**3. General features of neuronal energy metabolism**

Neuronal cells are strictly aerobic and require large quantities of ATP produced at high rate in order to function normally. Humans brain constitutes only 2% of the body's weight, but accounts for 20% of its resting O2 metabolism (Attwell & Laughlin, 2001). However, even in humans, heart and kidney consume significantly more oxygen per unit of the organ's mass than the brain (Rolfe & Brown, 1997). In rats, liver, kidney and heart consume correspondingly 2, 3 and 3.9 times more oxygen than the brain (Rolfe & Brown, 1997). Thus it is not the necessity for the high rate of oxidative metabolism that is unique for the central nerve system, but how the oxidative metabolism is organized in neuronal cells.

Cortical tissue is composed mainly of two types of cells: the nerve cells (or neurons) and the neuroglial cells (or glia). Glial cells greatly outnumber the neurons (Abeles, 1991; Attwell & Laughlin, 2001; Stolzenburg et al., 1989). Glial cells do not directly take part in the interactions between neurons, but play an important role in maintaining neuronal metabolism and in directing the development and growth of the brain. The classical categorization of neurons was based mainly on morphology of the cell body and its dendritic tree, and to a lesser extent on the morphology of the axon and its branches (Abeles, 1991).

There are two main categories of the cortical cells according to their physiological effects on each other: excitatory and inhibitory cells. Excitatory cells release transmitters (glutamate) at their synaptic ends that, on contact with the postsynaptic membrane, create ion currents that depolarize the postsynaptic cell. Inhibitory cells release transmitters, such as γ-aminobutyric acid (GABA), that tend to hyperpolarize the postsynaptic cell or to increase the conductance of the postsynaptic membrane to chloride, thereby diminishing the effects of the depolarizing currents generated by the excitatory synapses. Excitatory synapses dominate the brain's grey matter because excitatory neurons outnumber inhibitory cells by a factor of 9 to 1, and 90% of synapses release glutamate (Abeles, 1991; Braitenberg & Schüz, 1998). The distribution of mitochondria points to these glutamatergic synapses as the major users of metabolic energy (Wong-Riley, 1989; Attwell & Laughlin, 2001).

More than 80% of neuronal mitochondria are located in the dendrites and axonal terminals (Wong-Riley, 1989; Attwell & Laughlin, 2001). With high synaptic density of 8 x 108 per 1

from presymptomatic tgSOD1 rats showed almost normal rates of oxidative phosphorylation. Panov et all. (2011b) purified mitochondria in a Percoll gradient, and thus eliminated damaged mitochondria. This resulted in significantly lower yields of mitochondria per 1 gram of tissue of tgSOD1 rats: the yields were diminished by 27% for the brain and by 58% for spinal cord as compared with the normal Sprague Dawley rats (Panov et al., 2011b). Avossa et al. (2006) used immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard *in vitro* conditions. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motor neurons, and there were no signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. Together, the results of Panov et al. (2011b) and Avossa et al. (2006) suggest that in tgSOD1 rats the BM and SCM are primarily normal but suffer from some pathological events, which

In order to better understand the metabolic events involved in putative pathological mechanisms of neurodegeneration, we have to discuss the distinctive features of brain and

Neuronal cells are strictly aerobic and require large quantities of ATP produced at high rate in order to function normally. Humans brain constitutes only 2% of the body's weight, but accounts for 20% of its resting O2 metabolism (Attwell & Laughlin, 2001). However, even in humans, heart and kidney consume significantly more oxygen per unit of the organ's mass than the brain (Rolfe & Brown, 1997). In rats, liver, kidney and heart consume correspondingly 2, 3 and 3.9 times more oxygen than the brain (Rolfe & Brown, 1997). Thus it is not the necessity for the high rate of oxidative metabolism that is unique for the central

Cortical tissue is composed mainly of two types of cells: the nerve cells (or neurons) and the neuroglial cells (or glia). Glial cells greatly outnumber the neurons (Abeles, 1991; Attwell & Laughlin, 2001; Stolzenburg et al., 1989). Glial cells do not directly take part in the interactions between neurons, but play an important role in maintaining neuronal metabolism and in directing the development and growth of the brain. The classical categorization of neurons was based mainly on morphology of the cell body and its dendritic tree, and to a lesser extent

There are two main categories of the cortical cells according to their physiological effects on each other: excitatory and inhibitory cells. Excitatory cells release transmitters (glutamate) at their synaptic ends that, on contact with the postsynaptic membrane, create ion currents that depolarize the postsynaptic cell. Inhibitory cells release transmitters, such as γ-aminobutyric acid (GABA), that tend to hyperpolarize the postsynaptic cell or to increase the conductance of the postsynaptic membrane to chloride, thereby diminishing the effects of the depolarizing currents generated by the excitatory synapses. Excitatory synapses dominate the brain's grey matter because excitatory neurons outnumber inhibitory cells by a factor of 9 to 1, and 90% of synapses release glutamate (Abeles, 1991; Braitenberg & Schüz, 1998). The distribution of mitochondria points to these glutamatergic synapses as the major users of

More than 80% of neuronal mitochondria are located in the dendrites and axonal terminals (Wong-Riley, 1989; Attwell & Laughlin, 2001). With high synaptic density of 8 x 108 per 1

nerve system, but how the oxidative metabolism is organized in neuronal cells.

rapidly deteriorate mitochondria and cause death of neurons.

