including dopamine-β-hydroxylase and peptidylglycine α-amidating monooxygenase which are required for the biosynthesis of neurotransmitter (norepinephrine) and amidated neuropeptides, respectively. The clearance of copper from brain cells occurs via ATP7A, which promotes the excretion of copper through blood cerebrospinal fluid barrier [13].

Intracellular copper distribution

Upon cellular import, copper is immediately bound to either a copper chaperone(s) or to a non-proteinaceous ligand such as glutathione, that shuttles copper to various cuproenzymes present in different subcellular compartments [14, 15]. CCS is one such metallochaperone (see Glossary) that escorts copper from the plasma membrane localized CTR1 to copper-zinc superoxide dismutase, SOD1, which is mainly found in the cytoplasm [16]. Similarly, the cytosolic copper metallochaperone ATOX1 delivers copper to ATP7A and ATP7B, which are present on the Golgi membrane in the trans-Golgi network, where they pump copper into the Golgi lumen for its incorporation into several secretory cuproenzymes [1, 2]. Excess cytoplasmic copper can be sequestered by metallothioneins, cysteine-rich proteins that provide resistance to cellular copper toxicity (Figure 1) [17]. A recent study has uncovered an unexpected role of the nuclear protein complex, histone H3–H4 tetramer, in reducing Cu²⁺ to Cu¹⁺ and in maintaining mitochondrial and cytosolic cuproenzymes activity [18].

Mitochondrial copper homeostasis

Mitochondria serve as a major copper storage site, containing a pool of copper whose molecular identity is unknown [19, 20]. Typically, mammalian mitochondria contain 30–50 nanograms of copper per milligram of mitochondrial protein [21]. Under copper deficiency, cells prioritize mitochondrial copper homeostasis suggesting its critical requirement for this organelle [22]. A significant fraction (~25%) of the mitochondrial copper pool is utilized by cytochrome c oxidase (CcO), the most critical cuproenzyme of mitochondria [23]. As the terminal enzyme of the mitochondrial respiratory chain, CcO catalyzes the electron transfer from reduced cytochrome c to molecular oxygen, while simultaneously pumping protons from the matrix to the intermembrane space (IMS) to contribute to the proton gradient that powers mitochondrial ATP synthesis. This multi-subunit complex is localized to the inner mitochondrial membrane (IMM) and contains copper and heme as cofactors [24]. Three copper ions are bound within the catalytic core of CcO as Cu_A and Cu_B sites, which are present in COX2 and COX1 subunits, respectively [24]. Another important cuproenzyme present in mitochondria is SOD1, which is localized to the IMS, where it neutralizes superoxides produced by the mitochondrial respiratory chain.

For a long time, the evolutionarily conserved protein, COX17, was thought to be the mitochondrial copper shuttle due to its dual localization in the cytoplasm and mitochondria [14]. However, COX17 enters mitochondria in an unfolded state which is unlikely to carry copper into mitochondria [25]. Another piece of evidence that questioned the copper-shuttling role of COX17 was a creatively designed experiment where COX17 was tethered to the IMM using the transmembrane domain of SCO2, a known IMM localized protein [26]. The IMM-tethered COX17 was exclusively localized to mitochondrial IMS and was fully functional, a finding inconsistent with the copper-shuttling role of COX17 [26]. The current hypothesis is that copper is transported into mitochondria via a non-proteinaceous anionic ligand (CuL) [19, 20]. This non-proteinaceous anionic ligand has been identified in the cytosol in both the copper-bound and unbound forms, however, the molecular identity of this ligand has remained unknown to date [20]. Recent advances in chromatographic and mass spectrometry techniques which were used to identify low molecular mass labile metal pools in E. coli [27], could prove to be extremely useful in addressing this long-standing question.

