Motor Dysfunction Caused By Manganism In Mice And Related Molecular Mechanism

Manganese is one of essential trace elements. However, the excessive intake of Mn may produce adverse effects on human health. In the recent years, the widespread use of lead-free gasoline and pesticides, such as manganese ethylenebisdithiocarbamate, has increased the content of Mn in the environment and the exposure to Mn in the common population. In the occupational surroundings, some Mn-exposed operations may even cause the acute manganism. Therefore, more attention has been paid to the effects of over-exposure to Mn on the huaman health.Nervous system is one of the main target organs of manganism. Its main diseased regions include pallidum, subthalamic nucleus, putamen and ubstantia nigra. In clinic, neurotoxicity of Mn features general bradykinesia and muscle rigidity, on which the treatment of L-dopa has no significant effect. So it is of great significance to provide effective medical prevention based on illuminated mechanism of Mn neurotoxicity. In recent years, some researches showed that abnormal cerebral metabolism and lesion of DAergic neuron caused by Mn are the main factors of shaking palsy. Although a number of studies on the mechanism of Mn neurotoxicity have been conducted, the exact mechanism is still unknown. In this study, with the animal model of manganism, the change in motor ethology was observed to investigate mechanisms of motor dysfunction caused by manganism for the theoretical beses for its prevention. The study mainly includes: 1) the effect of manganism on motor behavior of mice; 2) the effect of manganism on morphology of GABAergic neuron and Dopaminergic neuron and their activity in substantia nigra (SN); 3) the effect of manganism on protein expression of KCC2 and NKCC1; 4) effects of Mn exposure on morphology of Dopaminergic neuron and microtubule; and protein expression of tau and phosphorylated tau.Methods:1. One mol/L MnCl2·4H2O was injected into left caudate putamen at the coordinate of striatum following the stereotaxic atlas of mouse brain.2. The behavioral changes of mice were observed in the behavioral experiments 24 h after Mn exposure, including rotarod test, open field test and pole test.3. Double immunofluorescences were conducted for GFP and Fos, TH and Fos in SN respectively; Western blot was used for the expression of Fos, GFP, TH, KCC2 and NKCC1 in SN.4. Immunohistochemistry was used to investigate the effects of manganism on Dopaminergic neuron in SN; double immunofluorescence was conducted to observe coexpression ofβ-tubulin3 and TH; Western blot was employed to determine the expression ofβ-tubulin3, tau and phosphorylated tau in SN.Results:1. Results of mice exposed to Mn Behavioral in behavioral tests: The results of rotarod test showed 24 h after Mn treatment, the time spent on the rotarod was significantly reduced compared with naive and sham (P <0.05); The results of open field test showed that the total horizontal distance was significantly decreased in Mn group compared with na?ve and sham (P <0.01), and the horizontal activity (motion times) showed no significant difference compared with na?ve and sham (P >0.05); the results of pole test showed mice the time from top to headdown and time from headdown to bottom were both increased significantly in Mn group compared with na?ve and sham (P <0.001).2. Morphological changes of DAergic neuron and GABAergic neuron and their activity 24 h after Mn injection by using double immunofluorescence: Fos-positive neurons were significantly increased in Mn group compared with controls, while DAergic neurons showed no significant change and there was no coexistence of Fos and DAergic neuron in SN; Fos-positive neurons were significantly increased in Mn group, while GABAergic neurons showed no significant change and there were significant coexistence of Fos and GABAergic neurons in SN; The Western blot showed expression of Fos was significantly increased in Mn group compared with controls, while the expression of GFP and TH was not significantly changed.3. Effect of KCC2 inhibitor on the motor behavior of mice:The protein expression of KCC2 in SN was significantly increased in Mn group, and that of NKCC1 showed no significant change. The treatment of KCC2 inhibitor reduced the protein expression of KCC2 and partially reversed the time spent on the rotarod of mice exposed to Mn; the treatment of KCC2 inhibitor could in part reverse the reduced total horizontal distance.4. Effects of manganism on morphology of Dopaminergic neuron and microtubule in SN: Immunohistochemistry showed the significant decrease in DAergic neurons in SN in Mn group compared with controls, and the decreased coexistence ofβ-tubulin3-positive neurons and Dopaminergic neurons; Western blot showed the significant decrease in the protein expression ofβ-tubulin3 and tau1 in Mn group, and the significant decrease in phosphorylated Ser199, phosphorylated Ser202 and significant decrease phosphorylated Ser396 and phosphorylated Ser404.Conclusions:1. Manganism can cause the significant motor behavioral changes in mice, such as hypokinesia, bradykinesia and rigid muscle.2. Manganism can not cause the alternation in the numbers of Dopaminergic neurons and GABAergic neurons, and GABAergic neuron is activated in SN reticular part, while Dopaminergic neuron is not activated in SN compact part. These results suggest that GABAergic neuron may be one of the target cells in the nervous lesion in the manganism. Besides, some expressions of Fos don't colocate with either GABAergic neuron or Dopaminergic neuron, suggesting neurotoxicity of manganism may be caused by other neurons falling into neither Dopaminergic neuron nor GABAergic neuron category.3. The treatment of KCC2 inhibitor can improve the motor behavior to some extent after manganism. The possible mechanism is that KCC2, existing in SN reticular part, specifically affects Dopaminergic neuron in SN compact part through the regulation of GABAergic neuron in SN reticular part, which in time influences the motor behavior of mice.4. The significantly decreased number of Dopaminergic neurons in SN after manganism, coupled with the slight change of microtubular structure in Dopaminergic neuron suggests that the structure change of microtubule may be one of the factors causing the decrease in the number of Dopaminergic neurons. In addition, the significantly increased expression of phosphorylated tau suggests that hyperphosphorylation of tau may contribute to the structure change of microtubule.