**3. General features of neuronal energy metabolism** 

on the morphology of the axon and its branches (Abeles, 1991).

metabolic energy (Wong-Riley, 1989; Attwell & Laughlin, 2001).

spinal cord energy metabolism and their mitochondria.

mm3 in all cortices (the density of synapses is not very much different among species) it is clear that in the brain most energy expenditures are associated with synaptic activity, where most mitochondria are located (Abeles, 1991). The close spacing of terminals along axons (5 μm, implying a diffusion time of only 25 milliseconds) make terminal and axonal mitochondria functionally indistinguishable (Attwell & Laughlin, 2001).

Neurons communicate by means of signals that lead to depolarization of their excitable membranes. The degree of depolarization of the dendritic or soma membranes is determined by complex interactions between excitatory and inhibitory transmitters. The depolarization spreads to the axon, leading to release of an excitatory or inhibitory transmitter at the axonal endings of the cell, thus allowing information to be transferred to the next cell. When impulses are propagated, the cell loses K+ and gains Na+, and in order to preserve excitability the cell must extrude Na+ and accumulate K+, using metabolic energy derived from oxidative metabolism (Abeles, 1991). Postsynaptic ion fluxes (Na+, K+, Cl- ) and presynaptic Ca2+ influx consume in grey matter approximately 75% of the brain's energy (Abeles, 1991; Attwell & Laughlin, 2001). Because depolarization and restoration of ionic composition across the neuronal membrane occur in the millisecond scale, neuronal mitochondria must provide large quantities of ATP in a very short time. This distribution of energy use reflects the fact that a chemical synapse is an amplifier: for each glutamate released approximately 100 ions enter the postsynaptic terminal (Abeles, 1991). The rest (25%) of the brain's energy is used for the basic "housekeeping" functions, such as maintenance of pH gradients, asymmetrical distributions of phospholipids in membrane bilayers, protein synthesis (about 2%), anaplerotic functions and recycling of glutamate from synaptic clefts (Attwell & Laughlin, 2001). It was suggested that white and grey matter require similar amounts of energy on basic (nonsignaling) cellular activities (Attwell & Laughlin, 2001).

It is widely accepted that nerve tissues of adult mammals use glucose as the major source of energy. To fully understand the specifics of energy metabolism in brain, it is important to take into account that, in dendrites and synaptic terminals, there is limited space for glycolytic enzymes that would provide mitochondria with enough pyruvate derived from glucose. Therefore, neurons cannot be functionally and metabolically regarded without astroglia. Astrocytes provide neurons with lactate, as the major source of energy, replenish the neuronal pool of glutamine and recycle glutamate (Ebert et al., 2003; Pellerin et al., 2007; Suzuki et al., 2011). Of the total energy consumed by the brain, approximately 80% of energy expenditure is satisfied by oxidation of glucose and 20% by oxidation of fatty acids (Ebert et al., 2003). As we will see later, neurons also oxidize significant amounts of glutamate and GABA as a source of energy. The neurotransmitter GABA is formed from glutamate by the action of glutamate decarboxylase. It appears that glutamine serves as the precursor for glutamate and GABA. Therefore, the pool of glutamine must be constantly replenished in anaplerotic reactions, which occur predominantly in astroglia.

$$\begin{array}{ccccc} \mathsf{H}\mathsf{O}\mathsf{-}\mathsf{C}^{\mathsf{I}}\mathsf{-}\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{C}\mathsf{H} & \xrightarrow{\mathsf{C}\mathsf{O}\mathsf{D}} & \mathsf{O}\mathsf{D}^{\mathsf{I}}\\ \mathsf{O}\mathsf{N}\mathsf{-}^{\mathsf{I}}\mathsf{N}\mathsf{-}^{\mathsf{I}} & \mathsf{O}\mathsf{N}\mathsf{-}^{\mathsf{I}} & \mathsf{O}\mathsf{N}\mathsf{-}^{\mathsf{I}}\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{C}\mathsf{H}\_{2}^{\mathsf{I}}-\mathsf{N}\mathsf{H}\_{2}^{\mathsf{I}} \\ \end{array}$$

Fig. 1. Formation of GABA from glutamate. GAD – glutamate decarboxylase.

Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS 233

In order to rapidly oxidize lactate to pyruvate, the cytosolic NADH must be oxidized back to NAD+. In neurons the task of reoxidation of the cytosolic NADH and transport of hydrogen into mitochondria occur via a number of functionally coupled cytosolic and mitochondrial enzymes, which form the malate-aspartate shuttle (MAS) (Figure 2). Glutamate is a necessary component of MAS. Because nonsynaptic and presynaptic neuronal mitochondria effectively oxidize glutamate (Yudkoff et al. 1994), neuronal cells

Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 (GLT-1) and EAAT2 (GLAST) located on presynaptic termini and glial cells (Furness et al., 2008). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (Auger & Attwell., 2000; Brasnjo& Otis., 2004). EAAT4 protein was found to be omnipresent throughout the fore- and midbrain (Massie et al., 2008). These data suggest that postsynaptic transport of glutamate into nerve terminals, where mitochondria are located (Abeles, 1991), may occur in all brain regions. According to calculations of Brasnjo and Otis (2004), in a single synapse EAAT4 bind and transport postsynaptically about 1.3 ± 0.1 x 106 glutamate molecules. In the brain, on average, 1 mm3 of tissue contains 1 x 108 synapses (Abeles, 1991; Nicholls, 1993). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus in a narrow space of spines and dendrites several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in a low mM range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions may temporarily metabolize, in addition to pyruvate, some amounts of glutamate (Yudkoff et al. 1994; Panov et al. 2009). Besides, GABA is also transported postsynaptically where it is catabolized in mitochondria to succinate (Tillakaratne et al.,

As we have mentioned, for rapid conversion of lactate to pyruvate, postsynaptic neurons must receive a certain amount of glutamate to fuel MAS to recycle cytosolic NAD+ (Berkich et al., 2007). Importantly, the activity of the brain mitochondrial isoform of aspartate glutamate transporter is controlled by Ca2+ (Pardo et al., 2006). Therefore increased neuronal activity through small changes in the extramitochondrial Ca2+ activates MAS, whereas increased mitochondrial Ca2+ activates mitochondrial respiration

There is evidence (Yudkoff et al., 1994), that in the presence of glutamate + pyruvate, the tricarboxylic acid (TCA) cycle in brain mitochondria operates as two coupled cycles: one (cycle A) leads from α-KG to OAA, and another (cycle B) from OAA to a-KG that includes the citrate synthase reaction (see figure 3). According to Yudkoff et al. (1994), the flux of substrates through cycle A is 3-5-fold faster than that through the cycle B. Thus, with pyruvate + glutamate + malate, activation of α-ketoglutarate dehydrogenase complex (α-KGDHC) and succinate dehydrogenase (SDH) may significantly increase the rates of the tricarboxylic acid cycle (TCA) cycle and respiratory chain in state 3 and state 3U (Fig. 3). A high turnover of cycle A with activated SDH would increase reverse electron transport

constantly have to replenish the cytosolic pool of glutamate.

1995).

(Nicholls, 2009).

(RET) and the associated ROS production.

Because anaplerotic functions of astrocytes require energy, oxidation of fatty acids occurs in glial mitochondria (Ebert et al., 2003) providing energy and saving glucose for production of lactate in aerobic glycolysis and α-ketoglutarate for synthesis *de novo* neuromediators glutamate and GABA (Waagepetersen et al., 1999; Ebert et al., 2003). Glutamine synthetase is an astrocytespecific enzyme (Norenberg & Martinez-Hernandez, 1979), and during octanoate oxidation in astrocytes most of the anaplerotic flux in brain is occurring in the TCA cycle associated with glutamine production (Ebert et al., 2003). In order to fulfill the task of metabolic provision of neuronal functions there are several mechanisms that ensure functional and metabolic compartmentalization and complementation between neurons and astroglia, which thus exclude competition for energy substrates. Neuronal mitochondria do not possess the full range activity of β-oxidation enzymes, whereas glial mitochondria readily oxidize fatty acids (Ebert et al., 2003). On the other hand, glial mitochondria do not have active glutamate-aspartate translocase (Berkich et al., 2007), which is the key enzyme of the malate aspartate shuttle and the major pathway for oxidation of glutamate by neuronal mitochondria. Thus astrocytes do not utilize glutamate as a respiratory substrate. The distribution of lactate dehydrogenase isoforms between glial cells and neurons promotes aerobic glycolysis in astroglia and utilization of lactate by neurons as the major respiratory substrate (Pellerin et al., 2007).

