Publications by authors named "Martina Ralle"

48 Publications

Single-cell visualization and quantification of trace metals in lysosome-related organelles.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
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http://dx.doi.org/10.1073/pnas.2026811118DOI Listing
April 2021

Transcription factor-driven alternative localization of Cryptococcus neoformans superoxide dismutase.

J Biol Chem 2021 Feb 7:100391. Epub 2021 Feb 7.

Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, United States of America; Department of Biochemistry, Duke University, Durham, North Carolina, United States of America; Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America. Electronic address:

Cryptococcus neoformans is an opportunistic fungal pathogen whose pathogenic lifestyle is linked to its ability to cope with fluctuating levels of copper (Cu), an essential metal involved in multiple virulence mechanisms, within distinct host niches. During lethal cryptococcal meningitis in the brain, C. neoformans senses a Cu deficient environment and is highly dependent upon its ability to scavenge trace levels of Cu from its host and adapt to Cu scarcity to successfully colonize this niche. In this study we demonstrate for this critical adaptation, the Cu-sensing transcription factor Cuf1 differentially regulates the expression of the SOD1 and SOD2 superoxide dismutases in novel ways. Genetic and transcriptional analysis reveals Cuf1 specifies 5'-truncations of the SOD1 and SOD2 mRNAs through specific binding to copper responsive elements (CuREs) within their respective promoter regions. This results in Cuf1-dependent repression of the highly abundant SOD1 and simultaneously induces expression of two isoforms of SOD2 from a single alternative transcript produced specifically under Cu limitation; the canonical mitochondrial targeted isoform and a novel alternative cytosolic isoform. The generation of cytosolic Sod2 during Cu limitation is required to maintain cellular antioxidant defense against superoxide stress both in vitro and in vivo. Further, decoupling Cuf1 regulation of Sod2 localization compromises the ability of C. neoformans to colonize organs in murine models of cryptococcosis. Our results provide a link between transcription factor-mediated alteration of protein localization and cell proliferation under stress, which could impact tissue colonization by a fungal pathogen.
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http://dx.doi.org/10.1016/j.jbc.2021.100391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961099PMC
February 2021

Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation.

Metallomics 2020 12;12(12):1995-2008

Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.
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http://dx.doi.org/10.1039/d0mt00156bDOI Listing
December 2020

Analysis of Wilson disease mutations revealed that interactions between different ATP7B mutants modify their properties.

Sci Rep 2020 08 10;10(1):13487. Epub 2020 Aug 10.

Department of Physiology, Johns Hopkins Medical Institute, Baltimore, MD, USA.

Wilson disease (WD) is an autosomal-recessive disorder caused by mutations in the copper (Cu)-transporter ATP7B. Thus far, studies of WD mutations have been limited to analysis of ATP7B mutants in the homozygous states. However, the majority of WD patients are compound-heterozygous, and how different mutations on two alleles impact ATP7B properties is unclear. We characterized five mutations identified in Indian WD patients, first by expressing each alone and then by co-expressing two mutants with dissimilar properties. Mutations located in the regulatory domains of ATP7B-A595T, S1362A, and S1426I-do not affect ATP7B targeting to the trans-Golgi network (TGN) but reduce its Cu-transport activity. The S1362A mutation also inhibits Cu-dependent trafficking from the TGN. The G1061E and G1101R mutations, which are located within the ATP-binding domain, cause ATP7B retention in the endoplasmic reticulum, inhibit Cu-transport, and lower ATP7B protein abundance. Co-expression of the A595T and G1061E mutations, which mimics the compound-heterozygous state of some WD patients, revealed an interaction between these mutants that altered their intracellular localization and trafficking under both low and high Cu conditions. These findings highlight the need to study WD variants in both the homozygous and compound-heterozygous states to better understand the genotype-phenotype correlations and incomplete penetrance observed in WD.
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http://dx.doi.org/10.1038/s41598-020-70366-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418023PMC
August 2020

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

Sci Rep 2020 05 12;10(1):7856. Epub 2020 May 12.

The Genetics Area Program, University of Missouri, Columbia, MO, 65211, USA.

