**6. Differences between BM and SCM isolated in 2007 and 2010 in the rates of ROS production with different substrates**

ROS production by mitochondria strongly depends on the metabolic state of the mitochondria, type of substrates, and is also tissue-specific (Kwong, Sohal, 1998; Muller et al., 2008, Panov et al., 2007). There are two types of ROS production by mitochondria – energy-dependent and non-energy-dependent (Panov et al., 2007). The energy-dependent ROS production is associated with the reverse electron transport (Kwong, Sohal, 1998; Muller et al., 2008, Panov et al., 2007). ROS production, which does not depend on energization and the metabolic state of the mitochondria may occur on complexes I, II and III and depends on the presence of inhibitors of the electron transport (Kwong, Sohal, 1998; Panov et al., 2007; St-Pierre et al. 2002). In the absence of respiratory inhibitors the rate of the non-energy dependent ROS production is relatively slow (Panov et al., 2007). We have shown that in BM and SCM the major source of ROS correlates with the reverse electron transport, which occurs during oxidation of succinate (Kwong, Sohal, 1998; Panov et al., 2007, 2009, 2011a, b), fatty acids (Panov et al., 2010c), or α-glycerophosphate (Tretter et al., 2007). Thus respiration rates in State 4 in the well coupled mitochondria oxidizing succinate correlates with the rates of ROS production (Panov et al., 2007, 2011a). Therefore changes in the rates of ROS production presented in Figure 4 closely reflect metabolic pattern of the mitochondria. Another reason why we do not present respiratory data is that in 2010 we had limited number of tgSOD1 rats, which precluded obtaining statistically significant data with all substrates and their mixtures.

In our studies on mitochondrial dysfunctions in the rat model of ALS (tgSOD1), we used as a reference control Sprague Dawley rats from Taconic (Germantown, NY). In the years of 2005-2007 we have discovered, that in this strain both BM and SCM had a unique metabolic attribute, which was absent in mitochondria from other organs: during simultaneous oxidation of pyruvate + glutamate + malate the rates of oxidative phosphorylation and the State 4 ROS production were significantly higher than with either glutamate or pyruvate alone (Panov 2009, 2011a). This substrate mixture corresponded to the metabolic situation in activated neurons (Panov et al., 2009). Addition of malonate, a potent inhibitor of SDH, attenuated the rates of respiration and ROS production with pyruvate + malate and glutamate + pyruvate + malate to the level with glutamate + malate.

With succinate as a substrate, ROS production was several times higher than with glutamate + pyruvate + malate, and was further increased in the presence of glutamate + pyruvate (Fig. 5A, 5B). The stimulation of the succinate-supported State 4 respiration and ROS production by glutamate and pyruvate was attributed to removal of endogenous oxaloacetate, which inhibits succinate dehydrogenase (SDH) (Panov et al. 2009, 2010). SCM from the control rats produced significantly more ROS than BM, particularly with the succinate containing substrate mixtures (Figure 5A, 5B; see also Panov et al. 2009, 2011a). We have concluded that in BM the activity of SDH was primordially inhibited by endogenous oxaloacetate (Panov 2009, 2010). This intrinsic inhibition of SDH by oxaloacetate was less evident in SCM, and therefore SCM produced more ROS with substrate mixtures that

Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS 239

In contrast to WT-BM isolated in 2007, the WT-BM isolated in 2010 produced ROS supported by succinate at much lower rates than with glutamate + pyruvate + malate (Fig. 5A). Addition of glutamate + pyruvate + malate to the WT-BM -2010 oxidizing succinate stimulated ROS production only to the level with glutamate + malate (Fig. 5A). Similar metabolic features were also observed with the WT-SCM isolated in 2010, although the differences from 2007 WT-SCM were smaller than for the BM (Fig 5B). It is important to note that in 2010 WT-SCM generated ROS with different substrates at the same levels as WT-BM, whereas in 2007 the WT-SCM generated significantly more ROS than the corresponding

Incubation conditions and designations as described in Fig. 5. Malonate 5 mM was added before mitochondria. Dark gray – BM isolated in 2007, light gray columns – BM isolated in 2010. Statistics: \*\*\* *p* < 0.001, NS – not significant difference. The dark columns and grey columns were compared with the corresponding column representing ROS production with glutamate and malate, taken as 100%. Fig. 6. Effect of malonate on ROS production by wild type brain mitochondria, isolated in 2007 and 2010, oxidizing glutamate + malate, pyruvate + malate, and glutamate + pyruvate