### **4. In activated neurons mitochondria utilize a mixture of substrates**

Mitochondria are devices for burning hydrogen producing water and releasing energy, a large part of which is accumulated as electrochemical transmembrane potential (ΔμH+) (Mitchell, 1977; Nicholls, 2008). ΔμH+ is utilized for production of ATP, and other useful work in the mitochondria and cell. Therefore lactate, which has additional hydrogen, as compared with pyruvate, has higher energetic value for neuronal mitochondria. In the cytosol of a neuron, lactate is oxidized to pyruvate in the reaction catalyzed by lactate dehydrogenase (LDH): NAD+ + Lactate NADH + H+ + Pyruvate. Pyruvate is transported into mitochondria and metabolized.

Abbreviations: AST – aspartate aminotransferase; GAT – glutamate-aspartate transporter, MDH – malate dehydrogenase; MKgT – malate- α -ketoglutarate transporter; OAA – oxaloacetate

Fig. 2. **The malate aspartate shuttle.** In a cell with respiring energized mitochondria the malate aspartate shuttle serves for transport and oxidation of hydrogen from cytosolic NADH to mitochondrial respiratory chain. The process is unidirectional because GAT is electrogenic and the matrix NADH is rapidly oxidized by respiratory chain.

Because anaplerotic functions of astrocytes require energy, oxidation of fatty acids occurs in glial mitochondria (Ebert et al., 2003) providing energy and saving glucose for production of lactate in aerobic glycolysis and α-ketoglutarate for synthesis *de novo* neuromediators glutamate and GABA (Waagepetersen et al., 1999; Ebert et al., 2003). Glutamine synthetase is an astrocytespecific enzyme (Norenberg & Martinez-Hernandez, 1979), and during octanoate oxidation in astrocytes most of the anaplerotic flux in brain is occurring in the TCA cycle associated with glutamine production (Ebert et al., 2003). In order to fulfill the task of metabolic provision of neuronal functions there are several mechanisms that ensure functional and metabolic compartmentalization and complementation between neurons and astroglia, which thus exclude competition for energy substrates. Neuronal mitochondria do not possess the full range activity of β-oxidation enzymes, whereas glial mitochondria readily oxidize fatty acids (Ebert et al., 2003). On the other hand, glial mitochondria do not have active glutamate-aspartate translocase (Berkich et al., 2007), which is the key enzyme of the malate aspartate shuttle and the major pathway for oxidation of glutamate by neuronal mitochondria. Thus astrocytes do not utilize glutamate as a respiratory substrate. The distribution of lactate dehydrogenase isoforms between glial cells and neurons promotes aerobic glycolysis in astroglia and utilization of

lactate by neurons as the major respiratory substrate (Pellerin et al., 2007).

into mitochondria and metabolized.

**4. In activated neurons mitochondria utilize a mixture of substrates** 

Mitochondria are devices for burning hydrogen producing water and releasing energy, a large part of which is accumulated as electrochemical transmembrane potential (ΔμH+) (Mitchell, 1977; Nicholls, 2008). ΔμH+ is utilized for production of ATP, and other useful work in the mitochondria and cell. Therefore lactate, which has additional hydrogen, as compared with pyruvate, has higher energetic value for neuronal mitochondria. In the cytosol of a neuron, lactate is oxidized to pyruvate in the reaction catalyzed by lactate dehydrogenase (LDH): NAD+ + Lactate NADH + H+ + Pyruvate. Pyruvate is transported

Abbreviations: AST – aspartate aminotransferase; GAT – glutamate-aspartate transporter, MDH –

Fig. 2. **The malate aspartate shuttle.** In a cell with respiring energized mitochondria the malate aspartate shuttle serves for transport and oxidation of hydrogen from cytosolic NADH to mitochondrial respiratory chain. The process is unidirectional because GAT is

malate dehydrogenase; MKgT – malate- α -ketoglutarate transporter; OAA – oxaloacetate

electrogenic and the matrix NADH is rapidly oxidized by respiratory chain.

In order to rapidly oxidize lactate to pyruvate, the cytosolic NADH must be oxidized back to NAD+. In neurons the task of reoxidation of the cytosolic NADH and transport of hydrogen into mitochondria occur via a number of functionally coupled cytosolic and mitochondrial enzymes, which form the malate-aspartate shuttle (MAS) (Figure 2). Glutamate is a necessary component of MAS. Because nonsynaptic and presynaptic neuronal mitochondria effectively oxidize glutamate (Yudkoff et al. 1994), neuronal cells constantly have to replenish the cytosolic pool of glutamate.

Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 (GLT-1) and EAAT2 (GLAST) located on presynaptic termini and glial cells (Furness et al., 2008). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (Auger & Attwell., 2000; Brasnjo& Otis., 2004). EAAT4 protein was found to be omnipresent throughout the fore- and midbrain (Massie et al., 2008). These data suggest that postsynaptic transport of glutamate into nerve terminals, where mitochondria are located (Abeles, 1991), may occur in all brain regions. According to calculations of Brasnjo and Otis (2004), in a single synapse EAAT4 bind and transport postsynaptically about 1.3 ± 0.1 x 106 glutamate molecules. In the brain, on average, 1 mm3 of tissue contains 1 x 108 synapses (Abeles, 1991; Nicholls, 1993). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus in a narrow space of spines and dendrites several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in a low mM range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions may temporarily metabolize, in addition to pyruvate, some amounts of glutamate (Yudkoff et al. 1994; Panov et al. 2009). Besides, GABA is also transported postsynaptically where it is catabolized in mitochondria to succinate (Tillakaratne et al., 1995).

As we have mentioned, for rapid conversion of lactate to pyruvate, postsynaptic neurons must receive a certain amount of glutamate to fuel MAS to recycle cytosolic NAD+ (Berkich et al., 2007). Importantly, the activity of the brain mitochondrial isoform of aspartate glutamate transporter is controlled by Ca2+ (Pardo et al., 2006). Therefore increased neuronal activity through small changes in the extramitochondrial Ca2+ activates MAS, whereas increased mitochondrial Ca2+ activates mitochondrial respiration (Nicholls, 2009).

There is evidence (Yudkoff et al., 1994), that in the presence of glutamate + pyruvate, the tricarboxylic acid (TCA) cycle in brain mitochondria operates as two coupled cycles: one (cycle A) leads from α-KG to OAA, and another (cycle B) from OAA to a-KG that includes the citrate synthase reaction (see figure 3). According to Yudkoff et al. (1994), the flux of substrates through cycle A is 3-5-fold faster than that through the cycle B. Thus, with pyruvate + glutamate + malate, activation of α-ketoglutarate dehydrogenase complex (α-KGDHC) and succinate dehydrogenase (SDH) may significantly increase the rates of the tricarboxylic acid cycle (TCA) cycle and respiratory chain in state 3 and state 3U (Fig. 3). A high turnover of cycle A with activated SDH would increase reverse electron transport (RET) and the associated ROS production.

Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS 235

also part of the TCA cycle – succinate dehydrogenase (SDH), feeds electrons into the mitochondrial pool of CoQ10 (ubiquinone, Q). The reduced ubiquinone (QH2) in its turn reduces the sites on Complex I that can generate superoxide radicals at high rate. This process, known as reverse electron transport, is energy-dependent and inhibited by rotenone. Some researchers deny the significance of succinate in ROS production on the pretext that succinate concentration is too low in mitochondria (Starkov, 2008; Stowe & Camara, 2009; Zoccarato et al., 2007). As we have shown recently, however, brain and spinal cord mitochondria may produce succinate even in the presence of pyruvate + malate (Panov et al., 2009) , and as we show in this article (see Fig. 6) even with glutamate + malate a large part of ROS production may be associated with oxidation of succinate. However a particularly large production of the succinate-dependent ROS was observed in the presence of glutamate + pyruvate + malate (Panov et al., 2009, 2011a, 2011b). With these substrates the increased succinate oxidation was caused by a dramatic increase in mitochondrial α-ketoglutarate due to high activities of aminotransferases (Balasz, 1965a, 1965b). Besides, GABA is catabolized with formation of succinate in postsynaptic mitochondria (Tillakaratne et al. 1995). Thus, in the excited neurons succinate is an indispensable mitochondrial metabolite. In this paper we provide evidence that ROS generation associated with the succinate-dependent reverse electron transport is a subject

Another objection to the importance of reverse electron transport (RET) in ROS generation argues that, because RET is energy-dependent, in the functioning cell the diminished mitochondrial energization will inhibit production of ROS (Starkov, 2008). The latter objection is valid for the most perpetually functioning organs, such as heart, kidney and liver. But it is only partially applicable to brain and spinal cord where a very large number of mitochondria are located at axonal and dendritic junctions (Wong-Riley, 1989). At the narrow spaces of synaptic junctions there is no other task for mitochondria beside provision of ATP for restoration of ionic composition in excited synapses. If the neurons are not excited, mitochondria become fully energized and produce ROS at high rate. Therefore axonal and synaptic junctions, including neuro muscular junctions, are particularly

Another possible reason why the succinate-dependent ROS production was underrated could be the fact that most researchers use "classical" mixtures of substrates, such as glutamate + malate, pyruvate + malate, succinate alone, or, very often, in the presence of

Figure 4 shows a representative experiment with the BM and SCM isolated in 2007 from tg SOD1 rats. One can see that with glutamate + malate, the rate of ROS production, measured as H2O2, there were no differences between tgBM and tgSCM, wtBM or wtSCM (not shown). With pyruvate + malate as substrates, tgSCM generated significantly more ROS than tgBM (see Fig. 5). This was because SCM in general oxidize succinate at higher rate than BM, presumably due to the lower intrinsic inhibition of SDH by oxaloacetate (Panov et al., 2011a). However, when tgBM and tgSCM oxidized a mixture of glutamate + pyruvate + malate, there was a several-fold increase in ROS production. TgSCM produced much more ROS than tgBM (Fig. 4, see also Fig. 6 in Panov et al., 2011b). Thus the usage of physiologically relevant mixtures of substrates is far more informative for understanding the role of mitochondria in pathogenesis of diseases, than the "classical"

vulnerable to oxidative damage if neurons are not excited for some reason.

for phenotypic variations.

rotenone.

approach.

