Macular Mouse Research Articles

Pharmacokinetics of CuGTSM, a Novel Drug Candidate, in a Mouse Model of Menkes Disease.

Menkes disease is a rare hereditary disease in which systemic deficiency of copper due to mutation of the ATP7A gene causes severe neurodegenerative disorders. The present parenteral drugs have limited efficacy, so there is a need for an efficacious drug that can be administered orally. This study focused on glyoxal-bis (N(4)-methylthiosemicarbazonato)-copper(II (CuGTSM), which has shown efficacy in macular mice, a murine model of Menkes disease, and examined its pharmacokinetics. In addition, nanosized CuGTSM (nCuGTSM) was prepared, and the effects of nanosizing on CuGTSM pharmacokinetics were investigated. CuGTSM or nCuGTSM (10mg/kg) was administered orally to male macular mice or C3H/HeNCrl mice (control), and plasma was obtained by serial blood sampling. Plasma concentrations of CuGTSM and GTSM were measured by LC-MS/MS and pharmacokinetic parameters were calculated. When CuGTSM was administered orally, CuGTSM and GTSM were both detected in the plasma of both mouse strains. When nCuGTSM was administered, the Cmax was markedly higher, and the mean residence time was longer than when CuGTSM was administered for both CuGTSM and GTSM in both mouse strains. With macular mice, the AUC ratio (GTSM/CuGTSM) was markedly higher and the plasma CuGTSM concentration was lower than with C3H/HeNCrl mice when either CuGTSM or nCuGTSM was administered. Absorption of orally administered CuGTSM was confirmed in macular mice, and the nano-formulation improved the absorption and retention of CuGTSM in the body. However, the plasma concentration of CuGTSM was lower in macular mice than in control mice, suggesting easier dissociation of CuGTSM.

Menkes disease: Oral administration of glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) rescues the macular mouse.

Menkes disease is a copper metabolism disorder caused by mutations in ATP7A, a copper-transporting P-type ATPase. In this study, oral copper supplementation via glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) (CuGTSM), a lipophilic copper complex, was investigated in male hemizygous macular (MoMl/y) mice, a mouse model of Menkes disease. CuGTSM was administered by oral gavage on postnatal days 5, 8, 11, 17, 23, and 32. The copper levels in the organs and serum, copper-dependent enzyme activities in the brain, and ceruloplasmin (Cp) activity in the serum were measured at 15 days and 3 and 8 months of age. Histological analysis of the intestines and the rotarod test were also performed. CuGTSM treatment extended the lifespan of MoMl/y mice and partly restored the copper concentrations and cytochrome oxidase and DBH activities in the brain; however, the rotarod test showed impaired motor performance. The treatment also increased copper concentrations and Cp activity in the serum. In suckling MoMl/y mice, CuGTSM treatment transiently induced diarrhea accompanied by copper accumulation and altered villus morphology in the ileum. Oral administration of CuGTSM extended the lifespan of MoMl/y mice. Oral administration is attractive, but pharmaceutical studies are needed to reduce the adverse enteral effects.

Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain

Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.

PET Imaging Analysis with 64Cu in Disulfiram Treatment for Aberrant Copper Biodistribution in Menkes Disease Mouse Model