CuL likely enters mitochondria by passive diffusion through porin, a large outer mitochondrial membrane channel (Figure 2). Once in the IMS, copper could be used for the metallation of CcO and SOD1 because the molecular machinery for copper insertion into these proteins resides in the IMS (Figure 2). Curiously, the cytosolic copper is first transported into the mitochondrial matrix via the IMM localized transporter SLC25A3 in mammals and Pic2 in yeast [28, 29]. Pic2-deleted yeast cells do not display a robust mitochondrial copper-deficient phenotype [29]. Moreover, copper supplementation in the growth media can overcome CcO deficiency in SLC25A3 knockout cells [28]. Together, these results suggest an alternate mechanism of copper import into mitochondria. Consistent with this idea, mitochondrial iron transporters from yeast (Mrs3) and fish (MFRN1) have been shown to transport copper as well [30, 31]. A more recent phylogenetic analysis of Pic2 and SLC25A3 identified specific amino acid requirements for copper transport establishing their important role in mitochondrial copper import [32]. Within the mitochondrial matrix, copper is stored in its ligand-bound form and is made bioavailable for the metallation of CcO when required [20]. Interestingly, the matrix copper must be exported back via an unidentified transporter into the IMS for its delivery to CcO as well as SOD1 (Figure 2) [33]. Apart from serving as a feedstock for CcO and SOD1, the biochemical role of this matrix copper pool remains to be elucidated (Figure 2).

Figure 2.

Copper trafficking within mammalian mitochondria.

Copper bound to a ligand (CuL) likely enters mitochondria via OMM localized porin and is then transported into the matrix by the IMM localized transporters SLC25A3 and MFRN1. The function of copper within the matrix is not known but this copper is transported back across the IMM by an unknown transporter. Once in the IMS, copper binds metallochaperones CCS and COX17, for its delivery to SOD1 and CcO, respectively. COX17 transfers copper to metallochaperones, COX11 and SCO1, which in turn inserts it into CcO subunits COX1 and COX2, respectively. COX11, COX19, and COA4 are components of the copper delivery pathway to the Cu_B site within the COX1 subunit. SCO1, SCO2, and COA6 are components of the copper delivery pathway to the Cu_A site within the COX2 subunit. The IMS-localized proteins COX23, CMC2, and COA5 are essential for CcO assembly but their involvement in the copper delivery pathway remains unknown. Abbreviations: OMM, outer mitochondrial membrane; IMS, intermembrane space; IMM, inner mitochondrial membrane. The figure was created with Biorender.com.

The matrix copper is mobilized back into the IMS, where it becomes available to metallochaperones involved in the copper delivery to SOD1 and the copper-containing subunits of CcO (Figure 2). SOD1 receives its copper cofactor from CCS, which is also found in the IMS, though the immediate donor of copper to mitochondrial CCS remains to be determined. Mobilization of copper from the mitochondrial matrix to the IMS for its delivery to copper sites in CcO subunits requires several evolutionarily conserved proteins (Table 1). The delivery of copper to CcO is a very complex process, driven by copper affinity gradients and dedicated copper metallochaperones that facilitate the sequential transfer of copper [34, 35]. The function of these CcO assembly factors can be broadly classified into two types: 1) copper metallochaperones such as COX17 and SCO1, that directly bind and deliver copper through specific protein-protein interactions, or 2) accessory factors such as COA6 and SCO2, that do not transfer copper themselves but rather facilitate this process (Figure 2). Copper delivery to COX1 and COX2 occurs as a modular process, requiring distinct sets of proteins for copper insertion into each subunit [35] (Figure 2). The IMS-localized CX₉C copper metallochaperone COX17 receives copper from the matrix and transfers it to metallochaperones SCO1, SCO2, and COX11 [36]. SCO1 functions as the COX2-specific metallochaperone that inserts copper into the Cu_A site of CcO [37]. COX11 functions as the COX1-specific metallochaperone that delivers copper to the Cu_B site of CcO [38, 39]. A prerequisite for copper binding by these cysteine-based copper metallochaperones is the presence of their cysteines in a reduced form to enable copper binding. This function is provided by SCO2 and COA6, which have been shown to function as disulfide reductases of SCO1 and COX2 proteins [37, 40–42].

Table 1.

Genes involved in mitochondrial copper biology.

