Elesclomol restores mitochondrial function in genetic models of copper deficiency.

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

X-linked spinal muscular atrophy in mice caused by autonomous loss of ATP7A in the motor neuron.

ATP7A is a copper-transporting P-type ATPase that is essential for cellular copper homeostasis. Loss-of-function mutations in the ATP7A gene result in Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia and failure to thrive, due to systemic copper deficiency. Most recently, rare missense mutations in ATP7A that do not impact systemic copper homeostasis have been shown to cause X-linked spinal muscular atrophy type 3 (SMAX3), a distal hereditary motor neuropathy. An understanding of the mechanistic and pathophysiological basis of SMAX3 is currently lacking, in part because the disease-causing mutations have been shown to confer both loss- and gain-of-function properties to ATP7A, and because there is currently no animal model of the disease. In this study, the Atp7a gene was specifically deleted in the motor neurons of mice, resulting in a degenerative phenotype consistent with the clinical features in affected patients with SMAX3, including the progressive deterioration of gait, age-dependent muscle atrophy, denervation of neuromuscular junctions and a loss of motor neuron cell bodies. Taken together, these data reveal autonomous requirements for ATP7A that reveal essential roles for copper in the maintenance and function of the motor neuron, and suggest that SMAX3 is caused by a loss of ATP7A function that specifically impacts the spinal motor neuron.

Autonomous requirements of the Menkes disease protein in the nervous system.

Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7a(Nes) mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7a(Nes) mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7a(Nes) mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients.

Kinesin family member 6 (kif6) is necessary for spine development in zebrafish.

BACKGROUND: Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. RESULTS: We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6(gw326) ) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6(gw326) mutants. CONCLUSIONS: Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.

Maternofetal and neonatal copper requirements revealed by enterocyte-specific deletion of the Menkes disease protein.

The essential requirement for copper in early development is dramatically illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by loss-of-function mutations in the gene encoding the copper transporting ATPase ATP7A. In this study, we generated mice with enterocyte-specific knockout of the murine ATP7A gene (Atp7a) to test its importance in dietary copper acquisition. Although mice lacking Atp7a protein within intestinal enterocytes appeared normal at birth, they exhibited profound growth impairment and neurological deterioration as a consequence of copper deficiency, resulting in excessive mortality prior to weaning. Copper supplementation of lactating females or parenteral copper injection of the affected offspring markedly attenuated this rapid demise. Enterocyte-specific deletion of Atp7a in rescued pregnant females did not restrict embryogenesis; however, copper accumulation in the late-term fetus was severely reduced, resulting in early postnatal mortality. Taken together, these data demonstrate unique and specific requirements for enterocyte Atp7a in neonatal and maternofetal copper acquisition that are dependent on dietary copper availability, thus providing new insights into the mechanisms of gene-nutrient interaction essential for early human development.

Atp7a determines a hierarchy of copper metabolism essential for notochord development.

The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.

Zebrafish mutants calamity and catastrophe define critical pathways of gene-nutrient interactions in developmental copper metabolism.

Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism.

In vivo correction of a Menkes disease model using antisense oligonucleotides.

Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish orthologue of the ATP7A gene. Embryonic-recessive lethal mutants have impaired copper homeostasis that results in absent melanin pigmentation, impaired notochord formation, and hindbrain neurodegeneration. In this current study, we have attempted to rescue these striking phenotypic alterations by using a series of antisense morpholino oligonucleotides directed against the splice-site junctions of two mutant calamity alleles. Our findings reveal a robust and complete correction of the copper-deficient defects of calamity in association with the generation of the WT Menkes protein in all rescued mutants. Interestingly, a quantitative analysis of atp7a-specific transcripts suggests that competitive translational regulation may account for the synthesis of WT protein in these embryos. This in vivo correction of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the potential for zebrafish models of human genetic disease in the development of treatments based on the principles of interactions of synthetic oligonucleotide analogues with mRNA.

Assembling the pieces.

Transition metals function as cofactors in specific proteins, catalyzing electron exchange reactions, binding substrates and stabilizing protein structure. Studies of human diseases and of model organisms have defined many of the molecular details of metal uptake, trafficking, and excretion. The current challenge is to integrate these details into a systematic view of metal content, speciation, localization and use within organisms and ecosystems.

Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading.

Copper-mediated oxidative damage is proposed to play a critical role in the pathogenesis of Cu/Zn superoxide dismutase (SOD1)-linked familial amyotrophic lateral sclerosis (FALS). We tested this hypothesis by ablating the gene encoding the copper chaperone for SOD1 (CCS) in a series of FALS-linked SOD1 mutant mice. Metabolic 64Cu labeling in SOD1-mutant mice lacking the CCS showed that the incorporation of copper into mutant SOD1 was significantly diminished in the absence of CCS. Motor neurons in CCS-/- mice showed increased rate of death after facial nerve axotomy, a response documented for SOD1-/- mice. Thus, CCS is necessary for the efficient incorporation of copper into SOD1 in motor neurons. Although the absence of CCS led to a significant reduction in the amount of copper-loaded mutant SOD1, however, it did not modify the onset and progression of motor neuron disease in SOD1-mutant mice. Hence, CCS-dependent copper loading of mutant SOD1 plays no role in the pathogenesis of motor neuron disease in these mouse models.

Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzyme.

The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam -/- mice and reveals additional defects. The pam -/- zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis.

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.

Characterization of trace metal content in the developing zebrafish embryo.

Trace metals are essential for health but toxic when present in excess. The maintenance of trace metals at physiologic levels reflects both import and export by cells and absorption and excretion by organs. The mechanism by which this maintenance is achieved in vertebrate organisms is incompletely understood. To explore this, we chose zebrafish as our model organism, as they are amenable to both pharmacologic and genetic manipulation and comprise an ideal system for genetic screens and toxicological studies. To characterize trace metal content in developing zebrafish, we measured levels of three trace elements, copper, zinc, and manganese, from the oocyte stage to 30 days post-fertilization using inductively coupled plasma mass spectrometry. Our results indicate that metal levels are stable until zebrafish can acquire metals from the environment and imply that the early embryo relies on maternal contribution of metals to the oocyte. We also measured metal levels in bodies and yolks of embryos reared in presence and absence of the copper chelator neocuproine. All three metals exhibited different relative abundances between yolks and bodies of embryos. While neocuproine treatment led to an expected phenotype of copper deficiency, total copper levels were unaffected, indicating that measurement of total metal levels does not equate with measurement of biologically active metal levels. Overall, our data not only can be used in the design and execution of genetic, physiologic, and toxicologic studies but also has implications for the understanding of vertebrate metal homeostasis.

NMDA receptor activation mediates copper homeostasis in hippocampal neurons.

Copper is an essential transition metal with a critical role in the CNS. This requirement is underscored by Menkes disease, a fatal neurodegenerative disorder of childhood resulting from the absence or dysfunction of a copper-transporting P-type ATPase. To elucidate the cell biological mechanisms of copper homeostasis in the CNS, a polyclonal antisera against Menkes ATPase was used in immunoblot and immunohistochemical studies, demonstrating abundant expression of this copper transporter in hippocampal neurons. Consistent with this observation, immunofluorescent analysis revealed Menkes ATPase in the late Golgi of hippocampal neurons in primary culture. Glutamate receptor activation was found to result in the rapid and reversible trafficking of Menkes ATPase to neuronal processes, independent of the intracellular copper concentration and specific for activation of the NMDA- but not AMPA/kainate-type glutamate receptors. Metabolic studies revealed that trafficking of Menkes ATPase after NMDA receptor activation is associated with rapid release of copper from hippocampal neurons. Menkes ATPase is directly required for this copper efflux, because similar studies in hippocampal neurons derived from mice lacking a functional Menkes ATPase demonstrated no copper release. Together, these data reveal a critical role for Menkes ATPase in the availability of an NMDA receptor-dependent, releasable pool of copper in hippocampal neurons and demonstrate a unique mechanism linking copper homeostasis and neuronal activation within the CNS.

Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.

Copper is an essential nutrient that plays a fundamental role in the biochemistry of the central nervous system, as evidenced by patients with Menkes disease, a fatal neurodegenerative disorder of childhood resulting from the loss-of-function of a copper-transporting P-type adenosine triphosphatase (ATPase). Despite clinical and experimental data indicating a role for copper in brain function, the mechanisms and timing of the critical events affected by copper remain poorly understood. A novel role for the Menkes ATPase has been identified in the availability of an N-methyl-D-aspartate (NMDA) receptor-dependent, releasable pool of copper in hippocampal neurons, suggesting a unique mechanism linking copper homeostasis and neuronal activation within the central nervous system. This article explores the evidence that copper acts as a modulator of neuronal transmission, and that the release of endogenous copper from neurons may regulate NMDA receptor activity. The relationship between impaired copper homeostasis and neuropathophysiology suggests that impairment of copper efflux could alter neuronal function and thus contribute to rapid neuronal degeneration.

The copper-iron connection: hereditary aceruloplasminemia.

Hereditary aceruloplasminemia is an autosomal recessive disorder of iron homeostasis due to loss-of-function mutations in the ceruloplasmin gene. Affected individuals may present in adulthood with evidence of hepatic iron overload, diabetes, peripheral retinal degeneration, dystonia, dementia, or dysarthria. Laboratory studies demonstrate microcytic anemia, elevated serum ferritin, and a complete absence of serum ceruloplasmin ferroxidase activity. Consistent with the observed neurologic findings, magnetic resonance imaging reveals iron accumulation within the basal ganglia. Histologic studies detect abundant iron in hepatocytes, reticuloendothelial cells of the liver and spleen, beta cells of the pancreas, and astrocytes and neurons throughout the central nervous system. Characterization of this disorder reveals an essential role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores and provides new insights into the mechanisms of human iron metabolism.

Copper homeostasis: specialized functions of the late secretory pathway.

Differentiated cells have evolved mechanisms to adapt the functions of the late secretory pathway to the specific needs of the organism. Reporting in this issue of Developmental Cell, Polishchuk et al. (2014) demonstrate that hepatocytes utilize a unique exocytic aspect of the late endosomal/lysosomal compartment to maintain organismal copper homeostasis.