The sensitivity of the State 4 ROS production supported by either pyruvate or glutamate to malonate (Fig, 6; Panov et al. 2009) demonstrates that at least with BM and SCM both glutamate and pyruvate cannot be regarded as "pure complex I substrates", as is commonly accepted. In BM and SCM during oxidation of glutamate or pyruvate, and particularly when glutamate and pyruvate are present simultaneously, significant amounts of α-ketoglutarate can be formed in transaminase reactions, which is further converted to succinate (Balasz,

**7. Effects of metabolic phenotype on ROS production by tgBM and tgSCM** 

Figure 7A shows generation of ROS by tgBM and tgSCM isolated from tgSOD1 rats in 2007, and figure 7B presents corresponding results for tgBM and tgSCM isolated from tgSOD1 rats in 2010. The figures clearly show that in spite of higher basic ROS production with

WT-BM (Fig. 5A and 5B. Note the scale difference between the figures).

+ malate.

1965; Panov et al. 2009).

produced or contained succinate (Panov 2011a). The described above metabolic features of BM and SCM were consistently observed in the wild type and tgSOD1 rats during period of 2005 – 2007 years (Panov et al. 2009, 2011a, 2011b).

**A.** Brain mitochondria; **B.** Spinal cord mitochondria. Dark grey – wild type rats in 2007, light gray – wild type rats in 2010. Mitochondria were incubated in a medium containing: 125 mM KCl, 10 mM MOPS, pH 7.2, 2 mM MgCl2, 2 mM KH2PO4, 10 mM NaCl, 1 mM EGTA, 0.7 mM CaCl2. At a Ca2+/EGTA ratio of 0.7, the free [Ca2+] is close to 1 M as determined using Fura-2. The substrate concentrations were: 5 mM succinate without rotenone, 5 mM glutamate, 2.5 mM pyruvate, and 2 mM malate. (Panov et al. 2009, 2011a, b). The results are presented as Mean ± SE, n = 3, and show the rates of ROS production in pmol H2O2 /minute/mg mitochondrial protein. Additions: 5 μM Amplex red, 1 U of horse radish peroxidase, 50 U of superoxide dismutase (Sigma), 0.05 mg mitochondria, glutamate 5 mM + malate 2 mM (G + M); pyruvate 2.5 mM + malate 2 mM (P + M); glutamate 5 mM + pyruvate 2.5 mM + malate 2 mM (G + P + M); succinate 5 mM (S); succinate 5 mM + glutamate 5 mM + pyruvate 2.5 mM + malate 2 mM (S + G + P + M). Statistics: \*\* *p* < 0.01; \*\*\* *p* < 0.001. For each year the data were compared with the rates of ROS production with glutamate + malate.

#### Fig. 5. **Comparison of ROS production by wild type brain and spinal cord mitochondria, isolated in 2007 and 2010, oxidizing different substrates and substrate mixtures.**

In 2008 we observed that BM and SCM from the wild type rats displayed a metabolic pattern that was different from the one described above. To highlight the differences in metabolic features of BM and SCM, we present data restricted to 2007, when significant mitochondrial dysfunctions in tgBM and tgSCM were observed as described in (Panov et al. 2011b), and 2010, when the tgSOD1 rat line failed to develop the disease features, and mitochondria showed little or no dysfunction as compared with the corresponding wild type animals. The related metabolic properties of the BM and SCM isolated from wild type animals in 2007 are presented and compared with those of 2010 in figures 5A and 5B.

Figure 5A compares ROS production by WT-BM isolated in 2007 and 2010. Of notice, the rate of ROS production by WT-BM isolated in 2010 was 3.5 fold higher than WT-BM isolated in 2007. However, in 2010 there was no differences in State 4 respiration (not shown) and ROS production (Fig. 5A) when BM oxidized glutamate + malate, pyruvate + malate or glutamate + pyruvate + malate. In contrast to WT-BM isolated in 2007, the much higher basic rate of ROS production supported by glutamate + malate in 2010 was inhibited by malonate to the level close to that in 2007 (Fig. 6). Similarly, in 2010 malonate inhibited ROS production in BM oxidizing glutamate + pyruvate + malate (Fig. 6) and pyruvate (not shown).

produced or contained succinate (Panov 2011a). The described above metabolic features of BM and SCM were consistently observed in the wild type and tgSOD1 rats during period of