Yeast Gene	Human Gene	Function	Refs
COX1	COX1	Subunit of the catalytic core of CcO, contains the CuB site, catalyzes electron transport to molecular oxygen	[24]
COX2	COX2	Subunit of the catalytic core of CcO, contains the CuA site, catalyzes electron transport to molecular oxygen	[24]
COX11	COX11	Metallochaperone, a component of the copper delivery pathway to the CuB site	[38, 39]
COX17	COX17	Metallochaperone, transfers copper to metallochaperones SCO1 and COX11	[36]
COX19	COX19	Precise function unknown, a component of the copper delivery pathway to the CuB site	[46]
COA4	COA4	Precise function unknown, a component of the copper delivery pathway to the CuB site	[45]
COA6	COA6	Disulfide reductase of COX2 and SCO1, a component of the copper delivery pathway to the CuA site	[41, 42, 97]
SCO1	SCO1	Metallochaperone, required for copper delivery to the CuA site	[37]
SCO2	SCO2	Copper-binding thiol-disulfide oxidoreductase of COX2, a component of the copper delivery pathway to the CuA site	[37, 40]
PIC2	SLC25A3	Mitochondrial copper importer	[28, 29]
CCS1	CCS	Metallochaperone, transfers copper to SOD1	[16]
SOD1	SOD1	Cytosol and mitochondria localized copper-zinc superoxide dismutase, detoxifies superoxides	[16, 98]

In addition to these proteins, several evolutionarily conserved twin CX₉C motif-containing IMS proteins including, Cox19, Cox23, Cmc1, Cmc2, Coa4, and Pet191 and have been implicated in the assembly of CcO [43]. Despite sharing a similar fold, these proteins lack the N-terminal cysteine-rich (CCXC) copper-binding motif found in Cox17, suggesting that they may not function as copper metallochaperones. Genetic suppression screens in yeast have linked some of these genes to the CcO copper delivery pathway, suggesting that these proteins may play a non-metallochaperone role in the delivery of copper to CcO. For example, suppression of the respiratory growth defect of yeast cox23 null mutant by COX17 overexpression links it to the mitochondrial copper delivery pathway to CcO [44]. A recent yeast genetic suppressor study has placed Coa4 upstream of Cox11 in the copper delivery pathway to the Cu_B site [45]. Along with genetic interaction studies, protein:protein interaction studies have also proved valuable in placing CX₉C proteins to the copper delivery pathway to CcO. For example, a SILAC-based quantitative proteomic study uncovered a dynamic physical interaction between Cox19 and Cox11, placing Cox19 in the copper delivery to the Cu_B site [46]. Another recent study characterizing human cell lines lacking COX11, COX19, and PET191 (COA5) showed that CcO copper chaperones form macromolecular assemblies and cooperate with several twin CX₉C proteins to coordinate copper transfer sequentially to the Cu_A and Cu_B sites [47]. Despite this progress, the precise molecular function of these proteins in copper delivery to CcO remains unknown.

Another example of the importance of maintaining mitochondrial copper homeostasis comes from a recent study investigating the role of copper ionophore-induced cell death. This study identified lipoylated proteins of the tricarboxylic acid cycle as the target for copper toxicity [48]. Utilizing genome-wide CRISPR screens, the authors showed that the deletion of components of the lipoic acid pathway or lipoylation target, the pyruvate dehydrogenase complex, confers resistance to the two copper ionophores tested [48]. Interestingly, cell death caused by copper overload occurred via a mechanism distinct from other forms of cell death including, apoptosis, ferroptosis, pyroptosis, and necroptosis. Therefore, the authors named this unique form of copper-dependent cell death “cuproptosis” [48]. These findings have important implications in conditions of mitochondrial copper overload, such as in Wilson disease where copper accumulation is observed in certain organs such as the liver.

Considering this finding, it is imperative to identify mitochondrial copper detoxification mechanisms. These would include mitochondrial copper export machinery, copper sequestering proteins, or antioxidant defense mechanisms that would counter copper-induced ROS production. As mentioned previously, mitochondrial matrix copper is exported for the metallation of CcO and SOD1 and it is expected that the same mitochondrial copper exporter plays a role in maintaining copper homeostasis under conditions of mitochondrial copper overload. Alternatively, copper-binding proteins or other ligands may be trafficked to mitochondria to sequester copper and counter its toxicity.

