Response of Fibroblasts from Menkes' and Wilson's Copper Metabolism-Related Disorders to Ionizing Radiation: Influence of the Nucleo-Shuttling of the ATM Protein Kinase.

Menkes' disease (MD) and Wilson's disease (WD) are two major copper (Cu) metabolism-related disorders caused by mutations of the ATP7A and ATP7B ATPase gene, respectively. While Cu is involved in DNA strand breaks signaling and repair, the response of cells from both diseases to ionizing radiation, a common DNA strand breaks inducer, has not been investigated yet. To this aim, three MD and two WD skin fibroblasts lines were irradiated at two Gy X-rays and clonogenic cell survival, micronuclei, anti-γH2AX, -pATM, and -MRE11 immunofluorescence assays were applied to evaluate the DNA double-strand breaks (DSB) recognition and repair. MD and WD cells appeared moderately radiosensitive with a delay in the radiation-induced ATM nucleo-shuttling (RIANS) associated with impairments in the DSB recognition. Such delayed RIANS was notably caused in both MD and WD cells by a highly expressed ATP7B protein that forms complexes with ATM monomers in cytoplasm. Interestingly, a Cu pre-treatment of cells may influence the activity of the MRE11 nuclease and modulate the radiobiological phenotype. Lastly, some high-passage MD cells cultured in routine may transform spontaneously becoming immortalized. Altogether, our findings suggest that exposure to ionizing radiation may impact on clinical features of MD and WD, which requires cautiousness when affected patients are submitted to radiodiagnosis and, eventually, radiotherapy.

Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain.

Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.

Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease.

Kidneys play an especial role in copper redistribution in the organism. The epithelial cells of proximal tubules perform the functions of both copper uptake from the primary urine and release to the blood. These cells are equipped on their apical and basal membrane with copper transporters CTR1 and ATP7A. Mosaic mutant mice displaying a functional dysfunction of ATP7A are an established model of Menkes disease. These mice exhibit systemic copper deficiency despite renal copper overload, enhanced by copper therapy, which is indispensable for their life span extension. The aim of this study was to analyze the expression of Slc31a1 and Slc31a2 genes (encoding CTR1/CTR2 proteins) and the cellular localization of the CTR1 protein in suckling, young and adult mosaic mutants. Our results indicate that in the kidney of both intact and copper-injected 14-day-old mutants showing high renal copper content, CTR1 mRNA level is not up-regulated compared to wild-type mice given a copper injection. The expression of the Slc31a1 gene in 45-day-old mice is even reduced compared with intact wild-type animals. In suckling and young copper-injected mutants, the CTR1 protein is relocalized from the apical membrane to the cytoplasm of epithelial cells of proximal tubules, the process which prevents copper transport from the primary urine and, thus, protects cells against copper toxicity.

The molecular mechanisms of copper metabolism and its roles in human diseases.

Copper is an essential element in cells; it can act as either a recipient or a donor of electrons, participating in various reactions. However, an excess of copper ions in cells is detrimental as these copper ions can generate free radicals and increase oxidative stress. In multicellular organisms, copper metabolism involves uptake, distribution, sequestration, and excretion, at both the cellular and systemic levels. Mammalian enterocytes take in bioavailable copper ions from the diet in a Ctr1-dependent manner. After incorporation, cuprous ions are delivered to ATP7A, which pumps Cu+ from enterocytes into the blood. Copper ions arrive at the liver through the portal vein and are incorporated into hepatocytes by Ctr1. Then, Cu+ can be secreted into the bile or the blood via the Atox1/ATP7B/ceruloplasmin route. In the bloodstream, this micronutrient can reach peripheral tissues and is again incorporated by Ctr1. In peripheral tissue cells, cuprous ions are either sequestrated by molecules such as metallothioneins or targeted to utilization pathways by chaperons such as Atox1, Cox17, and CCS. Copper metabolism must be tightly controlled in order to achieve homeostasis and avoid disorders. A hereditary or acquired copper unbalance, including deficiency, overload, or misdistribution, may cause or aggravate certain diseases such as Menkes disease, Wilson disease, neurodegenerative diseases, anemia, metabolic syndrome, cardiovascular diseases, and cancer. A full understanding of copper metabolism and its roles in diseases underlies the identification of novel effective therapies for such diseases.