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.
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http://dx.doi.org/10.1038/s41598-020-64521-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217913PMC
May 2020

Copper Modulation and Memory Impairment due to Hippocampal Tau Pathology

J Alzheimers Dis 2020 ;78(1):49-60

Department of Neurology, Oregon Health and Science University, Portland, OR, USA.

Background:Environmental copper has been implicated in the pathogenesis of Alzheimer’s disease based on evidence that: 1) brain copper levels increase with age, 2) copper promotes misfolding and toxicity of amyloid-β in vitro, 3) copper-modulating interventions reduce amyloid pathology in animal models. However, the effect of copper upon non-amyloid Alzheimer’s pathology is relatively under-explored.Objective:To determine if modulation of brain copper level affects brain tau pathology and/or associated cognitive impairment.Methods:We tested the hypothesis that brain copper modulates tau pathology by manipulating brain levels of copper in the PS19 transgenic mouse model of tau pathology. We treated PS19 and wild-type mice with oral zinc acetate, an established therapy for long term control of excess brain copper, and examined treatment effects upon brain copper, brain tau, NFT-like pathology, and spatial memory. We treated a second cohort of mice with exogenous dietary copper in order to evaluate whether excess environmental copper promotes brain tau pathology.Results:Copper-lowering with oral zinc attenuated spatial memory impairment in female but not male PS19 mice, without a significant effect upon tau pathology. Copper loading increased brain copper, but did not have an effect on brain tau pathology or spatial memory function.Conclusion:These findings suggest that a strategy to lower brain copper may be viable for symptomatic benefit in the setting of tau neuropathology, but unlikely to have robust effects on the underlying pathology. These findings are consistent with dietary or other exogenous copper being unlikely to promote tau pathology.
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http://dx.doi.org/10.3233/JAD-200002DOI Listing
January 2020

Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution.

J Am Chem Soc 2020 02 21;142(5):2145-2149. Epub 2020 Jan 21.

National Synchrotron Light Source II , Brookhaven National Laboratory , Upton , New York 11973 , United States.

We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM). This approach enables visualization of LBT-tagged proteins while simultaneously measuring the elemental distribution in cells at a spatial resolution necessary for visualizing cell membranes and eukaryotic subcellular organelles.
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http://dx.doi.org/10.1021/jacs.9b11571DOI Listing
February 2020

Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopy.

Curr Opin Chem Biol 2020 04 3;55:19-25. Epub 2020 Jan 3.

Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, United States. Electronic address:

Synchrotron-based X-ray fluorescence microscopy (XFM) has become an important imaging technique to investigate elemental concentrations and distributions in biological specimens. Advances in technology now permit imaging at resolutions rivaling that of electron microscopy, and researchers can now visualize elemental concentrations in subcellular organelles when using appropriate correlative methods. XFM is an especially valuable tool to determine the distribution of endogenous trace metals that are involved in neurodegenerative diseases. Here, we discuss the latest research on the unusual copper (Cu) storage vesicles that were originally identified in mouse brains and the involvement of Cu in Alzheimer's disease. Finally, we provide an outlook of how future improvements to XFM will drive current trace element research forward.
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http://dx.doi.org/10.1016/j.cbpa.2019.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237281PMC
April 2020

Copper and the brain noradrenergic system.

J Biol Inorg Chem 2019 12 5;24(8):1179-1188. Epub 2019 Nov 5.

Department of Physiology, Johns Hopkins Medical Institutes, 725 N Wolfe St, Baltimore, MD, 21205, USA.

Copper (Cu) plays an essential role in the development and function of the brain. In humans, genetic disorders of Cu metabolism may cause either severe Cu deficiency (Menkes disease) or excessive Cu accumulation (Wilson disease) in the brain tissue. In either case, the loss of Cu homeostasis results in catecholamine misbalance, abnormal myelination of neurons, loss of normal brain architecture, and a spectrum of neurologic and/or psychiatric manifestations. Several metabolic processes have been identified as particularly sensitive to Cu dis-homeostasis. This review focuses on the role of Cu in noradrenergic neurons and summarizes the current knowledge of mechanisms that maintain Cu homeostasis in these cells. The impact of Cu misbalance on catecholamine metabolism and functioning of noradrenergic system is discussed.
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http://dx.doi.org/10.1007/s00775-019-01737-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941745PMC
December 2019

4'-Phosphopantetheine corrects CoA, iron, and dopamine metabolic defects in mammalian models of PKAN.