Abbreviations: ALT – alanine aminotransferase; AST –aspartate aminotransferase; SDH – succinate dehydrogenase (Complex II).

Fig. 3. **The tricarboxylic acid cycle in the presence of pyruvate and glutamate operates as two coupled cycles A and B**. In the presence of glutamate and/or pyruvate as substrates high activities of AST and ALT may "short-circuit" oxaloacetate and α-ketoglutarate, thus converting the "normal" TCA cycle into two independently operating cycles A and B. Malonate is a quasi-irreversible inhibitor of SDH, whereas oxaloacetate (OAA) is a competitive but strong inhibitor. SDH has similar affinities for succinate and malate, but oxidation of malate results in formation of OAA on the enzyme's active center, which makes malate a strong inhibitor of SDH.

Thus in activated neurons mitochondria may oxidize simultaneously glutamate, pyruvate, and metabolites of the TCA cycle, such as malate, and succinate. Postsynaptically transported GABA is catabolized in mitochondria to succinate (Tillakaratne et al., 1995). Under these conditions, brain (BM) and spinal cord (SCM) mitochondria significantly increase the rate of oxidative phosphorylation and the succinate-dependent production of ROS (Panov et al., 2009, 2011a). With glutamate + pyruvate + malate as substrates, increased production of ATP and ROS was associated with dramatic increase in the matrix concentration of α-ketoglutarate (Balasz, 1965a) due to increased activities of mitochondrial aminotransferases AST and ALT (Fig. 3). Under these conditions glutamate dehydrogenase (GLDH) does not participate in production of α-KG (Balasz, 1965b). Since AST and ALT are present in great excess, compared with the respiration rate, the oxaloacetate (OAA) formed is continuously removed by the transamination reactions. Balazs (1965b) concluded that a competition takes place between the α-KGDHC and GLDH, probably for NAD+, resulting in preferential oxidation of α-oxoglutarate.

### **5. Distinctive properties of ROS generation in brain and spinal cord mitochondria**

In neuronal tissue mitochondria are the major source of ROS production (Stowe & Camara, 2009), which is indispensable feature of aerobic metabolism. It is generally accepted that during oxidation of the NAD-dependent substrates the limiting step is at the NADH-dehydrogenase site of Complex I (see Panov et al. 2007, 2009). With succinate as a substrate, production of ROS is significantly higher, because Complex II, which is

Abbreviations: ALT – alanine aminotransferase; AST –aspartate aminotransferase; SDH – succinate

Fig. 3. **The tricarboxylic acid cycle in the presence of pyruvate and glutamate operates as two coupled cycles A and B**. In the presence of glutamate and/or pyruvate as substrates high activities of AST and ALT may "short-circuit" oxaloacetate and α-ketoglutarate, thus converting the "normal" TCA cycle into two independently operating cycles A and B. Malonate is a quasi-irreversible inhibitor of SDH, whereas oxaloacetate (OAA) is a competitive but strong inhibitor. SDH has similar affinities for succinate and malate, but oxidation of malate results in formation of OAA on the enzyme's active center, which makes

Thus in activated neurons mitochondria may oxidize simultaneously glutamate, pyruvate, and metabolites of the TCA cycle, such as malate, and succinate. Postsynaptically transported GABA is catabolized in mitochondria to succinate (Tillakaratne et al., 1995). Under these conditions, brain (BM) and spinal cord (SCM) mitochondria significantly increase the rate of oxidative phosphorylation and the succinate-dependent production of ROS (Panov et al., 2009, 2011a). With glutamate + pyruvate + malate as substrates, increased production of ATP and ROS was associated with dramatic increase in the matrix concentration of α-ketoglutarate (Balasz, 1965a) due to increased activities of mitochondrial aminotransferases AST and ALT (Fig. 3). Under these conditions glutamate dehydrogenase (GLDH) does not participate in production of α-KG (Balasz, 1965b). Since AST and ALT are present in great excess, compared with the respiration rate, the oxaloacetate (OAA) formed is continuously removed by the transamination reactions. Balazs (1965b) concluded that a competition takes place between the α-KGDHC and GLDH, probably for NAD+, resulting

**5. Distinctive properties of ROS generation in brain and spinal cord** 

In neuronal tissue mitochondria are the major source of ROS production (Stowe & Camara, 2009), which is indispensable feature of aerobic metabolism. It is generally accepted that during oxidation of the NAD-dependent substrates the limiting step is at the NADH-dehydrogenase site of Complex I (see Panov et al. 2007, 2009). With succinate as a substrate, production of ROS is significantly higher, because Complex II, which is

dehydrogenase (Complex II).

malate a strong inhibitor of SDH.

in preferential oxidation of α-oxoglutarate.

