Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction

Abstract

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

Fig.1. Copper Status Differs in PAM^+/− and WT mice

A. Total liver copper was measured in PAM^+/− (Total N=13; N per group = 6,7) and WT mice (Total N=14; N per group=8,6) on the control or copper deficient diet. Copper deficiency reduced total copper (two-way deficiency main effect, p<0.0005; t-tests, p<0.0005). Liver copper was higher in PAM^+/− than WT mice on the control diet (t-test p=0.023) and the sensitivity of PAM^+/− mice to copper deficiency was increased (two-way interaction, p<0.0005). B. Serum ceruloplasmin activity was determined in PAM^+/− (Total N=22; N per group= 9,13) and WT mice (Total N=24; N per group = 11,13) on the two diets; levels were lower in copper deficient WT and PAM^+/− mice (t-tests, p<0.0005). CCS levels were quantified in separate liver samples by immunoblot (40 μg protein) in PAM^+/− (Total N=28; N per group =7) and WT mice (Total N=23; N per group = 5-6) with deficiency (C) or supplementation (D). CCS levels were normalized to γ-adaptin. CCS levels in WT mice were higher with the copper deficient vs. the control diet, where the PAM^+/− mice did not respond (two-way deficiency main effect, p=0.011; two-way interaction p=0.05; t-test, p=0.03). Hepatic CCS levels in PAM^+/− mice were slightly lower with copper supplementation (two-way interaction p=0.06; independent t-test p=0.06).

FIG. 2. Copper Deficiency Impairs Thermoregulation in WT Mice

(A) PAM^+/− (Total N=15; N per group =7,8) and WT mice (Total N=10; N per group=4,6) on the control or copper-deficient diet were exposed to a 4°C environment and core body temperature was assessed every 40 min. As observed previously, core temperature fell more in PAM^+/− mice than in WT mice on the control diet (multivariate ANOVA, p=0.014). Core temperature dropped more quickly and to a lower level when WT mice were kept on a copper deficient diet (multivariate ANOVA, p<0.0005). Core temperature was not altered with copper deficiency in PAM^+/− mice (two-way interaction, p<0.0005). (B) Laser Doppler velocimetry was used to measure tail blood flow velocity with cold exposure in WT (Total N=12; N per group=6) and PAM^+/− mice (Total N=13; N per group= 7,6) on the two diets. The robust cold-induced increase in blood flow velocity observed in WT mice on the control diet was eliminated by copper deficiency (multivariate ANOVA p<0.0005). The limited ability of PAM^+/− mice to increase flow velocity in response to cold exposure was further reduced by the copper deficient diet (multivariate ANOVA, p=0.004). The impairment of vasoconstriction was more pronounced in WT than PAM^+/− mice (two-way interaction, p=0.008). Error bars, SEM.

FIG. 3. Anxiety-like Behavior in WT Mice Is Sensitive to Copper Status

PAM^+/− (Total N=29; N per group=6-8) and WT mice (Total N =22; N per group=5-6) on the indicated diet were placed in the elevated zero maze for 5 min; the amount of time spent in the open areas was assessed by a blinded observer and is expressed as a percentage of the total time in the maze. Copper deficient WT mice spent less time in the open areas than WT mice on the control diet (two-way deficiency main effect p=0.025; independent t-test, p=0.004). On the control diet, PAM^+/− mice spent less time in the open arms than WT mice (two-way genotype main effect p=0.008; independent t-test, p=0.001), suggesting an anxiety-like phenotype. While PAM^+/− mice demonstrated a lower behavioral sensitivity to copper deficiency compared to WT mice (two-way interaction, p=0.001), PAM^+/− mice fed the copper supplemented diet spent more time in the open areas than PAM^+/− mice on the control diet (independent t-test, p=0.002). The behavior of WT mice did not change significantly with copper supplementation (two-way interaction p=0.005). Error bars, SEM.

FIG. 4. Copper Deficiency Increases Seizure Sensitivity in WT Mice

PAM^+/− (N=15 control; N=18 copper deficient) and WT (N= 14 control; N=16 copper deficient) mice were given an injection of PTZ (30 mg/kg, i.p.) and behavior was scored as described (83). The percentages of each group exhibiting seizures of each rating (0-5) are shown. Chi-square analyses revealed increased seizure severity in PAM^+/− mice compared to WT mice on either the copper supplemented diet (p=0.05) or the control diet (p =0.03). Compared to the control diet, copper deficiency (A) increased seizure severity in WT (chi square p = 0.001), but not in PAM^+/− mice. Data shown are for male mice; the same experiment was repeated with female mice [WT, N=8 control; N=10 copper deficient; PAM^+/−, N=8 control; N=11 copper deficient) with the same answer. Seizure severity did not differ with copper supplementation in either PAM^+/− (N=8 control; N=9 supplemented) or WT (N=8 control; N=11 supplemented) male mice compared to the control diet (B).

FIG. 5. Copper Deficiency Leads to Accumulation of TRH-Gly in WT Mice

Hypothalami from PAM^+/− and WT mice were extracted and the inactive precursor to amidated TRH, TRH-Gly, was quantified by radioimmunoassay with copper deficiency (A) and supplementation (B). For the copper deficiency experiment, 26 WT mice (11 control;15 deficient) and 37 PAM^+/− mice (17 control; 20 deficient) were used. For the copper supplementation experiment, 20 WT mice (9 control; 11 supplemented) and 14 PAM^+/− mice (7 for each group) were used. A significant accumulation of TRH-Gly was noted in PAM^+/− mice (two-way genotype main effect, p<0.0005; independent t-test p<0.0005) as previously described (Bousquet-Moore et al., 2009). Significantly higher levels of TRH-Gly were seen in copper deficient WT mice compared to WT mice on the control diet (two-way deficiency main effect, p = 0.036; independent t-test, p=0.03). Error bars, SEM.

FIG. 6. Copper Deficiency Affects PAM Processing

(A) The major cleavage products of PAM-1 are shown. (B) Serum levels of copper-optimized PAM activity were determined in WT and PAM^+/− mice on the indicated diet. For the copper deficiency experiment, 41 WT mice (19 control; 22 deficient) and 52 PAM^+/− (24 control; 28 deficient) were used. For the copper supplementation experiment, 19 WT mice (8 control;11 supplemented) and 16 PAM^+/− mice (8 per group) were used. An increase in serum activity with copper deficiency was noted in WT mice (independent t-test, p=0.048). Copper supplementation did not alter serum activity. Copper optimized PAM activity was also assessed in atria from PAM^+/− (Total N=14; N per group=7) and WT (Total N=11; N=5,6) mice on the control or copper deficient diet (C). Separate samples were subjected to Western blot analysis using antibody to PHM (9 WT and 10 PAM^+/−mice on either the control or deficient diets). Data for PAM-1 were quantified and normalized to γ-adaptin (D). Atrial PAM activity was lower in PAM^+/− vs. WT mice on both diets (two-way genotype main effect, p<0.0005; independent t-test, p=0.002) and declined with copper deficiency in WT mice (two-way interaction p=0.022; independent t-test p=0.01). Levels of intact PAM-1 protein were lower in PAM^+/− mice than in WT mice on the control diet (two-way genotype main effect, p=0.006; independent t-test, p=0.01) and only slightly decreased with copper deficiency in WT mice (independent t-test, p=0.07). Error bars, SEM.