ATP7B
Copper-transporting ATPase 2 (EC 7.2.2.8) (Copper pump 2) (Wilson disease-associated protein) [Contains: WND/140 kDa] [PWD] [WC1] [WND]
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Wilson disease is a hereditary disorder caused by mutations of the ATP7B gene, which leads to intoxication with copper as a result of an unbalance of copper homeostasis. The clinical manifestations resulting from this intoxication are related to the affectation of liver and the encephalon in most cases. Several animal models are currently available for the study of the malady. However, in such models no neurological symptoms are observed, which limits their use for the study of pathogenic effects of this disease on the central nervous system. The aim of the present study was to evaluate if copper feeding could induce a disease state in Drosophila melanogaster to model Wilson disease. The effect of the feeding of copper at the doses of 31 microM and 47 microM on the survival was initially evaluated. Next, behavioral experiments were conducted to determine whether the motor performance was altered by the 47 microM concentration. The results suggest that copper treatment decreases the viability of the flies. In addition, the decrease of viability was associated to an increase and decrease of spontaneous motor activity at early and late stages of the intoxication, respectively. Finally, the role of the dopaminergic neurotransmission system on the observed motor alterations was evaluated. The dopamine precursor L-dopa increased motor activity. In contrast, D2 receptor antagonist, Fluphenazine, was able to block both the increase and decrease of motor activity scores induced by copper. These results suggest that Drosophila melanogaster could be used as a model organism for the study of possible interventions with potential neuroprotective effects in Wilson disease.
MeSH Terms
- Age Factors
- Animals
- Copper Sulfate
- Disease Models, Animal
- Disease Progression
- Dopamine Agents
- Dopamine Antagonists
- Dopamine D2 Receptor Antagonists
- Dopaminergic Neurons
- Drosophila melanogaster
- Female
- Fluphenazine
- Hepatolenticular Degeneration
- Humans
- Levodopa
- Longevity
- Male
- Motor Activity
- Sampling Studies
- Synaptic Transmission
ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks. In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.
MeSH Terms
- Adenosine Triphosphatases
- Aging
- Animals
- Cation Transport Proteins
- Cell Size
- Copper Transporter 1
- Copper-Transporting ATPases
- Female
- Ganglia, Spinal
- Gene Expression Regulation
- Lumbar Vertebrae
- Neurons
- Organoplatinum Compounds
- Oxaliplatin
- RNA, Messenger
- Rats
- Rats, Wistar
- Reverse Transcriptase Polymerase Chain Reaction
In all living organisms trace element metabolism and transport are closely regulated at the genetic level. Copper is one of the essential microelements required for normal growth and development. The main organ in mammals involved in copper metabolism is the liver. It is known that copper metabolism in the liver is controlled by ATP7B, a P-type ATP-ase encoded by the Atp7b gene. However, little is known about the expression and function of the second important P-type ATP-ase, ATP7A encoded by the Atp7a gene. In this study we investigated the expression of the Atp7a gene in the liver during postnatal development in mice. We analyzed expression of Atp7a gene in the livers from neonatal (P.05), young (P14) and adult (P240) mice using RT-PCR and real-time PCR method. We found a transcript of the Atp7a gene in the liver of all investigated animals. Moreover, we found that the expression of the Atp7a gene in the liver in mice is age-dependent and decreases during postnatal development. Interestingly, the Atp7a expression in adult mice is very low in comparison with neonatal and young animals. Western blot analysis revealed that Atp7a is expressed not only at mRNA level but also at the protein level in the liver of all investigated animals. The expression of Atp7a gene and ATP7A protein was also confirmed in primary hepatocytes from adult mouse. Demonstration of the hepatic Atp7a gene expression may shed light on new aspects of copper metabolism in the liver in mammals.
