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Manganese targets m‐aconitase and activates iron regulatory protein 2 in AF5 GABAergic cells

Studies suggest that disturbances of amino acid metabolism and cellular iron regulation are important mechanisms underlying manganese (Mn) neurotoxicity, although the targets underlying these disturbances are poorly defined. Using the AF5 neural‐derived cell line, which displays GABAergic properties, we showed that Mn significantly increased glutamate release to 174%–214% of that of the control and that the effects of Mn exposure on the metabolism of glutamate, glutamine, alanine, and GABA resembled the effects of fluorocitrate, an inhibitor of aconitase, but not the effects of other toxicants including paraquat, rotenone, or 3‐nitropropionic acid. Consistent with this, Mn inhibited aconitase activity in AF5 cells, resulting in a 90% increase in intracellular citrate; an in vitro assay revealed that m‐aconitase was significantly more sensitive to inhibition by Mn than was c‐aconitase. RNA mobility shift assay and Western blot showed that Mn treatment caused c‐aconitase to be converted to iron regulatory protein 1 (IRP1) and increased the abundance of IRP2, leading to reduced H‐ferritin expression, increased transferrin receptor expression, and increased uptake of transferrin. To determine the relative contributions of IRP1 and IRP2 in mediating the effects of Mn on iron homeostasis, we exposed transgenic fibroblasts lacking either c‐aconitase/IRP1 or IRP2 to Mn. Manganese exposure minimally altered ferritin levels in cells possessing only c‐aconitase/IRP1, whereas cells possessing only IRP2 showed a robust decrease in ferritin, indicating a dominant role of IRP2 in Mn‐induced alteration of iron homeostasis. Together, these results demonstrate that m‐aconitase is an important target of Mn and thatMn‐induced alteration of iron homeostasis is mediated predominantly through IRP2. © 2007 Wiley‐Liss, Inc.