Restriction of Manganese Intake Prevents the Onset of Brain Manganese Overload in Zip14-/- Mice

Abstract

As a newly identified manganese transport protein, ZIP14 is highly expressed in the small intestine and liver, which are the two principal organs involved in regulating systemic manganese homeostasis. Loss of ZIP14 function leads to manganese overload in both humans and mice. Excess manganese in the body primarily affects the central nervous system, resulting in irreversible neurological disorders. Therefore, to prevent the onset of brain manganese accumulation becomes critical. In this study, we used Zip14^-/- mice as a model for ZIP14 deficiency and discovered that these mice were born without manganese loading in the brain, but started to hyper-accumulate manganese within 3 weeks after birth. We demonstrated that decreasing manganese intake in Zip14^-/- mice was effective in preventing manganese overload that typically occurs in these animals. Our results provide important insight into future studies that are targeted to reduce the onset of manganese accumulation associated with ZIP14 dysfunction in humans.

Keywords: SLC39A14; ZIP14; manganese; metal metabolism; nutrition.

Figure 1

Brain and blood manganese concentrations in mice from different age groups of wild-type (WT) and Zip14 knockout (Zip14^−/−) mice. (A,B) Brain and (C,D) blood manganese (Mn) contents measured by inductively coupled plasma mass spectrometry (ICP-MS) in male and female mice at newborn (NB), 1 week (1 wk), 3 weeks (3 wks), 6 weeks (6 wks), 12 weeks (12 wks) and 20 weeks (20 wks) of age (n = 5/group). Data were expressed as mean ± standard deviation (S.D.). Statistical analysis was performed using two-way ANOVA, followed by the Bonferroni post-hoc test to compare the age and sex-matched WT and Zip14^−/− mice. **** p < 0.0001.

Figure 2

Metal chelation did not prevent the development of brain manganese overload. CaNa₂EDTA was injected intraperitoneally into 1-week-old Zip14^−/− mice every other day for 2 weeks. Animals were sacrificed and tissues were collected when mice were 3 weeks old. Phosphate buffered saline (PBS) was used as the vehicle control. (A) Brain and (B) blood manganese concentrations were determined by ICP-MS analysis (n = 8, 4 males and 4 females). Data were expressed as mean ± S.D. Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni post-hoc test. **** indicates p < 0.0001; “ns” indicates not significant.

Figure 3

Liver manganese in mice from different age groups of WT and Zip14^−/− mice. Liver manganese concentrations measured by ICP-MS in (A) male and (B) female mice at newborn (NB), one week (1 wk), three weeks (3 wks), six weeks (6 wks), twelve weeks (12 wks) and twenty weeks (20 wks) of age (n = 4–5/group). Data were expressed as mean ± S.D. and statistical analysis was performed using two-way ANOVA, followed by the Bonferroni post-hoc test to compare results of WT and Zip14^−/− mice from the same age group. *** p < 0.001; **** p < 0.0001.

Figure 4

Adeno-associated virus (AAV)-mediated hepatic ZIP14 expression in Zip14^−/− mice. Zip14^−/− mice received a single dose of ZIP14-expressing AAV (AAV-ZIP14-FLAG) via intraperitoneal injection at 1 week old. At 3 weeks of age, mice were sacrificed and tissue samples were collected. PBS was used as the vehicle control. (A) Liver samples were analyzed by Western blotting (WB). Blots were probed for ZIP14 using both anti-mouse ZIP14 and anti-FLAG antibodies. β-Actin was used as a loading control. (B) The relative expression levels of ZIP14 in PBS-injected WT mice and AAV-ZIP14-injected Zip14^−/− mice were determined by normalizing to β-ACTIN (n = 5–7 female mice/group).

Figure 5

Restoration of hepatic ZIP14 expression increased liver manganese levels, but did not prevent brain manganese accumulation. (A) Liver, (B) blood and (C) brain manganese concentrations in PBS-injected WT and Zip14^−/− mice and AAV-ZIP14 injected Zip14^−/− mice were determined by ICP-MS (n = 5–7 female mice/group). Data were expressed as mean ± S.D. and statistical analysis was performed using one-way ANOVA, followed by the Bonferroni post-hoc test. * p < 0.05; ** p < 0.01 and **** p < 0.0001.

Figure 6

Maternal breast milk manganese content can be altered by dietary intervention. Zip14^+/− mating mice at 8–9 weeks old were fed diets containing 1 ppm, 20 ppm, 200 ppm, or 2000 ppm of manganese during breeding and lactation periods (for a total of ~6–7 weeks). Milk manganese contents were analyzed by ICP-MS (n = 3–4/group). Data were expressed as mean ± S.D. and statistical analysis was performed using one-way ANOVA, followed by the Bonferroni post-hoc test. * p < 0.05; ** p < 0.01, compared with the control group (20 ppm).

Figure 7

Restricting manganese intake prevents the onset of brain manganese loading in Zip14^−/− mice. (A) Schematic illustrating the experimental procedures. Heterozygous Zip14^+/− mating mice were fed with diets containing low to normal levels of Mn (1 ppm, 20 ppm and 200 ppm). Maternal mice were kept on the same diet during the mating and lactation period. Pups were sacrificed and brain tissues were collected at 3 weeks old after weaning. (B) Brain manganese was determined by ICP-MS analysis (n = 4/group, 2 males and 2 females). Data were expressed as mean ± S.D. and statistical analysis was performed using two-way ANOVA, followed by the Bonferroni post-hoc test to compare results of WT and Zip14^−/− mice. * p < 0.05; **** p < 0.0001; “ns” indicates not significant.