EMBO Mol Med 2019 12 29;11(12):e10489. Epub 2019 Oct 29.

Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR, USA.

Pantothenate kinase-associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease-vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4'-phosphopantetheine, normalized levels of the CoA-, iron-, and dopamine-related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4'-phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron-sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4'-phosphopantetheine as a candidate therapeutic for PKAN.
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http://dx.doi.org/10.15252/emmm.201910489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895607PMC
December 2019

Hemozoin produced by mammals confers heme tolerance.

Elife 2019 10 1;8. Epub 2019 Oct 1.

Department of Animal and Avian Sciences, University of Maryland, College Park, United States.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of combined with inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.
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http://dx.doi.org/10.7554/eLife.49503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773446PMC
October 2019

Manganese co-localizes with calcium and phosphorus in acidocalcisomes and is mobilized in manganese-deficient conditions.

J Biol Chem 2019 11 16;294(46):17626-17641. Epub 2019 Sep 16.

Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.
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http://dx.doi.org/10.1074/jbc.RA119.009130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873200PMC
November 2019

Obesity is associated with copper elevation in serum and tissues.

Metallomics 2019 08 28;11(8):1363-1371. Epub 2019 Jun 28.

Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Copper misbalance has been linked to fat accumulation in animals and experimental systems; however, information about copper homeostasis in human obesity is limited. In this study, the copper status of obese individuals was evaluated by measuring their levels of copper and cuproproteins in serum, adipose and hepatic tissues. The analysis of serum trace elements showed significant positive and element-specific correlation between copper and BMI after controlling for gender, age, and ethnicity. Serum copper also positively correlated with leptin, insulin, and the leptin/BMI ratio. When compared to lean controls, obese patients had elevated circulating cuproproteins, such as semucarbazide-sensitive amine oxidase (SSAO) and ceruloplasmin, and higher SSAO activity and copper levels in visceral fat. Although hepatic steatosis reduces copper levels in the liver, obese patients with no or mild steatosis have higher copper content in the liver compared to lean controls. In conclusion, obese patients evaluated in this study had altered copper status. Strong positive correlations of copper levels with BMI and leptin suggest that copper and/or cuproproteins may be functionally linked to fat accumulation.
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http://dx.doi.org/10.1039/c9mt00148dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753954PMC
August 2019

Localization of the Locus Coeruleus in the Mouse Brain.

J Vis Exp 2019 03 7(145). Epub 2019 Mar 7.

Department of Physiology, Johns Hopkins University, School of Medicine.

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-β-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).
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http://dx.doi.org/10.3791/58652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996810PMC
March 2019

ATP7A and ATP7B copper transporters have distinct functions in the regulation of neuronal dopamine-β-hydroxylase.

J Biol Chem 2018 12 19;293(52):20085-20098. Epub 2018 Oct 19.

From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,. Electronic address:

The copper (Cu) transporters ATPase copper-transporting alpha (ATP7A) and ATPase copper-transporting beta (ATP7B) are essential for the normal function of the mammalian central nervous system. Inactivation of ATP7A or ATP7B causes the severe neurological disorders, Menkes disease and Wilson disease, respectively. In both diseases, Cu imbalance is associated with abnormal levels of the catecholamine-type neurotransmitters dopamine and norepinephrine. Dopamine is converted to norepinephrine by dopamine-β-hydroxylase (DBH), which acquires its essential Cu cofactor from ATP7A. However, the role of ATP7B in catecholamine homeostasis is unclear. Here, using immunostaining of mouse brain sections and cultured cells, we show that DBH-containing neurons express both ATP7A and ATP7B. The two transporters are located in distinct cellular compartments and oppositely regulate the export of soluble DBH from cultured neuronal cells under resting conditions. Down-regulation of ATP7A, overexpression of ATP7B, and pharmacological Cu depletion increased DBH retention in cells. In contrast, ATP7B inactivation elevated extracellular DBH. Proteolytic processing and the specific activity of exported DBH were not affected by changes in ATP7B levels. These results establish distinct regulatory roles for ATP7A and ATP7B in neuronal cells and explain, in part, the lack of functional compensation between these two transporters in human disorders of Cu imbalance.
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http://dx.doi.org/10.1074/jbc.RA118.004889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311498PMC
December 2018

Altered zinc balance in the Atp7b mouse reveals a mechanism of copper toxicity in Wilson disease.

