**3. Mitochondrial dysfunction in ALS and in experimental motor neuron degeneration**

A convergent point of the deleterious mechanisms discussed above is the mitochondrion. This organelle is the main energy producer in eukaryotic cells and plays a fundamental role in normal cell physiology. Among the functions mitochondria carry out, besides ATP synthesis, intracellular Ca2+buffering has been recognized as another relevant factor for the protection against deleterious processes such as oxidative stress, excitotoxicity and necrotic and apoptotic death, thus playing a central role in neuronal survival.

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 203

mutation in the gene encoding subunit I of the mitochondrial respiratory chain complex IV (Comi et al., 1998). The electron transport chain proteins FAD synthetase, riboflavin kinase, cytochrome C1, and succinate dehydrogenase complex subunit B expression were

In the mSOD1 mice or cell culture familial ALS model, complexes I, II and IV of the electron transport chain exhibit decreased enzyme activities, even at early stages of the disease (Jung et al., 2002; Mattiazzi et al., 2002; Menzies et al., 2002a,b). In G93A-SOD1 mice the association of cytochrome c with the inner mitochondrial membrane was reduced and there was a significant decrease in respiratory chain complex IV (Kirkinezos et al., 2005). SODcontaining aggregates (Higgins et al., 2002; Jaarsma et al., 2001; Pasinelli et al., 2004) and decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential (Cassina et al., 2008), were observed in mitochondria from spinal cord

In neuronal cultures, glutamate-mediated excitotoxicity caused significant changes in mitochondrial function, such as decline in ATP levels, mitochondrial transmembrane potential collapse, decreased mitochondrial and cellular oxygen consumption, and oxidative phosphorylation uncoupling, all these events preceding cell death (Ankarcrona et al., 1995; Atlante et al., 1996; Maus et al., 1999; Monje et al., 2001). There is a link between excitotoxicity-induced intracellular Ca2+ overload and the collapse of m, since intracellular Ca2+ increase and its accumulation in mitochondria are sufficient to induce prominent and persistent depolarization, leading to mitochondrial dysfunction and to neuronal death in

Few studies on excitotoxicity have been carried out in vivo. In our laboratory we have developed two experimental models of spinal motor neurons degeneration by overactivation of AMPA receptors, both by infusing AMPA directly in the lumbar spinal cord of rats. In the first one AMPA is administered through microdialysis cannulas during short time periods (Corona & Tapia, 2004) and in the other AMPA is infused chronically during several days, using osmotic minipumps (Tovar-y-Romo et al., 2007). These models reproduce the main histopathological features of ALS: loss of lumbar motor neurons, astroglial activation and motor deficits that progresses to complete paralysis of the rear limbs. The main difference between the two models is the time required for the occurrence of motor neuron degeneration and the development of the paralysis. AMPA perfusion by microdialysis causes a rapid loss of motor neuron and paralysis, occurring within the initial 12 hours, while chronic AMPA infusion with osmotic minipumps triggers a progressive motor neuron loss and motor deficits throughout three to four days. For these reasons, the microdialysis model is defined as an acute model and the minipumps model as a chronic model of spinal motor neuron degeneration by excitotoxicity (Tovar-y-Romo et al., 2009a). The most important feature of both models is that motor neuron loss occurs without the influence of a genetic factor and thus presumably can be used to study the mechanisms that may be involved in motor neuron loss occurring in SALS, which accounts for over 90% of

We have recently assessed mitochondrial function in our acute model of spinal excitotoxic motor neuron degeneration, by studying mitochondrial oxygen consumption and transmembrane potential in mitochondria isolated from the lumbar spinal cord of rats perfused with AMPA. The AMPA-treated group showed decreased oxygen consumption, ADP-dependent respiratory control and transmembrane potential, as compared to control

significantly decreased in some ALS patients (Lin et al., 2009).

vitro (Schinder et al., 1996; White & Reynolds, 1996).

of transgenic mSOD1 rodents.

ALS cases.

Mitochondria are closely related to necrotic and apoptotic processes, which are the main cellular death mechanisms. During necrosis, mitochondria undergo rapid swelling and lysis. Although apoptosis is an energy-dependent active process, sometimes mitochondrial morphological alterations are associated with the intrinsic-apoptosis pathway. Furthermore, it is now recognized that apoptosis and necrosis are not two mutually exclusive processes, but they can occur simultaneously or one preceding the other (Kroemer et al., 2009; Martin, 2010; Martin et al., 2009; Shrivastava & Vivekanandhan, 2011).