Menkes disease (MD), an X-linked recessive disorder of copper metabolism caused by mutations in the copper-transporting ATP7A gene, results in growth failure and severe neurodegeneration in early childhood. Subcutaneous copper-histidine injection is the standard treatment for MD, but it has limited clinical efficacy. Furthermore, long-term copper injection causes excess copper accumulation in the kidneys, resulting in renal dysfunction. To attempt to resolve this issue, we used PET imaging with (64)Cu to investigate the effects of disulfiram on copper biodistribution in living mice serving as an animal model for MD (MD model mice). Macular mice were used as MD model mice, and C3H/He mice were used as wild-type mice. Mice were pretreated with 2 types of chelators (disulfiram, a lipophilic chelator, and d-penicillamine, a hydrophilic chelator) 30 min before (64)CuCl2 injection. After (64)CuCl2 injection, emission scans covering the whole body were performed for 4 h. After the PET scans, the brain and kidneys were analyzed for radioactivity with γ counting and autoradiography. After copper injection alone, marked accumulation of radioactivity ((64)Cu) in the liver was demonstrated in wild-type mice, whereas in MD model mice, copper was preferentially accumulated in the kidneys (25.56 ± 3.01 percentage injected dose per gram [%ID/g]) and was detected to a lesser extent in the liver (13.83 ± 0.26 %ID/g) and brain (0.96 ± 0.08 %ID/g). Copper injection with disulfiram reduced excess copper accumulation in the kidneys (14.54 ± 2.68 %ID/g) and increased copper transport into the liver (29.42 ± 0.98 %ID/g) and brain (5.12 ± 0.95 %ID/g) of MD model mice. Copper injection with d-penicillamine enhanced urinary copper excretion and reduced copper accumulation in most organs in both mouse groups. Autoradiography demonstrated that disulfiram pretreatment induced copper transport into the brain parenchyma and reduced copper accumulation in the renal medulla. PET studies with (64)Cu revealed that disulfiram had significant effects on the copper biodistribution of MD. Disulfiram increased copper transport into the brain and reduced copper uptake in the kidneys of MD model mice. The application of (64)Cu PET for the treatment of MD and other copper-related disorders may be useful in clinical settings.

Copper-trafficking efficacy of copper-pyruvaldehyde bis(N4-methylthiosemicarbazone) on the macular mouse, an animal model of Menkes disease

Menkes disease (MD) is a disorder of copper transport caused by ATP7A mutations. Although parenteral copper supplements are partly effective in treating MD, the copper level in the brain remains insufficient, whereas copper accumulates in the kidney. We investigated the copper-trafficking efficacy of copper-pyruvaldehyde bis(N4-methylthiosemicarbazone) (Cu-PTSM), a lipophilic copper complex, in macular mice, an animal model of MD. Macular mice were treated with cupric chloride (CuCl2) or Cu-PTSM on postnatal days 4, 10, and 17. At 4 wk of age, the copper levels in major organs and cytochrome oxidase (CO) activity in brain tissue were measured. Hematology, blood biochemistry, and urinary β2-microglobulin (β2-M) secretion were also assessed. The copper levels in the brains of the Cu-PTSM-treated group remained low, but CO activity in the cerebral and cerebellar cortices in the Cu-PTSM-treated group were higher than those in the CuCl2-treated group. There were no significant differences in hematological or biochemical findings or in urinary β2-M secretion among the groups. Although the copper-trafficking efficacy of Cu-PTSM was limited, the improved CO activity in the brain suggests that Cu-PTSM delivered copper more effectively to neuronal CO than did CuCl2. Reduced renal copper accumulation may be beneficial in prolonged copper supplementation.

Effect of copper and disulfiram combination therapy on the macular mouse, a model of Menkes disease

Menkes disease (MD) is a genetic neurodegenerative disorder characterized by copper deficiency due to a defect in ATP7A. Standard treatment involves parenteral copper–histidine administration. However, the treatment is ineffective if initiated after two months of age, because the administered copper accumulates in the blood–brain barrier and is not transported to neurons. To resolve this issue, we investigated the effects of a combination therapy comprising copper and disulfiram, a lipophilic chelator, in the macular mouse, an animal model of MD. Seven-day-old macular mice treated subcutaneously with 50μg of CuCl2 on postnatal day 4 were used. The mice were given a subcutaneous injection of CuCl2 (10μg) with oral administration of disulfiram (0.3mg/g body weight) twice a week until eight weeks of age, and then sacrificed. Copper concentrations in the cerebellum, liver, and serum of treated macular mice were significantly higher than those of control macular mice, which received only copper. Mice treated with the combination therapy exhibited higher cytochrome c oxidase activity in the brain. The ratios of noradrenaline and adrenaline to dopamine in the brain were also increased by the treatment, suggesting that dopamine β-hydroxylase activity was improved by the combination therapy. Liver and renal functions were almost normal, although renal copper concentration was higher in treated macular mice than in controls. These results suggest that disulfiram facilitates the passage of copper across the blood–brain barrier and that copper–disulfiram combination therapy may be an effective treatment for MD patients.