Metallomics 2018 11;10(11):1595-1606

Department of Biological Sciences, University of Alaska Anchorage, 3211 Providence Dr., Anchorage, AK 99508, USA.

Wilson disease (WD) is an autosomal recessive disorder caused by mutation in the ATP7B gene that affects copper transport in the body. ATP7B mutation damages copper transporter function, ultimately resulting in excessive copper accumulation and subsequent toxicity in both the liver and brain. Mechanisms of copper toxicity, however, are not well defined. The Atp7b-/- mouse model is well-characterized and presents a hepatic phenotype consistent with WD. In this study, we found that the untreated Atp7b-/- mice accumulate approximately 2-fold excess hepatic zinc compared to the wild type. We used targeted transcriptomics and proteomics to analyze the molecular events associated with zinc and copper accumulation in the Atp7b-/- mouse liver. Altered gene expression of Zip5 and ZnT1 zinc transporters indicated a transcriptional homeostatic response, while increased copper/zinc ratios associated with high levels of metallothioneins 1 and 2, indicated altered Zn availability in cells. These data suggest that copper toxicity in Wilson disease includes effects on zinc-dependent proteins. Transcriptional network analysis of RNA-seq data reveals an interconnected network of transcriptional activators with over-representation of zinc-dependent and zinc-responsive transcription factors. In the context of previous research, these observations support the hypothesis that mechanisms of copper toxicity include disruption of intracellular zinc distribution in liver cells. The translational significance of this work lies in oral zinc supplementation in treatment for WD, which is thought to mediate protective effects through the induction of metallothionein synthesis in the intestine. This work indicates broader impacts of altered zinc-copper balance in WD, including global transcriptional responses and altered zinc balance in the liver.
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http://dx.doi.org/10.1039/c8mt00199eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310031PMC
November 2018

Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes.

PLoS Biol 2018 09 10;16(9):e2006519. Epub 2018 Sep 10.

Department of Physiology, Johns Hopkins University, Baltimore, Maryland, United States of America.

Copper (Cu) has emerged as an important modifier of body lipid metabolism. However, how Cu contributes to the physiology of fat cells remains largely unknown. We found that adipocytes require Cu to establish a balance between main metabolic fuels. Differentiating adipocytes increase their Cu uptake along with the ATP7A-dependent transport of Cu into the secretory pathway to activate a highly up-regulated amino-oxidase copper-containing 3 (AOC3)/semicarbazide-sensitive amine oxidase (SSAO); in vivo, the activity of SSAO depends on the organism's Cu status. Activated SSAO oppositely regulates uptake of glucose and long-chain fatty acids and remodels the cellular proteome to coordinate changes in fuel availability and related downstream processes, such as glycolysis, de novo lipogenesis, and sphingomyelin/ceramide synthesis. The loss of SSAO-dependent regulation due to Cu deficiency, limited Cu transport to the secretory pathway, or SSAO inactivation shifts metabolism towards lipid-dependent pathways and results in adipocyte hypertrophy and fat accumulation. The results establish a role for Cu homeostasis in adipocyte metabolism and identify SSAO as a regulator of energy utilization processes in adipocytes.
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http://dx.doi.org/10.1371/journal.pbio.2006519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130853PMC
September 2018

Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model.

Mol Ther Methods Clin Dev 2018 Sep 9;10:165-178. Epub 2018 Jul 9.

Section on Translational Neuroscience, Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA.