As the organelle responsible for energy production in the cell, mitochondria possess the enzymatic machinery to catalyze the oxidation of various substrates generated inside and outside mitochondria, including pyruvate trough pyruvate dehydrogenase, fatty acids through β-oxidation, and carbon chains from amino acids. Energy is obtained by oxidation of all these biomolecules to finally CO2 and H2O through the tricarboxylic acid cycle and the respiratory chain. The tricarboxylic acid cycle is the converging point because the carbon skeletons of carbohydrates and fatty acids are metabolized to yield the acetyl group of acetyl-Coenzyme A, and many of the carbons of the amino acid skeleton also enter the cycle via its conversion to some cycle intermediates. The reducing equivalents generated in the tricarboxylic acid cycle reactions reduce pyridine and flavin nucleotides to NADH and FADH2. These electron transporters enter the respiratory chain, where electron flux through various redox carriers and centers in the enzyme complexes located in the inner mitochondrial membrane finally reduces O2 to H2O; this flux is coupled to ATP synthesis through oxidative phosphorylation.

The energy released by the electron flux through respiratory chain complexes is used to pump protons through the inner mitochondrial membrane, producing an alkaline and negatively charged mitochondrial matrix, as compared to the intermembrane space, thus creating a proton gradient. This proton gradient generates an electrochemical potential called proton-motive force (p), which supplies the energy to ATP synthase for ATP synthesis from ADP and inorganic phosphate. The p depends mainly on the mitochondrial transmembrane potential (m), which is the electric potential (negative inside), but it also depends on the transmembrane pH gradient (pH), which is the chemical potential (alkaline inside). Energy stored in the proton gradient can also transport solutes against concentration gradient across the membrane. The m is a central parameter that controls three fundamental and highly relevant cellular processes for neuronal survival: ATP synthesis, mitochondrial Ca2+ sequestration, and mitochondrial ROS generation. On the other hand, m is controlled by substrate availability, ATP demand, respiratory chain capacity, mitochondrial proton conductance, and mitochondrial Ca2+ sequestration (Nicholls & Budd, 2000). Therefore, mitochondrial bioenergetic status is crucial for controlling the susceptibility of neurons to chronic or acute stress and also in determining cellular fate (survival, apoptosis or necrosis).

Owing to the great relevance of mitochondria, their morphological, ultrastructural and functional characteristics have been studied in ALS patients. Deficits in respiratory chain complexes I and IV activities have been detected in the spinal cord and skeletal muscle (Borthwick et al., 1999; Crugnola et al., 2010; Vielhaber et al., 2000; Wiedemann et al., 2002; Wiedemann et al., 1998), and a temporal study of mitochondrial respiratory function in skeletal muscle in SALS demonstrated that respiratory complex IV activity is progressively altered as the disease develops (Echaniz-Laguna et al., 2006). Some cases of ALS have been described as a mitochondriopathy (Finsterer, 2002, 2003) including a mitochondrial DNA

Mitochondria are closely related to necrotic and apoptotic processes, which are the main cellular death mechanisms. During necrosis, mitochondria undergo rapid swelling and lysis. Although apoptosis is an energy-dependent active process, sometimes mitochondrial morphological alterations are associated with the intrinsic-apoptosis pathway. Furthermore, it is now recognized that apoptosis and necrosis are not two mutually exclusive processes, but they can occur simultaneously or one preceding the other (Kroemer et al., 2009; Martin,

As the organelle responsible for energy production in the cell, mitochondria possess the enzymatic machinery to catalyze the oxidation of various substrates generated inside and outside mitochondria, including pyruvate trough pyruvate dehydrogenase, fatty acids through β-oxidation, and carbon chains from amino acids. Energy is obtained by oxidation of all these biomolecules to finally CO2 and H2O through the tricarboxylic acid cycle and the respiratory chain. The tricarboxylic acid cycle is the converging point because the carbon skeletons of carbohydrates and fatty acids are metabolized to yield the acetyl group of acetyl-Coenzyme A, and many of the carbons of the amino acid skeleton also enter the cycle via its conversion to some cycle intermediates. The reducing equivalents generated in the tricarboxylic acid cycle reactions reduce pyridine and flavin nucleotides to NADH and FADH2. These electron transporters enter the respiratory chain, where electron flux through various redox carriers and centers in the enzyme complexes located in the inner mitochondrial membrane finally reduces O2 to H2O; this flux is coupled to ATP synthesis