**mitochondria** 

also part of the TCA cycle – succinate dehydrogenase (SDH), feeds electrons into the mitochondrial pool of CoQ10 (ubiquinone, Q). The reduced ubiquinone (QH2) in its turn reduces the sites on Complex I that can generate superoxide radicals at high rate. This process, known as reverse electron transport, is energy-dependent and inhibited by rotenone. Some researchers deny the significance of succinate in ROS production on the pretext that succinate concentration is too low in mitochondria (Starkov, 2008; Stowe & Camara, 2009; Zoccarato et al., 2007). As we have shown recently, however, brain and spinal cord mitochondria may produce succinate even in the presence of pyruvate + malate (Panov et al., 2009) , and as we show in this article (see Fig. 6) even with glutamate + malate a large part of ROS production may be associated with oxidation of succinate. However a particularly large production of the succinate-dependent ROS was observed in the presence of glutamate + pyruvate + malate (Panov et al., 2009, 2011a, 2011b). With these substrates the increased succinate oxidation was caused by a dramatic increase in mitochondrial α-ketoglutarate due to high activities of aminotransferases (Balasz, 1965a, 1965b). Besides, GABA is catabolized with formation of succinate in postsynaptic mitochondria (Tillakaratne et al. 1995). Thus, in the excited neurons succinate is an indispensable mitochondrial metabolite. In this paper we provide evidence that ROS generation associated with the succinate-dependent reverse electron transport is a subject for phenotypic variations.

Another objection to the importance of reverse electron transport (RET) in ROS generation argues that, because RET is energy-dependent, in the functioning cell the diminished mitochondrial energization will inhibit production of ROS (Starkov, 2008). The latter objection is valid for the most perpetually functioning organs, such as heart, kidney and liver. But it is only partially applicable to brain and spinal cord where a very large number of mitochondria are located at axonal and dendritic junctions (Wong-Riley, 1989). At the narrow spaces of synaptic junctions there is no other task for mitochondria beside provision of ATP for restoration of ionic composition in excited synapses. If the neurons are not excited, mitochondria become fully energized and produce ROS at high rate. Therefore axonal and synaptic junctions, including neuro muscular junctions, are particularly vulnerable to oxidative damage if neurons are not excited for some reason.

Another possible reason why the succinate-dependent ROS production was underrated could be the fact that most researchers use "classical" mixtures of substrates, such as glutamate + malate, pyruvate + malate, succinate alone, or, very often, in the presence of rotenone.

Figure 4 shows a representative experiment with the BM and SCM isolated in 2007 from tg SOD1 rats. One can see that with glutamate + malate, the rate of ROS production, measured as H2O2, there were no differences between tgBM and tgSCM, wtBM or wtSCM (not shown). With pyruvate + malate as substrates, tgSCM generated significantly more ROS than tgBM (see Fig. 5). This was because SCM in general oxidize succinate at higher rate than BM, presumably due to the lower intrinsic inhibition of SDH by oxaloacetate (Panov et al., 2011a). However, when tgBM and tgSCM oxidized a mixture of glutamate + pyruvate + malate, there was a several-fold increase in ROS production. TgSCM produced much more ROS than tgBM (Fig. 4, see also Fig. 6 in Panov et al., 2011b). Thus the usage of physiologically relevant mixtures of substrates is far more informative for understanding the role of mitochondria in pathogenesis of diseases, than the "classical" approach.

Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS 237

subject to phenotypic variations between species and in one species with time (Panov et al., 2010). Below we describe how changes in mitochondrial metabolism of BM and SCM of wild type Sprague Dawley rats affected mitochondrial dysfunctions in tgSOD1 rats in 2007 and

**6. Differences between BM and SCM isolated in 2007 and 2010 in the rates of** 

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

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

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

glutamate + pyruvate + malate to the level with glutamate + malate.

**ROS production with different substrates** 

with all substrates and their mixtures.

2010.

Fig. 4. **Production of ROS by brain and spinal cord mitochondria from tgSOD1 rats**. Mitochondrial production of ROS was registered as H2O2 by Amplex red method (Panov et al., 2009).

