**8. Hydrogen peroxide is the major species to cause oxidative stress in tgSOD1 mitochondria**

One of the distinctions of BM and SCM is a very high activity of MnSOD (SOD2) (Panov et al., 2009, 2011a, 2011b), which is distinct from the heart and skeletal muscle mitochondria (Muller et al., 2008). Even at very high rates of ROS production, such as shown in Figures 4 and 5A, 5B, addition of external SOD did not increase fluorescence of resorufin. The high activity of SOD2 means that BM and SCM effectively eliminate superoxide radicals from the matrix by converting them to H2O2. Therefore, we suggest that the major damaging factor in tgSOD1 motor neurons is H2O2. It was shown that H2O2 can interact with Cu,Zn-SOD (SOD1) resulting in peroxidase activity of SOD1 (Liochev, Fridovich, 2004), formation of strong oxidant CO3 radical (reviewed in Valentine et al., 2005), enzyme inactivation, release of Cu2+, protein oxidation and aggregation (Ramirez et al., 2009, Durer et al., 2009). At this point, the damaging effects of H2O2 will be increased dramatically because Cu2+ ions are several orders more active than Fe2+ in Fenton-like reaction with H2O2 (Dikalov et al., 2004), which will produce hydroxyl radical-like species and further drive lipid peroxidation,

glutamate and pyruvate by BM and SCM in 2010 (Fig. 5A and 5B), the overall rates of ROS generation with physiologically relevant substrate mixture - glutamate + pyruvate + malate were 5 times lower for tgBM and 10 times for tgSCM , when compared with transgenic

**A.** 2007; **B.** 2010. Dark grey – brain mitochondria, light gray – spinal cord mitochondria. Incubation conditions as in Fig. 5. Statistics: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. The data for tgSCM were compared

Similar multiple-fold differences in ROS production existed also with succinate and succinate containing substrate mixtures (Figs. 7A, 7B). In tgBM and tgSCM, the increases in ROS generation were substrate specific and depended on activity of SDH. These results led us to conclusion that changes in the metabolic phenotype of neuronal mitochondria, which occurred in 2008, resulted in a dramatic decrease in production of ROS in tgSOD1 rats.

One of the distinctions of BM and SCM is a very high activity of MnSOD (SOD2) (Panov et al., 2009, 2011a, 2011b), which is distinct from the heart and skeletal muscle mitochondria (Muller et al., 2008). Even at very high rates of ROS production, such as shown in Figures 4 and 5A, 5B, addition of external SOD did not increase fluorescence of resorufin. The high activity of SOD2 means that BM and SCM effectively eliminate superoxide radicals from the matrix by converting them to H2O2. Therefore, we suggest that the major damaging factor in tgSOD1 motor neurons is H2O2. It was shown that H2O2 can interact with Cu,Zn-SOD (SOD1) resulting in peroxidase activity of SOD1 (Liochev, Fridovich, 2004), formation of

of Cu2+, protein oxidation and aggregation (Ramirez et al., 2009, Durer et al., 2009). At this point, the damaging effects of H2O2 will be increased dramatically because Cu2+ ions are several orders more active than Fe2+ in Fenton-like reaction with H2O2 (Dikalov et al., 2004), which will produce hydroxyl radical-like species and further drive lipid peroxidation,

radical (reviewed in Valentine et al., 2005), enzyme inactivation, release

Fig. 7. **Generation of ROS by brain and spinal cord mitochondria from tgSOD1 rats,** 

**8. Hydrogen peroxide is the major species to cause oxidative stress in** 

mitochondria in 2007 (Fig. 7A and 7B).

with the corresponding results for tgBM.

**tgSOD1 mitochondria** 

strong oxidant CO3

**isolated in 2007 and 2010, oxidizing various substrates.**

protein damage and promote mitochondrial dysfunction. Loss of metals by mutant SOD1 leads to formation of amyloid-like aggregates (Durer et al., 2009). Mutations in SOD1 amplify reactions with H2O2, increase the lifetime of incorrectly folded states, and if exposed to even mild oxidative stress, incorrect disulfide links form and stabilize larger aggregates that may be resistant to the degradation by the quality control machinery of the cell, and thus increase association with mitochondria (Field et al., 2003; Furukawa, O'Halloran, 2008).
