Respiration and ROS production in brain and spinal cord mitochondria of transgenic rats with mutant G93a Cu/Zn-superoxide dismutase gene

Abstract Mitochondrial dysfunction is involved in the pathogenesis of motor neuron degeneration in the G93A mutant transgenic (tgmSOD1) animal model of ALS. However, it is unknown whether mitochondriopathy is a primary or secondary event. We isolated brain (BM) and spinal cord (SCM) mitochondria from 2 month old presymptomatic tgmSOD1 rats and studied respiration and generation of reactive oxygen species (ROS) using a new metabolic paradigm (Panov et al., Am. J. Physiol., Regul. Integr. Comp. Physiol., 2011). The yields of BM and SCM from tgmSOD1 rats were 27% and 58% lower than normal rats (WT). The rates of the State 3 and State 3U respiration of tgBM and tgSCM were normal with glutamate + pyruvate + malate as substrates but were inhibited with pyruvate + malate in tgBM and glutamate + malate in tgSCM. In tgSCM the State 4 respiration with all substrates was significantly (1.5–2 fold) increased as compared with WT-SCM. Western blot analysis showed that tgSCM had lower contents of complexes III (− 60%) and IV (− 35%), and the presence of mutated SOD1 protein in both tgBM and tgSCM. With glutamate + pyruvate + malate or succinate + glutamate + pyruvate + malate as substrates, tgBM and tgSCM generated 5–7 fold more ROS than normal mitochondria, and tgSCM generated two times more ROS than tgBM. We show that the major damaging ROS species in tgmSOD1 animals is H2 O2 . It is known that mutated SOD1, damaged by H2 O2 , associates with mitochondria, and we suggest that this further increases production of H2 O2 . We also show that the total tissue calcium content remained normal in the brain but was diminished by 26% in the spinal cord of presymptomatic tgmSOD1 rats. Conclusion: In tgSCM abnormally high rates of ROS generation, associated with reverse electron transport, result in accelerated mitochondriopathy, and the Ca2+ -dependent excitotoxic death of motor neurons. Thus mitochondrial dysfunction is a key early element in pathogenesis of motor neuron degeneration in tgmSOD1 rats.