Mitochondrial dysfunction in human genetic diseases of copper metabolism

Mitochondrial and non-mitochondrial cuproenzymes have many different biochemical functions and owing to their diverse roles in cellular and organismal physiology, loss-of-function mutations in genes required for copper absorption, elimination, or transport often result in fatal or severely debilitating disorders (Figure 3). For example, mutations in CcO assembly factors that are involved in copper delivery to CcO including SCO1, SCO2, and COA6, have been shown to cause fatal infantile mitochondrial disorders. These disorders are typically characterized by cardioencephalomyopathy, though significant genetic and clinical heterogeneity is observed in these patients. Mutations and clinical presentations for each of these genes are described in Box 1. Mutations in copper transporters – ATP7A and ATP7B – that lead to whole body, organ, and intracellular copper imbalance cause Menkes disease and Wilson disease, respectively [49, 50]. Although not prototypical mitochondrial disorders, perturbations in mitochondrial copper content in these disorders significantly contribute to the disease pathology. Genetics, epidemiology, and clinical pathology associated with these diseases are described below.

Figure 3.

Cuproenzymes and their role in human physiology and pathology.

The formal and abbreviated names of cuproenzymes as well as their enzymatic and physiological role(s) are listed. Typical clinical presentations of mitochondrial disorders, Menkes disease, and Wilson disease are depicted below, and causative enzyme(s) are indicated. Downward-facing arrows indicate reduced enzymatic activity. The figure was created with Biorender.com.

Box 1. Disorders of the mitochondrial copper delivery pathway.

SCO1:

Mutations in SCO1 have been identified in four infants [100–102]. These patients presented with a variety of cardiac, liver, and neurological ailments including ventricular hypertrophy, hepatomegaly, and brain atrophy. Metabolic acidosis with increased excretion of lactate, fumarate, succinate, and malate in urine has also been reported. All patients died before the age of 6 months. Molecular studies on patient DNA have revealed a variety of loss-of-function mutations including frameshift and point mutations (P174L, G132S, M294V, V93X) some of which introduce a premature stop codon. Biochemical studies on patient samples showed decreased CcO activity in the liver and skeletal muscles, and a reduction in CcO containing supercomplexes in skeletal muscles [100, 101].

SCO2:

As compared to SCO1, a much larger number of patients have been reported with pathogenic mutations in SCO2 [103–106]. These patients presented with hypertrophic cardiomyopathy, hepatomegaly, muscle hypotonia, spinal muscular atrophy, neuropathy, and increased lactate in blood and cerebrospinal fluid. The mutational spectrum in SCO2 patients is broad with some mutations leading to death within six months of age whereas other patients survive to adulthood. For example, patients that carry compound heterozygous mutations with E140K (the most common mutation reported in SCO2 patients) and one of the following mutations W36X, Q53X, R90X, C133S, L151P, M177T, S225F, R171W die within six months of birth [103–108]. There have been cases of compound heterozygous mutations (E140K/P169T and D135G/R171Q) in patients with axonal Charcot-Marie-Tooth disease where patients survived through infancy and developed neuropathy in early adolescence [109]. Biochemical studies on patient tissues revealed decreased CcO activity in skeletal muscles, heart, liver, and fibroblasts [103, 104].

COA6:

Mutations in COA6 have been identified in two patients with hypertrophic cardiomyopathy [110, 111]. A W59C mutation and an E87X mutation were identified in one patient, and a homozygous W66R mutation in another patient. Biochemical studies revealed decreased COX activity in patient heart and skin fibroblasts [110, 111]. Interestingly, copper supplementation was able to rescue the COX2 and CcO levels in cultured patient skin fibroblasts [111].