Menkes disease is a lethal neurodegenerative disorder of copper metabolism caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Based on our prior clinical and animal studies, we seek to develop a therapeutic approach suitable for application in affected human subjects, using the () mouse model that closely mimics the Menkes disease biochemical and clinical phenotypes. Here, we evaluate the efficacy of low-, intermediate-, and high-dose recombinant adeno-associated virus serotype 9 (rAAV9)-ATP7A delivered to the cerebrospinal fluid (CSF), in combination with subcutaneous administration of clinical-grade copper histidinate (sc CuHis, IND #34,166). Mutant mice that received high-dose (1.6 × 10 vg) cerebrospinal fluid-directed rAAV9-rsATP7A plus sc copper histidinate showed 53.3% long-term (≥300-day) survival compared to 0% without treatment or with either treatment alone. The high-dose rAAV9-rsATP7A plus sc copper histidinate-treated mutant mice showed increased brain copper levels, normalized brain neurochemical levels, improvement of brain mitochondrial abnormalities, and normal growth and neurobehavioral outcomes. This synergistic treatment effect represents the most successful rescue to date of the mouse model. Based on these findings, and the absence of a large animal model, we propose cerebrospinal fluid-directed rAAV9-rsATP7A gene therapy plus subcutaneous copper histidinate as a potential therapeutic approach to cure or ameliorate Menkes disease.
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http://dx.doi.org/10.1016/j.omtm.2018.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080355PMC
September 2018

The Function of ATPase Copper Transporter ATP7B in Intestine.

Gastroenterology 2018 01 25;154(1):168-180.e5. Epub 2017 Sep 25.

Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Background & Aims: Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver-the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues.

Methods: We collected intestinal tissues from ATP7B-knockout (Atp7b) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy.

Results: ATP7B maintains a Cu gradient along the duodenal crypt-villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons.

Conclusions: ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.
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http://dx.doi.org/10.1053/j.gastro.2017.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848507PMC
January 2018

Copper, zinc and calcium: imaging and quantification in anterior pituitary secretory granules.

Metallomics 2016 09 18;8(9):1012-22. Epub 2016 Jul 18.

Department of Molecular Biology and Biophysics, UConn Health, 263 Farmington Avenue, Farmington, CT 06030, USA.

The anterior pituitary is specialized for the synthesis, storage and release of peptide hormones. The activation of inactive peptide hormone precursors requires a specific set of proteases and other post-translational processing enzymes. High levels of peptidylglycine α-amidating monooxygenase (PAM), an essential peptide processing enzyme, occur in the anterior pituitary. PAM, which converts glycine-extended peptides into amidated products, requires copper and zinc to support its two catalytic activities and calcium for structure. We used X-ray fluorescence microscopy on rat pituitary sections and inductively coupled plasma mass spectrometry on subcellular fractions prepared from rat anterior pituitary to localize and quantify copper, zinc and calcium. X-ray fluorescence microscopy indicated that the calcium concentration in pituitary tissue was about 2.5 mM, 10-times more than zinc and 50-times more than copper. Although no higher than cytosolic levels, secretory granule levels of copper exceeded PAM levels by a factor of 10. Atp7a, which transports copper into the lumen of the secretory pathway, was enriched in endosomes and Golgi, not in secretory granules. If Atp7a transfers copper directly to PAM, this pH-dependent process is likely to occur in Golgi and endosomes.
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http://dx.doi.org/10.1039/c6mt00079gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976788PMC
September 2016

Systemic Delivery and Biodistribution of Cisplatin in Vivo.

Mol Pharm 2016 08 24;13(8):2677-82. Epub 2016 Jun 24.

Department of Chemistry, Portland State University , Portland, Oregon 97201, United States.

Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017860PMC
August 2016

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

J Biol Chem 2016 08 16;291(32):16644-58. Epub 2016 May 16.

the Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon 97239,

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).
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http://dx.doi.org/10.1074/jbc.M116.727248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974379PMC
August 2016

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

J Biol Chem 2015 Aug 13;290(35):21264-79. Epub 2015 Jul 13.

From the Departments of Molecular Biology and Biophysics and Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030,

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.
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http://dx.doi.org/10.1074/jbc.M115.641027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571858PMC
August 2015

Nutrigenomics analysis reveals that copper deficiency and dietary sucrose up-regulate inflammation, fibrosis and lipogenic pathways in a mature rat model of nonalcoholic fatty liver disease.

J Nutr Biochem 2015 Oct 15;26(10):996-1006. Epub 2015 May 15.

