Cu2+ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo
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Christian T Sheline1, Dennis W Choi
1Department of Neurology and Center for the Study of Nervous System Injury Washington University School of Medicine, St. Louis, MO 63110, USA. shelinec@neuro.wustl.edu
Abstract:
Wilson's disease results from mutations in the P-type Cu(2+)-ATPase causing Cu(2+) toxicity. We previously demonstrated that exposure of mixed neuronal/glial cultures to 20 microM Cu(2+) induced ATP loss and death that were attenuated by mitochondrial substrates, activators, and cofactors. Here, we show differential cellular sensitivity to Cu(2+) that was equalized to 5 microM in the presence of the copper exchanger/ionophore, disulfiram. Because Cu(2+) facilitates formation of oxygen radicals (ROS) which inhibit pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), we hypothesized that their inhibition contributed to Cu(2+)-induced death. Toxic CU(2+) exposure was accompanied by early inhibition of neuronal and hepatocellular PDH and KGDH activities, followed by reduced mitochondrial transmembrane potential, DeltaPsi(M). Thiamine (1-6 mM), and dihydrolipoic acid (LA, 50 microM), required cofactors for PDH and KGDH, attenuated this enzymatic inhibition and subsequent death in all cell types. Furthermore, liver PDH and KGDH activities were reduced in the Atp7b mouse model of Wilson's disease prior to liver damage, and were partially restored by oral thiamine supplementation. These data support our hypothesis that Cu(2+)-induced ROS may inhibit PDH and KGDH resulting in neuronal and hepatocellular death. Therefore, thiamine or lipoic acid may constitute potential therapeutic agents for Wilson's disease.
Insights
Wilson's disease involves copper toxicity. This study shows thiamine and lipoic acid may protect against copper-induced cell death by supporting key enzymes, offering potential new treatments.
Area of Science:
- Biochemistry
- Neuroscience
- Genetics
Background:
- Wilson's disease stems from P-type Cu(2+)-ATPase gene mutations, leading to copper toxicity.
- Previous work showed copper-induced ATP loss and cell death in neuronal/glial cultures, which mitochondrial substrates could mitigate.
- Copper ions (Cu(2+)) generate reactive oxygen species (ROS), inhibiting crucial enzymes like pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH).
Purpose of the Study:
- To investigate the role of PDH and KGDH inhibition in copper-induced neuronal and hepatocellular death.
- To evaluate the therapeutic potential of thiamine and dihydrolipoic acid (LA) in mitigating copper toxicity.
- To examine enzyme activity in the Atp7b mouse model of Wilson's disease.
Main Methods:
- Utilized mixed neuronal/glial cultures and hepatocellular models exposed to copper.
- Assessed cellular sensitivity to copper, enzyme activities (PDH, KGDH), and mitochondrial membrane potential.
- Administered disulfiram, thiamine, and dihydrolipoic acid (LA) to copper-exposed cells and in the Atp7b mouse model.
Main Results:
- Copper exposure (Cu(2+)) led to differential cellular sensitivity, equalized by disulfiram.
- Cu(2+) induced early inhibition of neuronal and hepatocellular PDH and KGDH, followed by reduced mitochondrial potential.
- Thiamine and LA attenuated enzyme inhibition and cell death; thiamine partially restored liver enzyme activity in Atp7b mice.
Conclusions:
- Copper-induced ROS likely inhibit PDH and KGDH, contributing to neuronal and hepatocellular death in Wilson's disease.
- Thiamine and dihydrolipoic acid show potential as therapeutic agents by supporting these critical enzymes.
- Targeting enzyme cofactors may offer a novel therapeutic strategy for Wilson's disease.