Menkes disease

Menkes disease is an X-linked multisystemic disorder caused by pathogenic mutations in ATP7A and typically results in death during early childhood [49]. Menkes is a relatively rare disease with incidence estimates ranging from 1/50,000 to 1/360,000 live births depending on the population surveyed, though these numbers may be an underestimate [49]. Clinical presentations for Menkes disease include failure to thrive, connective tissue defects, kinky hair, hypotonia, seizures, and progressive neurodegeneration [51] (Figure 3). As ATP7A is required for directional transport of copper across polarized epithelial cells of the intestine, kidney, and blood-brain barrier, complete or partial loss-of-function of this protein results in systemic copper deficiency in major organs of the body, including the brain, with copper accumulation observed in the polarized epithelial cells. Menkes patients exhibit decreased activity of multiple cuproenzymes and was historically connected to mitochondrial dysfunction due to reduced CcO activity [51, 52]. Ragged-red fibers, a typical feature of classical mitochondrial disorders, were also reported in the skeletal muscle of a Menkes disease patient [53]. Some of the defects observed in the brain of Menkes disease patients including neuronal cell loss in the cerebellum and severe demyelination are partly similar to those in patients with Leigh syndrome, a mitochondrial disorder characterized by biochemical defects in mitochondrial respiratory chain complexes including CcO [54]. The hypoactivity of brain CcO is the primary driver for neurodevelopmental defects observed in these patients. Mitochondrial redox imbalance could also significantly contribute to pathologies caused by the accumulation of copper [55]. Diminished activity of other brain cuproenzymes including dopamine b-hydroxylase and peptide α -amidating monooxygenase, also contributes to clinical presentations. The substrate and product of dopamine β-hydroxylase – dopamine and norepinephrine – are used as reliable diagnostic markers for this disease [56].

Wilson disease

Wilson disease is a rare autosomal recessive genetic disorder caused by mutations in ATP7B [50]. Unlike Menkes disease, which manifests in infancy, clinical presentations of Wilson disease typically become apparent during the teenage years [50]. The prevalence of Wilson disease is also higher than Menkes disease, at 1:30,000 live births [50]. As ATP7B is primarily tasked with eliminating surplus copper from the body, this disorder is characterized by copper accumulation in the liver, brain, kidney, and other tissues dependent on ATP7B function [57]. This disease is progressive and if left untreated results in liver failure, as well as neurological and psychiatric symptoms in a subset of patients [50] (Figure 3). Mitochondrial structural abnormalities have been reported in Wilson disease pathology [58]. As copper accumulates in Wilson disease patient cells and Wilson animal models, mitochondrial membrane integrity is compromised [59]. Critically, the disease status correlated with mitochondrial but not whole-liver copper content [59]. Interestingly, even in pre-symptomatic patients and animal models of Wilson disease, ultrastructural alterations of mitochondria in hepatocytes have been observed [59, 60]. The liver of Wilson disease patients exhibits increased levels of mitochondrial ROS and apoptotic markers [61]. This hepatic injury results in liver damage in the form of cirrhosis and chronic hepatitis, culminating in progressive liver failure [50]. Wilson disease patients with acute hepatic failure present with mitochondrial respiratory chain deficits [62]. In the Wilson disease mouse model tx-j, CcO activity progressively decreased as normalized to mitochondrial abundance [63]. A recent study reported mitochondrial DNA-depletion like syndrome in Wilson disease models and patients. Specifically, mitochondrial DNA copy number was found to be decreased in the blood of Wilson disease patients as well as in the liver of the tx-j mouse model. This finding was consistent with a metabolomic signature that highlighted enriched DNA synthesis/replication pathways in serum metabolomics of Wilson disease patients [64]. Thus, mitochondrial copper burden appears to be one of the key causes of Wilson disease pathology.

The expression of metallothioneins, MT1 and MT2, are highly upregulated in models of Wilson disease and provide protection against non-sequestered copper [65, 66]. Additionally, metallothioneins have been suggested to serve as highly sensitive and specific markers for the detection of Wilson disease in human patients [67]. Interestingly, a previous study had shown that MT1 can also localize to the IMS of mitochondria [68]. This finding raises the question of whether metallothioniens play a protective role in mitochondria of Wilson disease patients.