Menkes病モデル動物“macular mouse”の病態に関する研究

Menkes病モデル動物“macular mouse”の病態に関する研究

Effect of copper and diethyldithiocarbamate combination therapy on the macular mouse, an animal model of Menkes disease

Menkes disease (MD) is a neurodegenerative disorder characterized by a copper deficiency in the brain. It is caused by the defective intestinal absorption of copper resulting from a deficiency of a copper-transporting ATPase, ATP7A. This gives rise to an accumulation of copper in the intestine. The copper deficiency in the brain of MD patients cannot be improved by copper injections, because the administered copper accumulates at the blood-brain barrier and is not transported across to the neurons. To resolve this problem, we investigated the effect of a combination therapy of copper and sodium diethyldithiocarbamate (DEDTC), a lypophilic chelator, in an animal model of MD, the macular mouse. Four-week-old macular mice treated with 50 mug of CuCl2 on the 7th day after birth were used. Experimental mice were given a subcutaneous injection of CuCl2 (4 microg) and an intraperitoneal injection of DEDTC (0.2 mg/g body weight) twice a week for 4 weeks and then sacrificed. Copper concentrations and cytochrome-c oxidase activity in the brains of treated mice were higher than those of control macular mice, which received only copper or saline. The ratios of brain noradrenaline to dopamine and of adrenaline to dopamine were also increased by the treatment, suggesting that the activity of dopamine beta-hydroxylase, a copper-dependent enzyme, was improved by the treatment. Liver and renal function tests showed no abnormalities in the treated mice, although copper concentrations in the kidneys of treated mice were higher than those of control macular mice. These results suggest that DEDTC facilitates the passage of copper across the blood-brain barrier and that the combination therapy of copper and DEDTC may be an effective treatment for the neurological disturbances suffered by patients with MD.

X-ray Microanalysis of Melanin Granules of Retinal Pigment Epithelium and Choroid in Hereditary Copper Deficient Mice (Macular Mice)

The role of trace elements in vivo has not been completely clarified. Trace elements were studied in melanin granules in the retinal pigment epithelium (RPE) and choroid of hereditary copper-deficient macular mice as a model of Menkes' disease. The analysis of elements in these melanin granules was done by new methods: freeze-embedding and an energy dispersive X-ray microanalysis (EDX). We used 14-day- and 1-month-old male hemizygote macular mice for the experiments and normal litter-mates as controls. Melanin granules in RPE and choroid contained sulfur, chloride, calcium, iron, copper and zinc. Calcium and copper were especially abundant in 14-day-old hemizygote macular mice, although there were few melanin granules in their RPE. The fact that copper was most abundant in the melanin granules in the RPE of 14-day-old macular mice suggests that the synthesis of melanin granules in the RPE and choroid of the hemizygote macular mice cannot be completed because of the lower activity of copper-containing enzymes such as tyrosinase and the abnormal copper distribution in various organs. Therefore, the melanin granules in the RPE and choroid of hemizygote macular mice are irregular in shape and few in number. Large amounts of copper concentrated in melanin granules in the RPE and choroid of hemizygote macular mice might induce quantitative abnormalities of trace elements.

Mottled gene expression and copper distribution in the macular mouse, an animal model for Menkes disease.

Journal of Inherited Metabolic DiseaseVolume 21, Issue 3 p. 199-202 Article Mottled gene expression and copper distribution in the macular mouse, an animal model for Menkes disease Y. Murata, Y. MurataSearch for more papers by this authorH. Kodama, H. KodamaSearch for more papers by this authorY. Mori, Y. MoriSearch for more papers by this authorM. Kobayashi, M. KobayashiSearch for more papers by this authorT. Abe, T. AbeSearch for more papers by this author Y. Murata, Y. MurataSearch for more papers by this authorH. Kodama, H. KodamaSearch for more papers by this authorY. Mori, Y. MoriSearch for more papers by this authorM. Kobayashi, M. KobayashiSearch for more papers by this authorT. Abe, T. AbeSearch for more papers by this author First published: 01 June 1998 https://doi.org/10.1023/A:1005383114315Citations: 16AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume21, Issue3June 1998Pages 199-202 RelatedInformation

Copper-metallothionein in the kidney of macular mice: a model for Menkes disease.