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells.

Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2'-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease.

Copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. Here we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamitygw71 zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamitygw71 zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamitygw71 zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease.

Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher concentrations. Here we have analyzed the effect of 36 ATP7A missense mutations identified in phenotypically different MD patients. Nine mutations identified in patients with severe MD, virtually eliminated ATP7A synthesis, in most cases due to aberrant RNA splicing. A group of 21 predominantly severe mutations led to trapping of the protein in TGN and displayed essentially no activity in a yeast-based functional assay. These were predicted to inhibit the catalytic phosphorylation of the protein. Four mutants showed diffuse post-TGN localization, while two displayed copper dependent trafficking. These six variants were identified in patients with mild MD and typically displayed activity in the yeast assay. The four post-TGN located mutants were presumably affected in the catalytic dephosphorylation of the protein. Together these results indicate that the severity of MD correlate with cellular localization of ATP7A and support previous studies indicating that phosphorylation is crucial for the exit of ATP7A from TGN, while dephosphorylation is crucial for recycling back to TGN.

The role of insufficient copper in lipid synthesis and fatty-liver disease.

The essential transition metal copper is important in lipid metabolism, redox balance, iron mobilization, and many other critical processes in eukaryotic organisms. Genetic diseases where copper homeostasis is disrupted, including Menkes disease and Wilson disease, indicate the importance of copper balance to human health. The severe consequences of insufficient copper supply are illustrated by Menkes disease, caused by mutation in the X-linked ATP7A gene encoding a protein that transports copper from intestinal epithelia into the bloodstream and across the blood-brain barrier. Inadequate copper supply to the body due to poor diet quality or malabsorption can disrupt several molecular level pathways and processes. Though much of the copper distribution machinery has been described and consequences of disrupted copper handling have been characterized in human disease as well as animal models, physiological consequences of sub-optimal copper due to poor nutrition or malabsorption have not been extensively studied. Recent work indicates that insufficient copper may be important in a number of common diseases including obesity, ischemic heart disease, and metabolic syndrome. Specifically, marginal copper deficiency (CuD) has been reported as a potential etiologic factor in diseases characterized by disrupted lipid metabolism such as non-alcoholic fatty-liver disease (NAFLD). In this review, we discuss the available data suggesting that a significant portion of the North American population may consume insufficient copper, the potential mechanisms by which CuD may promote lipid biosynthesis, and the interaction between CuD and dietary fructose in the etiology of NAFLD. © 2016 IUBMB Life, 69(4):263-270, 2017.

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

Diagnostic copper imaging of Menkes disease by synchrotron radiation-generated X-ray fluorescence analysis.

Copper (Cu) is an indispensable metal for normal development and function of humans, especially in central nervous system (CNS). However, its redox activity requires accurate Cu transport system. ATP7A, a main Cu(2+) transporting-ATPase, is necessary to efflux Cu across the plasma membrane and synthesize cuproenzymes. Menkes disease (MD) is caused by mutations in ATP7A gene. Clinically, MD is Cu deficiency syndrome and is treated with Cu-histidine injections soon after definite diagnosis. But outcome of the most remains poor. To estimate the standard therapy, Cu distribution in the treated classic MD patients is analyzed by synchrotron-generated X-ray fluorescence technique (SR-XRF), which identifies and quantifies an individual atom up to at subcellular level of resolution with wide detection area. SR-XRF analysis newly reveals that Cu exists in spinal cord parenchyma and flows out via venous and lymph systems. By systemic analysis, excess Cu is detected in the proximal tubular cells of the kidney, the mucosal epithelial cells of the intestine, and the lymph and venous systems. The current study suggests that the standard therapy supply almost enough Cu for patient tissues. But given Cu passes through the tissues to venous and lymph systems, or accumulate in the cells responsible for Cu absorption.

Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells.