Additional human pathologies associated with disrupted mitochondrial copper homeostasis

Several neurodegenerative disorders have been linked to either the aberrant function of mitochondrial copper-binding proteins or perturbation in mitochondrial copper homeostasis. Specifically, a subset of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in SOD1, and mitochondrial accumulation of mutant SOD1 is an important contributor to the pathology of this disorder [69]. There is also a large body of literature suggesting that other common neurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease are also characterized by a disruption in copper homeostasis [reviewed in 70]. MEDNIK syndrome and Huppke-Brendel syndrome are rare disorders of copper metabolism that exhibit reduced serum copper and ceruloplasmin levels [71, 72]. MEDNIK syndrome is caused by pathogenic mutations in AP1S1. These patients exhibit symptoms and biochemical markers that are similar to both Menkes (hypotonia, developmental delay, cerebral atrophy, reduced CcO abundance) and Wilson disease (liver copper overload) [71, 73]. This is likely because AP1S1 is required for the proper intracellular trafficking of ATP7A and ATP7B proteins. Huppke-Brendel syndrome is caused by pathogenic mutations in SLC33A1, which encodes an acetyl-coA transporter. These patients exhibit hypotonia, developmental delay, seizures, and neurodevelopmental defects including cerebellar hypoplasia and severe demyelination, clinical presentations that are reminiscent of Menkes disease [72]. Reduced CcO activity has also been reported in one patient [74]. The molecular mechanism by which defective acetyl-coA transporter cause copper imbalance remains to be determined. Additionally, pathogenic mutations in the mitochondrial copper importer SLC25A3 have been reported and shown to cause muscular hypotonia and progressive hypertrophic cardiomyopathy [75].

Copper therapeutics

The therapeutic value of pharmacological agents that can restore cellular and tissue copper homeostasis is increasingly being realized [76, 77]. Many copper-transporting pharmacological agents were initially developed as anti-cancer drugs because cancer cells have a low threshold for tolerating oxidative agents like copper [78, 79]. Owing to their high efficiency in transporting copper across biological membranes, these chemotherapeutic drugs are attractive copper delivery agents in diseases of genetic copper deficiencies (Table 2). For example, our recent studies on elesclomol, a copper ionophore that was originally developed as a chemotherapeutic agent [80], have shown that the administration of low doses of elesclomol ameliorates disease phenotypes in yeast, zebrafish, and mouse models of copper deficiency, including a mouse model of Menkes disease [81, 82]. Elesclomol-copper complex (ES-Cu) restores mitochondrial function in copper deficient cells by making copper bioavailable to CcO [81, 82]. The unique ability of ES-Cu to deliver copper to mitochondria is likely dependent on mitochondrial reductase FDX1, which was recently shown to bind ES-Cu [83]. More recently, our lab has demonstrated that ES-Cu can also make copper bioavailable to the Golgi compartment in a Ccc2 (a yeast ortholog of ATP7A/ATP7B)-dependent manner [84]. This is an important observation as many cuproenzymes are metallated in the Golgi lumen [2]. Notably, other copper-binding drugs such as disulfiram or copper salts such as Cu-histidinate have shown limited efficacy in the treatment of Menkes disease patients [56, 76, 85]. Therefore, there is a dire need for better copper delivery agents. Our recent pre-clinical studies provide a strong motivation for repurposing ES-Cu for the treatment of copper deficiency disorders, including Menkes disease [81, 82, 86].

Table 2.

Copper therapeutics

Compound	Potential therapeutic application	Chemical structure	Refs
Copper ionophores
Elesclomol	Menkes disease, Mitochondrial disorders, Cancer		[80, 86]
CuATSM	ALS, Parkinson’s disease, Cancer		[87, 88, 99]
CuGTSM	Alzheimer’s disease, Cancer		[89, 99]
Disulfiram	Cancer, Alcohol abuse drug		[79]
Copper chelators
D-penicillamine	Wilson disease		[90, 92]
Trientine	Wilson disease		[90, 92]
Tetrathiomolybdate	Wilson disease		[90, 92, 93]

Other copper transporting complexes such as CuATSM and CuGTSM have also been investigated for their use in treating neurodegenerative diseases. For example, CuATSM has been shown to increase the life span of a mouse model of ALS [87] and improve disease phenotypes in animal models of Parkinson’s disease [88]. CuGTSM was reported to be neuroprotective in an animal model of Alzheimer’s disease [89]. While these copper-ionophores have shown early promise in the treatment of animal studies, they likely have a narrow therapeutic window. Therefore, the next challenge lies in expanding their therapeutic index.

