Interactions of peptide amidation and copper: Novel biomarkers and mechanisms of neural dysfunction
Introduction
In both Menkes disease, with low levels of copper, and Wilson's disease, with elevated levels of copper, neural function is impaired (Madsen and Gitlin, 2007, Schlief and Gitlin, 2006, Cunliffe et al., 2001, Tumer and Horn, 1997). These diseases are rare, caused by mutations in ATP7A and ATP7B, respectively. In addition to these two copper transporting P type ATPases, the uptake and tissue-specific distribution of dietary copper involves additional transporters and copper-binding chaperones (Madsen and Gitlin, 2007, Schlief and Gitlin, 2006). Menkes patients and mottled/brindled (ATP7aMo-br) mice, an animal model for Menkes Disease, exhibit increased susceptibility to seizures and neurodegeneration which can be partially reversed with copper supplementation (Sheela et al., 2005). Similar symptoms result from dietary copper deficiency; livestock consuming a copper-deficient diet develop progressive ataxic myelopathy (Madsen and Gitlin, 2007, Kumar, 2006). In humans, acquired copper deficiency can accompany long-term parenteral nutrition or malabsorption; in addition to hematological symptoms, sensory ataxia and spastic gait may occur (Madsen and Gitlin, 2007). Biomarkers that accurately reflect mild changes in copper status have not yet been established (Harvey and McArdle, 2008).
Copper-dependent enzymes, which evolved with the appearance of atmospheric molecular oxygen, are rare, with about a dozen in mammals (Critchton and Pierre, 2001, Ridge et al., 2008). Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DBM) are homologous copper-dependent monooxygenases, essential for the synthesis of α-amidated peptides and norepinephrine/epinephrine, respectively. The biosynthesis of amidated peptides, conserved from Planaria to humans, is compromised in mottled/brindled mice (Asada et al., 2005, Prigge et al., 2000, Steveson et al., 2003, Niciu et al., 2007). ATP7A must interact with Atox1, a cytoplasmic copper chaperone, to transport Cu(I) from the cytoplasm into the secretory pathway (Madsen and Gitlin, 2007, Prohaska and Gybina, 2004, Puig and Thiele, 2002).
Mutations of either PAM or ATP7A affect the vascular system and thermoregulation (Niciu et al., 2006, Niciu et al., 2007, Bousquet-Moore et al., 2009, Czyzyk et al., 2005, Al-Bitar et al., 2005, Ozawa et al., 2002, Maury et al., 2007). Mice lacking PAM do not survive past E13.5; thin arterial walls and edema are apparent at E12.5 (Czyzyk et al., 2005). Mice with a single copy of the PAM gene are viable (Czyzyk et al., 2005, Bousquet-Moore et al., 2009). Along with mild glucose intolerance, PAM+/− mice showed deficits in peripheral vasoconstriction; supplemental copper reversed this deficit (Bousquet-Moore et al., 2009). By coupling our analysis of functional impairments in PAM heterozygous mice (PAM+/−) to dietary manipulations of copper, we sought to identify indicators of peptidergic dysfunction and mild copper deficiency.
We exposed PAM+/− mice and their wildtype (WT) littermates to either a copper-deficient or copper-supplemented diet. Dietary copper deficiency in WT mice increased anxiety-like behavior and seizure sensitivity, deficits similar to those observed in PAM+/− mice. Supplementary copper ameliorated some, but not all, of the deficits observed in PAM+/− mice. In addition, we found that copper status differed in PAM+/− and WT mice on the control diet. This unexpected finding led to the hypothesis that PAM is linked to the ability of an organism to provide copper to tissues. Comparisons of gene expression profiles in PAM+/− vs. WT mice confirmed this hypothesis.
Section snippets
Animal care and use
Male and female PAM+/− and C57BL/6J WT littermates (2–5 months old) were used in these experiments; male PAM+/− mice were bred with female C57BL/6J WT mice from Jackson Labs (Bar Harbor ME). All had been backcrossed more than 12 generations. Tail clips and/or ear tags taken at the time of weaning were used to determine the genotype (Bousquet-Moore et al., 2009). All protocols were approved by the Animal Care and Use Committee at the University of Connecticut Health Center.
Manipulation of dietary copper
WT and PAM+/− mice
Copper status in WT mice on copper-deficient and supplemented diets
Because our previous work demonstrated that copper supplementation restored the ability of PAM+/− mice to maintain body temperature in the cold (Bousquet-Moore et al., 2009), we wanted to assess the effects of marginal copper deficiency on WT mice. Copper deficiency was created by feeding adult male mice a diet containing 0.6 ppm Cu/kg instead of 15 ppm Cu/kg for 9 weeks. Copper status was assessed by measuring total liver copper (Fig. 1A), serum ceruloplasmin (Fig. 1B) and liver levels of
Effects of PAM heterozygosity are widespread and diverse
In organisms as simple as Planaria and Hydra, amidated peptides play multiple roles in normal physiology (Asada et al., 2005). Since PAM is the only enzyme known to synthesize amidated peptides, we used mice with a single functional copy of PAM to identify functions that were most sensitive to a decrease in PAM and dietary copper. While haploinsufficiency of many genes is without effect, a single functional copy of the vesicular monoamine transporter 2 gene (Fukui et al., 2007) or various ion
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