Menkes disease is an X-linked disorder of copper metabolism. Excess amounts of copper in the kidney of Macular mice, a model for this disease, were found as copper-metallothionein (Cu-MT) from kidney of the mice. Histochemical studies of Cu-MT based on its autofluorescent emission properties showed that the protein was predominant in the proximal convoluted tubule (PCT) cells of the cortex. PCT cells are known to be the primary site of the nephrotoxicity caused by heavy metals. MT mRNA was also observed in the cortex, indicating that the protein was biosynthesized in this region. On the basis of these results, we suggest that biosynthesis and degradation of Cu-MT occur repeatedly in the PCT cells of the cortex. We also compared the histochemical localization of Cu-MT in Macular mice and Long-Evans cinnamon rats, a model for Wilson's disease. The significance of this comparison is discussed.

Occurrence of two missense mutations in Cu-ATPase of the macular mouse, a Menkes disease model.

We have investigated the genetic defect of the Cu-ATPase gene (Atp7a) in the macular mouse, a genetic model of classical Menkes disease. Northern blot analysis showed that its placenta and kidney possess a normal amount of the Cu-ATPase mRNA of the normal size; sequencing analysis revealed two missense mutations, His674Arg and Ser1381 Pro, in a PCR-amplified cDNA for mutant Cu-ATPase. The latter mutation was suspected to affect the function of the ATPase, because it lies in the transmembrane segment that is thought to form a channel for the transportation of copper ions.

Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease.

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

A serine-to-proline mutation in the copper-transporting P-type ATPase gene of the macular mouse.

We have investigated the cDNA sequence of the copper-transporting P-type ATPase (Atp7a) gene of the macular mouse, a model for human Menkes disease. A point mutation (T to C) that results in substitution of proline for serine in a putative eighth transmembrane domain of the ATP7A was identified. This contrasts with abnormalities identified in the Atp7a of other mottled mouse strains: lack of expression of Atp7a mRNA in the dappled mouse, and a splicing mutation in the blotchy mouse.

Electron microscopic study of retinas of macular mice.

Menkes' disease may be due to a lack or deficiency of copper in various organs. The macular mouse is known as a model for Menkes' disease. We examined melanin granules in the retinal pigment epithelium and the activity of cytochrome oxidase, a copper-containing enzyme, in the retinas of macular mice by electron microscopy. In the retinas of hemizygote macular mice we demonstrated cytochemically (oxidative polymerization of diaminobenzidine to an osmophilic reaction product) the activity of cytochrome oxidase. The distribution of melanin granules in the retinal pigment epithelium related to the activity of another copper-containing enzyme, tyrosinase was also studied. Stereological methods were applied to obtain quantitative data. In the retinal photoreceptor inner segments of the macular mouse, the mitochondria were more numerous than in normal litermates and they appeared swollen. There were fewer melanin granules in the retinal pigment epithelium of macular mice than in that of normal littermates. The cytochrome oxidase activity was significantly lower in the macular mice than in the controls. Macular mice have lower activity of cytochrome oxidase and fewer melanin granules than do normal mice. Both changes may be related to copper deficiency. These results correspond to the retinal changes seen in patients with Menkes' disease.

Electron Microscopic Study of Optic Nerves of Macular Mice

The optic nerve of the macular mouse as a model of Menkes' disease was examined by electron microscopy. Since hemizygote macular mice die at 14 or 15 days of age, they were treated with 50μg CuCl 2per 0.1ml distilled water at 7 days of age. The optic nerves of 1-month-old hemizygote macular mice treated with copper showed hypomyelination and unmyelinated axons, while 1-month-old heterozygote macular mice had focal demyelination of axons. The number of myelinated axons in treated hemizygotes and heterozygotes was statistically significantly lower than that in the control littermates. Oligodendrocytes form myelin sheaths. Since oligodendrocytes of the hemizygote macular mice may have lower activities of cuproenzymes, such as cytochrome oxidase and superoxide dismutase, hypomyelination is assumed to be caused by the dysfunction of oligodendrocyte.