The energy released by the electron flux through respiratory chain complexes is used to pump protons through the inner mitochondrial membrane, producing an alkaline and negatively charged mitochondrial matrix, as compared to the intermembrane space, thus creating a proton gradient. This proton gradient generates an electrochemical potential called proton-motive force (p), which supplies the energy to ATP synthase for ATP synthesis from ADP and inorganic phosphate. The p depends mainly on the mitochondrial transmembrane potential (m), which is the electric potential (negative inside), but it also depends on the transmembrane pH gradient (pH), which is the chemical potential (alkaline inside). Energy stored in the proton gradient can also transport solutes against concentration gradient across the membrane. The m is a central parameter that controls three fundamental and highly relevant cellular processes for neuronal survival: ATP synthesis, mitochondrial Ca2+ sequestration, and mitochondrial ROS generation. On the other hand, m is controlled by substrate availability, ATP demand, respiratory chain capacity, mitochondrial proton conductance, and mitochondrial Ca2+ sequestration (Nicholls & Budd, 2000). Therefore, mitochondrial bioenergetic status is crucial for controlling the susceptibility of neurons to chronic or acute stress and also in determining cellular fate

Owing to the great relevance of mitochondria, their morphological, ultrastructural and functional characteristics have been studied in ALS patients. Deficits in respiratory chain complexes I and IV activities have been detected in the spinal cord and skeletal muscle (Borthwick et al., 1999; Crugnola et al., 2010; Vielhaber et al., 2000; Wiedemann et al., 2002; Wiedemann et al., 1998), and a temporal study of mitochondrial respiratory function in skeletal muscle in SALS demonstrated that respiratory complex IV activity is progressively altered as the disease develops (Echaniz-Laguna et al., 2006). Some cases of ALS have been described as a mitochondriopathy (Finsterer, 2002, 2003) including a mitochondrial DNA

2010; Martin et al., 2009; Shrivastava & Vivekanandhan, 2011).

through oxidative phosphorylation.

(survival, apoptosis or necrosis).

mutation in the gene encoding subunit I of the mitochondrial respiratory chain complex IV (Comi et al., 1998). The electron transport chain proteins FAD synthetase, riboflavin kinase, cytochrome C1, and succinate dehydrogenase complex subunit B expression were significantly decreased in some ALS patients (Lin et al., 2009).

In the mSOD1 mice or cell culture familial ALS model, complexes I, II and IV of the electron transport chain exhibit decreased enzyme activities, even at early stages of the disease (Jung et al., 2002; Mattiazzi et al., 2002; Menzies et al., 2002a,b). In G93A-SOD1 mice the association of cytochrome c with the inner mitochondrial membrane was reduced and there was a significant decrease in respiratory chain complex IV (Kirkinezos et al., 2005). SODcontaining aggregates (Higgins et al., 2002; Jaarsma et al., 2001; Pasinelli et al., 2004) and decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential (Cassina et al., 2008), were observed in mitochondria from spinal cord of transgenic mSOD1 rodents.

In neuronal cultures, glutamate-mediated excitotoxicity caused significant changes in mitochondrial function, such as decline in ATP levels, mitochondrial transmembrane potential collapse, decreased mitochondrial and cellular oxygen consumption, and oxidative phosphorylation uncoupling, all these events preceding cell death (Ankarcrona et al., 1995; Atlante et al., 1996; Maus et al., 1999; Monje et al., 2001). There is a link between excitotoxicity-induced intracellular Ca2+ overload and the collapse of m, since intracellular Ca2+ increase and its accumulation in mitochondria are sufficient to induce prominent and persistent depolarization, leading to mitochondrial dysfunction and to neuronal death in vitro (Schinder et al., 1996; White & Reynolds, 1996).

Few studies on excitotoxicity have been carried out in vivo. In our laboratory we have developed two experimental models of spinal motor neurons degeneration by overactivation of AMPA receptors, both by infusing AMPA directly in the lumbar spinal cord of rats. In the first one AMPA is administered through microdialysis cannulas during short time periods (Corona & Tapia, 2004) and in the other AMPA is infused chronically during several days, using osmotic minipumps (Tovar-y-Romo et al., 2007). These models reproduce the main histopathological features of ALS: loss of lumbar motor neurons, astroglial activation and motor deficits that progresses to complete paralysis of the rear limbs. The main difference between the two models is the time required for the occurrence of motor neuron degeneration and the development of the paralysis. AMPA perfusion by microdialysis causes a rapid loss of motor neuron and paralysis, occurring within the initial 12 hours, while chronic AMPA infusion with osmotic minipumps triggers a progressive motor neuron loss and motor deficits throughout three to four days. For these reasons, the microdialysis model is defined as an acute model and the minipumps model as a chronic model of spinal motor neuron degeneration by excitotoxicity (Tovar-y-Romo et al., 2009a). The most important feature of both models is that motor neuron loss occurs without the influence of a genetic factor and thus presumably can be used to study the mechanisms that may be involved in motor neuron loss occurring in SALS, which accounts for over 90% of ALS cases.