We suggested the following metabolic scenario for the neuronal mitochondria located at synaptic junctions. During neuronal activity, the postsynaptic mitochondria are exposed to increased levels of pyruvate and glutamate, which enhance ATP production due to specific interactions between the glutamate transforming enzymes and the TCA cycle enzymes. This effect of the substrate mixtures is specific for the BM, and SCM (Panov et al., 2009, 2011a). As soon as activation of a synapse comes to an end, transport of glutamate from the synaptic cleft would also stop. As a result, activation of respiration induced by the simultaneous presence of glutamate and pyruvate will also end because glutamate becomes exhausted. Thus the neuromediator glutamate controls energy metabolism in brain not only at the level of astrocytes by enhancing production of lactate, but also at the level of neurons by controlling the activity of MAS and specific interactions between aminotransferases.

In the absence of neuronal activation, SDH (Complex II) in the postsynaptic mitochondria is inhibited by OAA (Panov et al. 2010a). The physiological significance of this inhibition is to prevent excessive ROS production associated with reverse electron transport. This is particularly important for neurons because with pyruvate, which is the major mitochondrial substrate in resting neurons (Pellerin et al., 1994; Hertz, 2004), there is a substantial production of succinate and thus a possibility of increased RET-dependent ROS production (see Fig. 5A, 5B; Panov et al. 2009, 2011a, 2011b). During increased neuronal activity glutamate and pyruvate temporarily release the inhibition of SDH. Increased ATP production (state 3) prevents generation of ROS (Starkov, 2008; Votyakova & Reynolds, 2001). Because SCM in general oxidize succinate at higher rates than BM, due to lower intrinsic inhibition of SDH, they are more vulnerable to oxidative stress than BM (Panov et al., 2011a, 2011b).

Activation of oxidative phosphorylation and increased ROS generation with pyruvate + glutamate + malate was observed only with BM and SCM (Panov et al., 2009, 2011a). However, activation of succinate oxidation by pyruvate and glutamate may be a common phenomenon for mitochondria from many organs. Our experiments with mitochondria from different organs and different species of rats and mice, and with one species (Sprague Dawley) of rats over period of 6 years, have shown that mitochondrial metabolism is a

Fig. 4. **Production of ROS by brain and spinal cord mitochondria from tgSOD1 rats**. Mitochondrial production of ROS was registered as H2O2 by Amplex red method (Panov et

controlling the activity of MAS and specific interactions between aminotransferases.

In the absence of neuronal activation, SDH (Complex II) in the postsynaptic mitochondria is inhibited by OAA (Panov et al. 2010a). The physiological significance of this inhibition is to prevent excessive ROS production associated with reverse electron transport. This is particularly important for neurons because with pyruvate, which is the major mitochondrial substrate in resting neurons (Pellerin et al., 1994; Hertz, 2004), there is a substantial production of succinate and thus a possibility of increased RET-dependent ROS production (see Fig. 5A, 5B; Panov et al. 2009, 2011a, 2011b). During increased neuronal activity glutamate and pyruvate temporarily release the inhibition of SDH. Increased ATP production (state 3) prevents generation of ROS (Starkov, 2008; Votyakova & Reynolds, 2001). Because SCM in general oxidize succinate at higher rates than BM, due to lower intrinsic inhibition of SDH, they are more vulnerable to oxidative stress than BM (Panov et

Activation of oxidative phosphorylation and increased ROS generation with pyruvate + glutamate + malate was observed only with BM and SCM (Panov et al., 2009, 2011a). However, activation of succinate oxidation by pyruvate and glutamate may be a common phenomenon for mitochondria from many organs. Our experiments with mitochondria from different organs and different species of rats and mice, and with one species (Sprague Dawley) of rats over period of 6 years, have shown that mitochondrial metabolism is a

We suggested the following metabolic scenario for the neuronal mitochondria located at synaptic junctions. During neuronal activity, the postsynaptic mitochondria are exposed to increased levels of pyruvate and glutamate, which enhance ATP production due to specific interactions between the glutamate transforming enzymes and the TCA cycle enzymes. This effect of the substrate mixtures is specific for the BM, and SCM (Panov et al., 2009, 2011a). As soon as activation of a synapse comes to an end, transport of glutamate from the synaptic cleft would also stop. As a result, activation of respiration induced by the simultaneous presence of glutamate and pyruvate will also end because glutamate becomes exhausted. Thus the neuromediator glutamate controls energy metabolism in brain not only at the level of astrocytes by enhancing production of lactate, but also at the level of neurons by

al., 2009).

al., 2011a, 2011b).

subject to phenotypic variations between species and in one species with time (Panov et al., 2010). Below we describe how changes in mitochondrial metabolism of BM and SCM of wild type Sprague Dawley rats affected mitochondrial dysfunctions in tgSOD1 rats in 2007 and 2010.