Localization of Menkes gene expression in the mouse brain; its association with neurological manifestations in Menkes model mice.

Menkes gene (Mc1 or MNK, encoding putative copper-transporting ATPase) expression was investigated and compared in normal and macular mutant mouse brain. Northern blot analysis showed a distinct 8.3-kb transcript and no obvious difference in size or extent in normal mice and macular mutants on postnatal days 0, 4, 7, 10 or 13. In situ hybridization revealed that certain specific populations of cells in the brain express Menkes mRNA, and that their localization in normal and mutant mice did not differ and was conserved on days 4, 10 and 13. The most intense hybridization signals were observed in the hippocampal CA1 region and dentate gyrus, the olfactory bulb nuclei, the cerebellar granular cell layer, the choroid plexus and the ependyma, with less intense signals in the hippocampal CA3 region and cerebellar Purkinje cells. In addition, necrotic neuronal cell death was predominantly observed in the CA3 region and the Purkinje cells of macular mice after postnatal day 10. The finding that the regions that had lower expression level of Menkes mRNA corresponded to those showing neuronal necrosis suggests that the Menkes gene may be responsible for the neuronal degeneration in some specific portions of the brain and clinical manifestations in this mutant.

Nutritional State and Catecholamine Metabolism in Macular Mice

ABSTRACT Catecholamine and amino acid contents in the brain of the macular mutant mouse, a model mouse of Menkes kinky hair desease, were studied at days 4, 7, 10 and 13 postnatally. The contents of norepinephrine and epinephrine in the macular mutant mouse brain were significantly lower than those of controls at all ages. In contrast, the content of dopamine in the mutant mouse brain was significantly higher than that in the controls at days 4 and 7, and then it decreased rapidly to become less than that of age‐matched controls at days 10 and 13. Body weight gain of mutant mice was remarkably stunted, and weight loss was observed at day 7 of age. Body weight at day 13 postnatum was 56.1% that of the control. To examine the possible effects of malnutrition on the catecholamine metabolism in the mutant mouse brain, the contents of various amino acids in the brain and serum were measured. In the mutant mouse serum, tyrosine and phenylalanine, which play key roles in catecholamine metabolism, were significantly reduced compared to control, whereas only tyrosine showed a significant decrease in the brain. These results indicate that the rapid decrease in the catecholamine content in the macular mutant mouse brain is possibly influenced by the decrease in serum tyrosine subsequent to progressive malnutrition.

Histochemical study of mitochondrial enzymes in cerebellar cortex of macular mutant mouse, a model of Menkes kinky hair disease

Histochemical study of mitochondrial enzymes in cerebellar cortex of macular mutant mouse, a model of Menkes kinky hair disease

Copper Distribution in Fetus and Placenta of the Macular Mutant Mouse as a Model of Menkes Kinky Hair Disease

Menkes kinky hair disease (MKHD) in humans is caused by a disturbance in copper homeostasis. A mutant mouse shows clinical and biochemical features very close to MKHD. In an attempt to elucidate the defect in copper transport, the copper distribution in various organs of 18-gestational-day-old macular mouse embryos, following administration by a single injection of saline (control) or 50 micrograms of CuCl2 on day 16 of gestation or by two injections on days 15 and 17 of gestation to the dams, was examined both biochemically and histochemically. The copper content in the hemizygous fetus (Ml/y) born to the homozygous mother, who had no copper injection during gestation, was lower in the brain and liver but higher in the placenta than in the respective organs of the normal fetus. When 50 micrograms of CuCl2 was injected into heterozygous dams (Ml/+) on day 16 of gestation, their hemizygous fetuses showed a slight increase in the copper content in the brain and liver, but the amount of copper in these organs was still less than that of the normal fetus. Conversely, the copper content in the placenta of the hemizygous fetus was far higher than that of the normal fetus. In the copper staining of the fetuses harvested from heterozygous dams, some fetuses showed copper deposition in the placenta, but not in the liver. The others showed no copper deposit in both the placenta and liver, thus indicating that the former were hemizygous for the mutation and the latter were normal littermates.(ABSTRACT TRUNCATED AT 250 WORDS)