MeSH Terms
- Adenosine Triphosphatases
- Aging
- Animals
- Animals, Newborn
- Carcinoma, Hepatocellular
- Cation Transport Proteins
- Cell Line, Tumor
- Cells, Cultured
- Copper
- Copper-Transporting ATPases
- Hepatocytes
- Liver
- Liver Neoplasms
- Male
- Mice
- RNA, Messenger
Kidneys regulate their copper content more effectively than many other organs in diseases of copper deficiency or excess. We demonstrate that two copper-transporting ATPases, ATP7A and ATP7B, contribute to this regulation. ATP7A is expressed, to a variable degree, throughout the kidney and shows age-dependent intracellular localization. In 2-wk-old mice, ATP7A is located in the vicinity of the basolateral membrane, whereas in 20-wk-old mice, ATP7A is predominantly in intracellular vesicles. Acute elevation of serum copper, via intraperitoneal injection, results in the in vivo redistribution of ATP7A from intracellular compartments toward the basolateral membrane, illustrating a role for ATP7A in renal response to changes in copper load. Renal copper homeostasis also requires functional ATP7B, which is coexpressed with ATP7A in renal cells of proximal and distal origin. The kidneys of Atp7b(-/-) mice, an animal model of Wilson disease, show metabolic alterations manifested by the appearance of highly fluorescent deposits; however, in marked contrast to the liver, renal copper is not significantly elevated. The lack of notable copper accumulation in the Atp7b(-/-) kidney is likely due to the compensatory export of copper by ATP7A. This interpretation is supported by the predominant localization of ATP7A at the basolateral membrane of Atp7b(-/-) cortical tubules. Our results suggest that both Cu-ATPases regulate renal copper, with ATP7A playing a major role in exporting copper via basolateral membranes and protecting renal tissue against copper overload.
MeSH Terms
- Adenosine Triphosphatases
- Aging
- Animals
- Cation Transport Proteins
- Cell Membrane
- Copper
- Copper-Transporting ATPases
- Disease Models, Animal
- Female
- Hepatolenticular Degeneration
- Kidney
- Kidney Tubules, Distal
- Kidney Tubules, Proximal
- Male
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
Hepatic abnormalities in Long-Evans Cinnamon (LEC) rats, an animal model of Wilson disease (WD), were restored by the expression of the human ATP7B cDNA under the control of CAG promoter. Expression of ATP7B transcript and protein in the liver of the transgenic rats resulted in the restoration of biosynthesis of holoceruloplasmin and biliary copper excretion. Meanwhile, transgenic rats showed striking improvements in their hepatic abnormalities, i.e., rescue from fulminant hepatitis, late onset of hepatic cholangiofibrosis, suppression of hepatocellular carcinoma and much improved survival rates. Moreover, dramatic decreases were noted both in the levels of hepatic copper and iron in transgenic rats before the occurrence of hepatitis. These results indicated that the human ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by the excretion of copper into the bile, and that ATP7B is critical to hepatic dysfunctions in WD. This first successful transgenic rescue has important implications for the gene therapy of WD.
MeSH Terms
- Adenosine Triphosphatases
- Aging
- Animals
- Animals, Genetically Modified
- Carcinoma, Hepatocellular
- Cation Transport Proteins
- Ceruloplasmin
- Copper
- Copper-Transporting ATPases
- Disease Models, Animal
- Female
- Gene Expression
- Hepatitis, Animal
- Hepatolenticular Degeneration
- Humans
- Iron
- Liver
- Rats
- Rats, Inbred LEC
- Rats, Sprague-Dawley
- Survival Rate
- Transgenes
We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD). PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day. PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development. Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal. In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway. PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes. These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.
MeSH Terms
- Adenosine Triphosphatases
- Aging
- Alternative Splicing
- Animals
- Biological Clocks
- Biological Transport
- Carrier Proteins
- Cation Transport Proteins
- Circadian Rhythm
- Copper
- Copper-Transporting ATPases
- Cyclic AMP
- Photoreceptor Cells, Vertebrate
- Pineal Gland
- Rats
- Rats, Sprague-Dawley
- Signal Transduction