Involvement of MEK5/ERK5 signaling pathway in manganese-induced cell injury in dopaminergic MN9D cells

Background: Over-exposure to manganese (Mn) causes irreversible movement disorders with signs and symptoms similar, but not identical, to idiopathic Parkinson's disease (IPD). Recent data suggest that Mn toxicity occurs in dopaminergic (DA) neurons, although the mechanism remains elusive. This study was designed to investigate whether Mn interfered the apoptotic signaling transduction cascade in DA neurons. Methods: Mouse midbrain dopaminergic MN9D cells were exposed to Mn in a concentration range of 0, 400, 800, or 1200 mu M as designated as control, low, medium, and high exposure groups, respectively. The flow cytometry with Annexin V/PI double staining and immunohistochemistry were used to assess the apoptosis. Results: Data indicated that Mn exposure caused morphological alterations typical of apoptosis, increased apoptotic cells by 2-8 fold, and produced reactive oxidative species (ROS) by 1.5-2.2 fold as compared to controls (p < 0.05). Studies by qPCR and Western blot revealed that Mn exposure significantly increased the protein expression of extracellular signal-regulated kinase-5 (ERK5) and mitogen-activated ERK kinase-5 (MEK5) (p < 0.05). The presence of BIX02189, a specific inhibitor of MER/ERK, caused a much greater cytotoxicity, i.e., higher cell death, more ROS production, and worsened apoptosis, than did the treatment with Mn alone. Following Mn exposure, the expression of a downstream effector Bcl-2 was reduced by 48 % while those of Box and Caspase-3 were increased by 266.7 % and 90.1 %, respectively, as compared to controls (p < 0.05). Conclusion: Taken together, these data provide the initial evidence that the signaling transduction cascade mediated by MEK5/ERK5 is responsible to Mn-induced cytotoxicity; Mn exposure, by suppressing anti-apoptotic function while facilitating pro-apoptotic activities, alters neuronal cell's survival and functionally inhibits DA production by MN9D cells.