In contrast to copper delivery agents, copper chelation therapy aims to remove excess copper accumulation in tissues. The development of multiple copper chelators has allowed for the reduction of copper burden in Wilson disease patients, significantly improving the survival and quality of life (Table 2). For example, D-penicillamine has classically been utilized as a Wilson disease therapeutic as it binds excess free copper forming a copper complex that is excreted through the urine, thereby lowering blood copper levels and preventing hepatic copper accumulation when administered chronically [90]. Though D-penicillamine improves liver symptoms, it has been linked to the worsening of neurologic symptoms in a subset of Wilson disease patients [91]. The subsequent copper chelator developed was trientine, which has an improved safety profile [92]. Tetrathiomolybdate, a more recently developed therapeutic for Wilson disease, is deemed the safest for preserving neurological function and is an effective agent for reducing hepatic copper accumulation [92, 93].

Concluding remarks

Studies from yeast genetics and rare human genetic disorders have highlighted the essential role of mitochondrial copper in cellular and organismal health. Mitochondrial copper deficiency or disruption in copper delivery to CcO underlies a variety of clinical presentations typically affecting neurodevelopment and often resulting in fatal infantile disorders with no Food and Drug Administration (FDA)-approved treatment. Though, exciting studies with elesclomol have opened new possibilities for repurposing this cancer drug for the treatment of copper deficiency disorders such as Menkes disease and associated syndromes [86]. It is also becoming increasingly apparent that the mitochondrial copper pool influences mitochondrial redox homeostasis, iron-sulfur cluster biogenesis, and TCA cycle flux via lipoylation [48, 55, 94]. However, whether matrix copper engages with these pathways under normal physiological conditions remains to be seen. The presence of a copper pool within the mitochondrial matrix raises an important question - Are there yet to be discovered mitochondrial cuproproteins or copper-regulated enzymes that function in the matrix of mitochondria? (See Outstanding Questions). With the ability to make precise genetic lesions through CRISPR-Cas9, the availability of targeted copper delivery through elesclomol, advances in analytical methods such as mass spectrometry-based proteomics and metabolomics, and the emergence of powerful in vivo imaging techniques including nanoscale secondary ion mass spectrometry (nanoSIMS) and X-ray fluorescence microscopy (XFM) [95, 96], we are poised to find answers to some of these outstanding questions and advance our knowledge of mitochondrial copper biology.

Outstanding questions.

What are the cellular and organismal biomarkers of copper deficiency and excess?

What is the chemical identity of copper-ligand that transports copper to the mitochondria?

What is the role of the mitochondrial copper pool?

What are the molecular players involved in mitochondrial copper export?

How sensitive are currently available methods for in vivo copper detection?

Highlights.

Copper is an essential trace element that is crucial for the activity of several cuproenzymes.

Mitochondria play a key role in cellular copper homeostasis and house critical cuproenzymes including, cytochrome c oxidase and superoxide dismutase.

Perturbations in mitochondrial copper homeostasis cause human genetic disorders.

Copper therapeutics targeting mitochondria are gaining interest in the treatment of rare genetic disorders.

Glossary

CcO assembly factor

A broad term used to describe any proteins that are required for the assembly of mitochondrial cytochrome c oxidase

Chelator

Small molecule that binds metal ions, making it non-bioavailable

Ionophore

Small molecule that reversibly binds and transports ions through biological membranes

Metallochaperone

Metal-binding protein that inserts metal ions into target protein(s) via specific protein-protein interactions

Mitochondrial disorders

Mitochondrial disorders are defined by a biochemical defect in the mitochondrial respiratory chain and are caused by mutations in proteins that localize to mitochondria