Effect of Hfe Deficiency on Memory Capacity and Motor Coordination after Manganese Exposure by Drinking Water in Mice

Abstract

Excess manganese (Mn) is neurotoxic. Increased manganese stores in the brain are associated with a number of behavioral problems, including motor dysfunction, memory loss and psychiatric disorders. We previously showed that the transport and neurotoxicity of manganese after intranasal instillation of the metal are altered in Hfe-deficient mice, a mouse model of the iron overload disorder hereditary hemochromatosis (HH). However, it is not fully understood whether loss of Hfe function modifies Mn neurotoxicity after ingestion. To investigate the role of Hfe in oral Mn toxicity, we exposed Hfe-knockout (Hfe (-/-)) and their control wild-type (Hfe (+/+)) mice to MnCl2 in drinking water (5 mg/mL) for 5 weeks. Motor coordination and spatial memory capacity were determined by the rotarod test and the Barnes maze test, respectively. Brain and liver metal levels were analyzed by inductively coupled plasma mass spectrometry. Compared with the water-drinking group, mice drinking Mn significantly increased Mn concentrations in the liver and brain of both genotypes. Mn exposure decreased iron levels in the liver, but not in the brain. Neither Mn nor Hfe deficiency altered tissue concentrations of copper or zinc. The rotarod test showed that Mn exposure decreased motor skills in Hfe (+/+) mice, but not in Hfe (-/-) mice (p = 0.023). In the Barns maze test, latency to find the target hole was not altered in Mn-exposed Hfe (+/+) compared with water-drinking Hfe (+/+) mice. However, Mn-exposed Hfe (-/-) mice spent more time to find the target hole than Mn-drinking Hfe (+/+) mice (p = 0.028). These data indicate that loss of Hfe function impairs spatial memory upon Mn exposure in drinking water. Our results suggest that individuals with hemochromatosis could be more vulnerable to memory deficits induced by Mn ingestion from our environment. The pathophysiological role of HFE in manganese neurotoxicity should be carefully examined in patients with HFE-associated hemochromatosis and other iron overload disorders.

Keywords: Barnes maze; Hemochromatosis; Iron overload; Learning; Neurotoxicity; Rotarod.

Fig. 1.. Effect of Mn exposure on physiological characteristics in Hfe-deficient mice. (A) Body weights (n = 19~24 per group) of mice 5 weeks post-exposure to Mn in drinking water. (B) Mean levels of water consumption (n = 7~10 per group) throughout the exposure period. (C) Hematocrit values (n = 5~7 per group). (D) Non-heme iron levels in serum (n = 5~7 per group). (E) Non-heme iron levels in the liver (n = 13~20 per group). Empty and closed bars represent water-drinking and Mn-drinking mice, respectively. Data were presented as means ± SEM and were analyzed using two-way ANOVA followed by post-hoc comparisons.

Fig. 2.. Liver metal levels after Mn exposure in drinking water. Levels of iron, manganese, copper and zinc in the liver were measured by inductively coupled plasma mass spectrometry (ICP-MS). Empty and closed bars represent water-drinking and Mn-drinking mice, respectively. Data were presented as means ± SEM (n = 6 per group) and were analyzed using two-way ANOVA followed by post-hoc comparisons.

Fig. 3.. Brain metal levels after Mn exposure in drinking water. Levels of iron, manganese, copper and zinc in the brain were measured by ICP-MS. Empty and closed bars represent water-drinking and Mn-drinking mice, respectively. Data were presented as means ± SEM (n = 6 per group) and were analyzed using two-way ANOVA followed by post-hoc comparisons.

Fig. 4.. Effect of Mn exposure on motor coordination in Hfe-deficient mice. Motor function was determined by the rotarod test. Shown are (A) the time spent before the first footing loss each day over four days among the four groups and (B) the percentage of the best time over four days between Mn-treated mice and water-treated mice. Data were presented as means ± SEM (n = 15~17 per group) and were analyzed using repeated two-way ANOVA followed by post-hoc comparisons (A) or t-test (B). * p < 0.05 between Mndrinking Hfe^+/+ and Hfe^−/− mice.

Fig. 5.. Effect of Mn exposure on spatial memory capacity in Hfe-deficient mice. Spatial learning and memory capacity were determined by the Barnes maze test. Shown are (A) the latency to the target hole during the learning period over three days of training and (B) the percentage of the latency to the target hole on the test day (day 4) between Mn-treated mice and water-treated mice. Data were presented as means ± SEM (n = 15~17 per group) and were analyzed using two-way ANOVA followed by post-hoc comparisons (A) or t-test (B). * p < 0.05 between Mn-drinking Hfe^+/+ and Hfe^−/− mice. ^† p < 0.05 between water-drinking Hfe^−/− and Mn-drinking Hfe^−/− mice.