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

Abstract

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 50 μg of CuCl₂ on postnatal day 4 were used. The mice were given a subcutaneous injection of CuCl₂ (10 μg) with oral administration of disulfiram (0.3 mg/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.

Introduction

Menkes disease (MD) is an X-linked recessive disorder caused by a defect in a copper-transporting ATPase (ATP7A). In humans, ATP7A is expressed in almost all cell types except hepatocytes. In normal cells, ATP7A is localized in the trans-Golgi network and transports copper from the cytosol into the Golgi apparatus, where copper is incorporated into secretory copper enzymes [1]. In MD-affected cells, however, copper accumulates in the cytosol and cannot be excreted. Copper accumulation in the intestines results in copper absorption failure, leading to overall copper deficiency in the body, except in the kidney. Copper also accumulates in the cells of the blood–brain barrier. Thus, copper cannot be delivered from the bloodstream to neurons after barrier maturation [2], [3], [4]. Copper concentrations in the serum, liver, and brain of MD patients are significantly lower, resulting in reduced activities of copper-dependent enzymes such as cytochrome c oxidase, dopamine β-hydroxylase, and lysyl oxidase. Currently, parenteral administration of copper–histidine is the standard treatment for MD [1], [5]. 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. Therefore, copper delivery to neurons is the most important objective in the treatment of MD-associated neurological degeneration [6].

We previously reported that a combination therapy comprising copper and diethyldithiocarbonate (DEDTC) improved copper concentrations, cytochrome c oxidase activity, and catecholamine metabolism in the brain of macular mice [7]. A dimer of DEDTC, disulfiram, has been used for the treatment of alcoholism and cocaine addiction and as a modulator of cisplatin-induced toxicity [8], [9], [10]; thus, oral disulfiram is easily applicable in the clinical setting. Here, we report the effects of a combination therapy comprising copper injection and oral disulfiram on the macular mouse, an animal model of MD [11].