**A.** Brain mitochondria; **B.** Spinal cord mitochondria. Dark grey – wild type rats in 2007, light gray – wild type rats in 2010. Mitochondria were incubated in a medium containing: 125 mM KCl, 10 mM MOPS, pH 7.2, 2 mM MgCl2, 2 mM KH2PO4, 10 mM NaCl, 1 mM EGTA, 0.7 mM CaCl2. At a Ca2+/EGTA ratio of 0.7, the free [Ca2+] is close to 1 M as determined using Fura-2. The substrate concentrations were: 5 mM succinate without rotenone, 5 mM glutamate, 2.5 mM pyruvate, and 2 mM malate. (Panov et al. 2009, 2011a, b). The results are presented as Mean ± SE, n = 3, and show the rates of ROS production in pmol H2O2 /minute/mg mitochondrial protein. Additions: 5 μM Amplex red, 1 U of horse radish peroxidase, 50 U of superoxide dismutase (Sigma), 0.05 mg mitochondria, glutamate 5 mM + malate 2 mM (G + M); pyruvate 2.5 mM + malate 2 mM (P + M); glutamate 5 mM + pyruvate 2.5 mM + malate 2 mM (G + P + M); succinate 5 mM (S); succinate 5 mM + glutamate 5 mM + pyruvate 2.5 mM + malate 2 mM (S + G + P + M). Statistics: \*\* *p* < 0.01; \*\*\* *p* < 0.001. For each year the data were

Fig. 5. **Comparison of ROS production by wild type brain and spinal cord mitochondria,** 

In 2008 we observed that BM and SCM from the wild type rats displayed a metabolic pattern that was different from the one described above. To highlight the differences in metabolic features of BM and SCM, we present data restricted to 2007, when significant mitochondrial dysfunctions in tgBM and tgSCM were observed as described in (Panov et al. 2011b), and 2010, when the tgSOD1 rat line failed to develop the disease features, and mitochondria showed little or no dysfunction as compared with the corresponding wild type animals. The related metabolic properties of the BM and SCM isolated from wild type

**isolated in 2007 and 2010, oxidizing different substrates and substrate mixtures.**

animals in 2007 are presented and compared with those of 2010 in figures 5A and 5B.

oxidizing glutamate + pyruvate + malate (Fig. 6) and pyruvate (not shown).

Figure 5A compares ROS production by WT-BM isolated in 2007 and 2010. Of notice, the rate of ROS production by WT-BM isolated in 2010 was 3.5 fold higher than WT-BM isolated in 2007. However, in 2010 there was no differences in State 4 respiration (not shown) and ROS production (Fig. 5A) when BM oxidized glutamate + malate, pyruvate + malate or glutamate + pyruvate + malate. In contrast to WT-BM isolated in 2007, the much higher basic rate of ROS production supported by glutamate + malate in 2010 was inhibited by malonate to the level close to that in 2007 (Fig. 6). Similarly, in 2010 malonate inhibited ROS production in BM

2005 – 2007 years (Panov et al. 2009, 2011a, 2011b).

compared with the rates of ROS production with glutamate + malate.

In contrast to WT-BM isolated in 2007, the WT-BM isolated in 2010 produced ROS supported by succinate at much lower rates than with glutamate + pyruvate + malate (Fig. 5A). Addition of glutamate + pyruvate + malate to the WT-BM -2010 oxidizing succinate stimulated ROS production only to the level with glutamate + malate (Fig. 5A). Similar metabolic features were also observed with the WT-SCM isolated in 2010, although the differences from 2007 WT-SCM were smaller than for the BM (Fig 5B). It is important to note that in 2010 WT-SCM generated ROS with different substrates at the same levels as WT-BM, whereas in 2007 the WT-SCM generated significantly more ROS than the corresponding WT-BM (Fig. 5A and 5B. Note the scale difference between the figures).

Incubation conditions and designations as described in Fig. 5. Malonate 5 mM was added before mitochondria. Dark gray – BM isolated in 2007, light gray columns – BM isolated in 2010. Statistics: \*\*\* *p* < 0.001, NS – not significant difference. The dark columns and grey columns were compared with the corresponding column representing ROS production with glutamate and malate, taken as 100%.

Fig. 6. Effect of malonate on ROS production by wild type brain mitochondria, isolated in 2007 and 2010, oxidizing glutamate + malate, pyruvate + malate, and glutamate + pyruvate + malate.

The sensitivity of the State 4 ROS production supported by either pyruvate or glutamate to malonate (Fig, 6; Panov et al. 2009) demonstrates that at least with BM and SCM both glutamate and pyruvate cannot be regarded as "pure complex I substrates", as is commonly accepted. In BM and SCM during oxidation of glutamate or pyruvate, and particularly when glutamate and pyruvate are present simultaneously, significant amounts of α-ketoglutarate can be formed in transaminase reactions, which is further converted to succinate (Balasz, 1965; Panov et al. 2009).