INTRODUCTION: Bone abnormalities, one of the primary manifestations of Menkes disease (MD), include a weakened bone matrix and low mineral density. However, the molecular and cellular mechanisms underlying these bone defects are poorly understood. METHODS: We present in vitro modeling for impaired osteogenesis in MD using human induced pluripotent stem cells (iPSCs) with a mutated ATP7A gene. MD-iPSC lines were generated from two patients harboring different mutations. RESULTS: The MD-iPSCs showed a remarkable retardation in CD105 expression with morphological anomalies during development to mesenchymal stem cells (MSCs) compared with wild-type (WT)-iPSCs. Interestingly, although prolonged culture enhanced CD105 expression, mature MD-MSCs presented with low alkaline phosphatase activity, reduced calcium deposition in the extracellular matrix, and downregulated osteoblast-specific genes during osteoblast differentiation in vitro. Knockdown of ATP7A also impaired osteogenesis in WT-MSCs. Lysyl oxidase activity was also decreased in MD-MSCs during osteoblast differentiation. CONCLUSIONS: Our findings indicate that ATP7A dysfunction contributes to retardation in MSC development and impairs osteogenesis in MD.

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.

Small amounts of functional ATP7A protein permit mild phenotype.

Mutations in ATP7A lead to at least three allelic disorders: Menkes disease (MD), Occipital horn syndrome and X-linked distal motor neuropathy. These disorders are mainly seen in male individuals, but a few affected females have been described. More than 400 different mutations have been identified in the ATP7A gene. We have conducted several studies in the hope of uncovering the relationship between genotype and phenotype. We have examined the X-inactivation pattern in affected females, the effect of exon-deletions and--duplications, and splice-site mutations on the composition and amount of ATP7A transcript, and we have examined the structural location of missense mutations. The X-inactivation pattern did not fully explain the manifestation of MD in a small fraction of carriers. Most of the affected females had preferential inactivation of the X-chromosome with the normal ATP7A gene, but a few individuals exhibited preferential inactivation of the X-chromosome with the mutated ATP7A gene. The observed mild phenotype in some patients with mutations that effect the composition of the ATP7A transcript, seems to be explained by the presence of a small amount of normal ATP7A transcript. The location of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with copper, whereas at higher concentrations, ATP7A shifts to the post-Golgi compartments or to the plasma membrane to export copper out of the cell. Impaired copper-regulation trafficking has been observed for ATP7A mutants, but its impact on the clinical outcome is not clear. The major problem in patients with MD seems to be insufficient amounts of copper in the brain. In fact, prenatal treatment of mottled mice as a model for human MD with a combination of chelator and copper, produces a slight increase in copper levels in the brain which perhaps leads to longer survival and more active behavior. In conclusion, small amounts of copper at the right location seem to relieve the symptoms.

Molecular and biochemical characterization of Mottled-dappled, an embryonic lethal Menkes disease mouse model.

Mottled-dappled (Mo-dp) is a mouse model of Menkes disease caused by a large, previously uncharacterized deletion in the 5' region of Atp7a, the mouse ortholog of ATP7A. Affected mutants die in utero at embryonic day 17, and show bending and thickening of the ribs and distortion of the pectoral and pelvic girdles and limbs. To characterize this allele, we designed a custom 4x180K microarray on the mouse X chromosome and performed comparative genomic hybridization using extracted DNA from normal and carrier Mo-dp females, and identified an approximately 9 kb deletion. We used PCR to fine-map the breakpoints and amplify a junction fragment of 630 bp. Sequencing of the junction fragment disclosed the exact breakpoint locations and that the Mo-dp deletion is precisely 8990 bp, including approximately 2 kb in the promoter region of Atp7a. Western blot analysis of Mo-dp heterozygous brains showed diminished amounts of Atp7a protein, consistent with reduced expression due to the promoter region deletion on one allele. In heterozygous females, brain copper levels tended to be lower compared to wild type whereas neurochemical analyses revealed higher dihydroxyphenylacetic acid:dihydroxyphenylglycol (DOPAC:DHPG) and dopamine:norepinephrine (DA:NE) ratios compared to normal (P=0.002 and 0.029, respectively), consistent with partial deficiency of dopamine-beta-hydroxylase, a copper-dependent enzyme. Heterozygous females showed no significant differences in body weight compared to wild type females. Our results delineate the molecular details of the Mo-dp mutation for the first time and define novel biochemical findings in heterozygous female carriers of this allele.