Department of Biological Sciences, University of Alaska Anchorage, Anchorage, AK, 99508. Electronic address:

Nonalcoholic fatty liver disease (NAFLD) prevalence is increasing worldwide, with the affected US population estimated near 30%. Diet is a recognized risk factor in the NAFLD spectrum, which includes nonalcoholic steatohepatitis (NASH) and fibrosis. Low hepatic copper (Cu) was recently linked to clinical NAFLD/NASH severity. Simple sugar consumption including sucrose and fructose is implicated in NAFLD, while consumption of these macronutrients also decreases liver Cu levels. Though dietary sugar and low Cu are implicated in NAFLD, transcript-level responses that connect diet and pathology are not established. We have developed a mature rat model of NAFLD induced by dietary Cu deficiency, human-relevant high sucrose intake (30% w/w) or both factors in combination. Compared to the control diet with adequate Cu and 10% (w/w) sucrose, rats fed either high-sucrose or low-Cu diet had increased hepatic expression of genes involved in inflammation and fibrogenesis, including hepatic stellate cell activation, while the combination of diet factors also increased ATP citrate lyase and fatty acid synthase gene transcription (fold change > 2, P < 0.02). Low dietary Cu decreased hepatic and serum Cu (P ≤ 0.05), promoted lipid peroxidation and induced NAFLD-like histopathology, while the combined factors also induced fasting hepatic insulin resistance and liver damage. Neither low Cu nor 30% sucrose in the diet led to enhanced weight gain. Taken together, transcript profiles, histological and biochemical data indicate that low Cu and high sucrose promote hepatic gene expression and physiological responses associated with NAFLD and NASH, even in the absence of obesity or severe steatosis.
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http://dx.doi.org/10.1016/j.jnutbio.2015.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578989PMC
October 2015

Modulation of tau phosphorylation by environmental copper.

Transl Neurodegener 2014 17;3(1):24. Epub 2014 Nov 17.

Department of Neurology and Parkinson's Disease Research Education and Clinical Care Center (PADRECC), Portland Veterans Affairs Medical Center, Portland, OR USA ; Department of Neurology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201 USA.

Background: The transition metal copper enhances amyloid β aggregation and neurotoxicity, and in models of concomitant amyloid and tau pathology, copper also promotes tau aggregation. Since it is not clear if the effects of environmental copper upon tau pathology are dependent on the presence of pathological amyloid β, we tested the effects of copper overload and complexing in disease models which lack pathological amyloid β.

Methods: We used cell culture and transgenic murine models to test the effects of environmental copper on tau phosphorylation. We used oral zinc acetate as a copper lowering agent in mice and examined changes in blood and brain metals through inductively coupled plasma mass spectroscopy. Behavioral effects of copper lowering were assessed with Morris water maze and novel object recognition tasks. Changes in tau phosphorylation were examined by phosphorylation specific antibodies on Western blots.

Results: In human neuroblastoma cells, excess copper promoted tau phosphorylation and a copper complexing agent, tetrathiomolybdate, attenuated tau phosphorylation. In a transgenic mouse model expressing wild type human tau, copper-lowering by oral zinc suppressed plasma and brain levels of copper, and resulted in a marked attenuation of tau phosphorylation. No significant changes in behavior were observed with copper lowering, but a trend to improved recognition of the novel object was observed in zinc acetate treated mice.

Conclusions: We propose that reduction of brain copper by blocking uptake of copper from the diet may be a viable strategy for modulating tau pathology in Alzheimer's disease. The potential benefits of this approach are tempered by the absence of a behavioral benefit and by the health risks of excessive lowering of copper.
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http://dx.doi.org/10.1186/2047-9158-3-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322670PMC
February 2015

Genetic determinants of amidating enzyme activity and its relationship with metal cofactors in human serum.

BMC Endocr Disord 2014 Jul 15;14:58. Epub 2014 Jul 15.

Department of Neuroscience, University of Connecticut Health Center, 06030 Farmington, CT, USA.

Background: α-amidation is a final, essential step in the biosynthesis of about half of all peptide hormones and neurotransmitters. Peptidylglycine α-amidating monooxygenase (PAM), with enzymatic domains that utilize Cu and Zn, is the only enzyme that catalyzes this reaction. PAM activity is detected in serum, but its significance and utility as a clinical biomarker remain unexplored.

