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Manganese targets m-aconitase and activates iron regulatory protein 2 in AF5 GABAergic cells
Daniel R Crooks1, Manik C Ghosh, Michelle Braun-Sommargren
1Department of Environmental Toxicology, University of California, Santa Cruz, California 95064, USA.
Abstract:
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.
Insights
Manganese neurotoxicity involves amino acid and iron metabolism disruptions. This study identifies mitochondrial aconitase as a key manganese target, with iron regulatory protein 2 (IRP2) primarily mediating iron homeostasis changes.
Area of Science:
- Neuroscience
- Biochemistry
- Toxicology
Background:
- Manganese (Mn) neurotoxicity is linked to amino acid metabolism and iron regulation disturbances.
- The specific molecular targets of Mn toxicity remain poorly understood.
Purpose of the Study:
- To investigate the molecular mechanisms of manganese neurotoxicity, focusing on aconitase and iron homeostasis.
- To identify the primary iron regulatory protein involved in Mn-induced cellular changes.
Main Methods:
- Utilized AF5 neural cells and transgenic fibroblasts lacking specific iron regulatory proteins (IRP1, IRP2).
- Assessed glutamate release, amino acid metabolism, aconitase activity, and iron regulatory protein expression (IRP1, IRP2) and function.
- Analyzed ferritin and transferrin receptor expression and transferrin uptake.
Main Results:
- Manganese exposure increased glutamate release and altered amino acid metabolism, mimicking fluorocitrate effects.
- Manganese inhibited mitochondrial aconitase (m-aconitase) significantly more than cytoplasmic aconitase (c-aconitase), leading to increased intracellular citrate.
- Mn treatment induced conversion of c-aconitase to IRP1 and increased IRP2, decreasing ferritin and increasing transferrin receptor expression.
- IRP2, not IRP1, was found to be the dominant mediator of Mn-induced alterations in iron homeostasis, evidenced by ferritin level changes in knockout cells.
Conclusions:
- Mitochondrial aconitase is a critical molecular target of manganese toxicity.
- Manganese-induced disruption of cellular iron homeostasis is primarily mediated by iron regulatory protein 2 (IRP2).