Catecholamine metabolites affected by the copper-dependent enzyme dopamine-beta-hydroxylase provide sensitive biomarkers for early diagnosis of menkes disease and viral-mediated ATP7A gene therapy.

Menkes disease is a lethal X-linked recessive disorder of copper metabolism caused by mutations in ATP7A, a copper-transporting ATPase with diverse and important biological functions. Partial deficiency of dopamine-beta-hydroxylase is a biochemical hallmark of this illness due to the normal role of ATP7A in delivery of copper as an enzymatic cofactor. We exploited this fact to develop a diagnostic test for Menkes disease, which proved highly sensitive and specific. The assay has enabled early identification of affected patients, leading to enhanced survival and improved neurodevelopment after early copper treatment, including some completely normal outcomes. In preclinical efforts to develop improved therapies for patients with non-copper-responsive ATP7A mutations, we used brain-directed adeno-associated viral gene therapy to rescue a murine model of the disease. Statistically significant improvement in brain catechol ratios correlated with enhanced survival, and cerebrospinal fluid catechols represent candidate surrogate markers of treatment effect in a future gene therapy clinical trial.

L-threo-dihydroxyphenylserine corrects neurochemical abnormalities in a Menkes disease mouse model.

OBJECTIVE: Menkes disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting adenosine triphosphatase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine beta hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes disease, mottled-brindled (mo-br), we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology. METHODS: At 8, 10, and 12 days of age, wild-type and mo-br mice received intraperitoneal injections of 200µg/g body weight of L-DOPS, or mock solution. Five hours after the final injection, the mice were euthanized, and brains were removed. We measured catecholamine metabolites affected by DBH via high-performance liquid chromatography with electrochemical detection, and assessed brain histopathology. RESULTS: Compared to mock-treated controls, mo-br mice that received intraperitoneal L-DOPS showed significant increases in brain norepinephrine (p < 0.001) and its deaminated metabolite, dihydroxyphenylglycol (p < 0.05). The ratio of a non-beta-hydroxylated metabolite in the catecholamine biosynthetic pathway, dihydroxyphenylacetic acid, to the beta-hydroxylated metabolite, dihydroxyphenylglycol, improved equivalently to results obtained previously with brain-directed ATP7A gene therapy (p < 0.01). However, L-DOPS treatment did not arrest global brain pathology or improve somatic growth, as gene therapy had. INTERPRETATION: We conclude that (1) L-DOPS crosses the blood-brain barrier in mo-br mice and corrects brain neurochemical abnormalities, (2) norepinephrine deficiency is not the cause of neurodegeneration in mo-br mice, and (3) L-DOPS treatment may ameliorate noradrenergic hypofunction in Menkes disease.

Mutation in the CPC motif-containing 6th transmembrane domain affects intracellular localization, trafficking and copper transport efficiency of ATP7A protein in mosaic mutant mice--an animal model of Menkes disease.

Copper is an essential micronutrient for all living organisms. ATP7A protein is a copper-transporting ATPase which plays a vital role in the maintenance of cellular copper homeostasis in mammals. This protein is retained within the trans-Golgi network, but after binding copper it can be translocated to the cell membrane to participate in the efflux of excess Cu. Mutation of the ATP7A gene in humans results in the severe neurodegenerative disorder, Menkes disease. The mouse ATP7A homolog encodes a protein that plays the same role in copper transport. Mosaic mutant mice display a lethal phenotype which resembles Menkes disease, although the underlying molecular defect has not been characterized until now. In the present study we identified a G to C nucleotide exchange in exon 15 of the Atp7a gene in mosaic mutants, which resulted in an arginine to proline substitution in the highly conserved 6th transmembrane domain of the ATP7A protein. This mutated protein was mislocalized in kidney cells isolated from mosaic mutant mice, and following exposure of these cells to increased copper concentrations it was not translocated to the plasma membrane. Disturbance of ATP7A function in mosaic mice results in increased copper accumulation in the small intestine and kidneys, and in Cu deficiency in the brain, liver and heart. Mouse models of Menkes disease belong to the mottled mutant group. The mosaic mutant represents another interesting animal model for Menkes disease that will be of value in research on copper metabolism and transport in mammals.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.