Methods: We used well-established enzymatic assays specific for the peptidylglycine-α -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domains of PAM to quantify amidating activity in the sera of 144 elderly men. Relationships between PHM and PAL activity and serum levels of their respective active-site metals, Cu and Zn, were analyzed. Study participants were also genotyped for eight non-coding single nucleotide polymorphisms (SNPs) in PAM, and relationships between genotype and serum enzyme activity and metal levels were analyzed.

Results: Serum PHM and PAL activities were normally distributed and correlated linearly with each other. Serum PAL activity, but not serum PHM activity, correlated with serum Cu; neither activity correlated with serum Zn. Study subjects possessing the minor alleles for rs32680 had lower PHM and PAL activities, and subjects with minor alleles for rs11952361 and rs10515341 had lower PHM activities.

Conclusions: Our results characterize large variation in serum amidating activity and provide unique insight into its potential origin and determinants. Common non-coding polymorphisms affect serum amidating activity and Cu levels. Serum amidating activity should be explored as a biomarker for functionality in the elderly and in additional study groups.
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http://dx.doi.org/10.1186/1472-6823-14-58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113131PMC
July 2014

Oral zinc reduces amyloid burden in Tg2576 mice.

J Alzheimers Dis 2014 ;41(1):179-92

Department of Neurology and Parkinson's Disease Research Education and Clinical Care Center (PADRECC), Portland Veterans Affairs Medical Center, Portland, OR, USA Department of Neurology, Oregon Health and Sciences University, Portland, OR, USA.

The aggregation of amyloid-β in Alzheimer's disease can be affected by free transition metals such as copper and zinc in the brain. Addition of copper and zinc with amyloid acts to increase aggregation and copper additionally promotes the formation of reactive oxygen species. We propose that reduction of brain copper by blocking uptake of copper from the diet is a viable strategy to regulate the formation of insoluble amyloid-β in the brain of Tg2576 mice. Mice were treated with regimens of zinc acetate, which acts with metallothionein to block copper uptake in the gut, at various times along their lifespan to model prevention and treatment paradigms. We found that the mice tolerated zinc acetate well over the six month course of study. While we did not observe significant changes in cognition and behavior, there was a reduction in insoluble amyloid-β in the brain. This observation coincided with a reduction in brain copper and interestingly no change in brain zinc. Our findings show that blocking copper uptake from the diet can redistribute copper from the brain and reduce amyloid-β aggregation.
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http://dx.doi.org/10.3233/JAD-131703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051494PMC
January 2015

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain.

Proc Natl Acad Sci U S A 2013 Nov 28;110(46):E4279-88. Epub 2013 Oct 28.

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27708.

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.
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http://dx.doi.org/10.1073/pnas.1311749110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831961PMC
November 2013

Peptidylglycine α-amidating monooxygenase heterozygosity alters brain copper handling with region specificity.

J Neurochem 2013 Dec 13;127(5):605-19. Epub 2013 Oct 13.

Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, USA.

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.
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http://dx.doi.org/10.1111/jnc.12438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938575PMC
December 2013

Opportunities in multidimensional trace metal imaging: taking copper-associated disease research to the next level.

Anal Bioanal Chem 2013 Feb 19;405(6):1809-20. Epub 2012 Oct 19.

X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA.

Copper plays an important role in numerous biological processes across all living systems predominantly because of its versatile redox behavior. Cellular copper homeostasis is tightly regulated and disturbances lead to severe disorders such as Wilson disease and Menkes disease. Age-related changes of copper metabolism have been implicated in other neurodegenerative disorders such as Alzheimer disease. The role of copper in these diseases has been a topic of mostly bioinorganic research efforts for more than a decade, metal-protein interactions have been characterized, and cellular copper pathways have been described. Despite these efforts, crucial aspects of how copper is associated with Alzheimer disease, for example, are still only poorly understood. To take metal-related disease research to the next level, emerging multidimensional imaging techniques are now revealing the copper metallome as the basis to better understand disease mechanisms. This review describes how recent advances in X-ray fluorescence microscopy and fluorescent copper probes have started to contribute to this field, specifically in Wilson disease and Alzheimer disease. It furthermore provides an overview of current developments and future applications in X-ray microscopic methods.
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http://dx.doi.org/10.1007/s00216-012-6437-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566297PMC
February 2013