We have recently assessed mitochondrial function in our acute model of spinal excitotoxic motor neuron degeneration, by studying mitochondrial oxygen consumption and transmembrane potential in mitochondria isolated from the lumbar spinal cord of rats perfused with AMPA. The AMPA-treated group showed decreased oxygen consumption, ADP-dependent respiratory control and transmembrane potential, as compared to control

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 205

to the fact that ATP synthesis requires a thermodynamic threshold for m, while Ca2+ transport can proceed at much lower m and excessive Ca2+ concentrations reduce <sup>m</sup> dramatically. When Ca2+ concentration does not recover below the critical point, excessive Ca2+ overload in the mitochondrial matrix can lead to mitochondrial swelling, loss of respiratory control, increased mitochondrial ROS generation, m collapse (depolarization) diminished ATP synthesis, and Ca2+ release from the mitochondrial matrix caused by inner mitochondrial membrane permeabilization through the mitochondrial permeability transition pore (MPTP, a large protein complex forming a non-selective pore through the inner mitochondrial membrane) (Al-Nasser & Crompton, 1986; Nicholls & Budd, 2000; Peng & Jou, 2010). When mitochondrion depolarizes, accumulated Ca2+ goes back into the cytoplasm, either through the Ca2+ uniporter, the Na+/Ca2+ exchanger, or through the MPTP. Since p depends mainly on m, its collapse causes p collapse, which results not only in halting ATP synthesis but also in a rapid cytoplasmic ATP hydrolysis because ATP

In motor neurons, the damage produced by these alterations may be enhanced because they do not have sufficient mitochondrial Ca2+-buffering capacity, due in part to a lower mitochondrial density per volume compared to non-motor neurons (Grosskreutz et al., 2007). In addition, other buffering mechanisms are deficient in spinal and cortical motor neurons because they lack the Ca2+-binding proteins calbindin D-28K and parvalbumin. This may explain why other motor neurons that express these proteins, such as those located in oculomotor and Onuf's nuclei, are not usually affected in ALS (Alexianu et al., 1994; Celio, 1990; Ince et al., 1993; Palecek et al., 1999). For all these reasons, mitochondrial Ca2+ overload plays a key role in glutamatergic excitotoxicity (Nicholls et al., 2003), given that overactivation of Ca2+-permeable AMPA receptors, which are abundant in spinal motor neurons, confers to these cells a special vulnerability to AMPA receptor-mediated excitotoxicity (Corona & Tapia, 2007; Grosskreutz et al., 2010). AMPA exposure to spinal motor neuron cultures results in an intracellular Ca2+ concentration increase that triggers mitochondrial Ca2+ overload, depolarization and ROS generation (Carriedo et al., 2000). So, there is abundant evidence that suggest that mitochondrial damage, probably related to Ca2+ homeostasis disturbances, is involved in SALS and FALS (Manfredi & Xu, 2005;

Due to the large size of motor neurons and their long processes reaching muscles, they have an expensive energy cost and this renders them very vulnerable to energy deficits. Much of the ATP demand in neurons is used in the ion pumping through plasma membrane to maintain membrane potential. Thus, Na+/K+-ATPase is the most demanding ATP process in neurons (Scott & Nicholls, 1980) in order to expel Na+ excess resulting from excitation. Intracellular Ca2+ regulation by Ca-ATPases is also highly energy consuming, as previously

There is abundant evidence both in vitro and in vivo that any restriction in the ability of the cell to generate ATP can exacerbate or even induce glutamatergic excitotoxicity. The energylinked excitotoxic hypothesis (Beal et al., 1993; Greene & Greenamyre, 1996; Novelli et al., 1988) proposes that the correlation between excitotoxic damage and energy restriction is due to plasma membrane depolarization. Diminished ATP levels cause a decrease in Na+/K+- ATPase and Ca2+-ATPase functions, lessening Na+ and Ca2+ removal. This triggers plasma

synthase catalytic function reverses in an attempt to restore p.

Menzies et al., 2002a; Swerdlow et al., 1998; von Lewinski & Keller, 2005).

**3.2 Energy deficits** 

discussed.

rats perfused only with Krebs-Ringer medium (Santa-Cruz and Tapia, in preparation). These results suggest that mitochondrial dysfunction plays a crucial role in spinal motoneuron degeneration induced by overactivation of AMPA receptors in vivo. These mechanisms could be involved in ALS motoneuron degeneration.

### **3.1 Ca2+, mitochondria and motor neuron degeneration**

Under physiological conditions, Ca2+ participates as intracellular messenger in many normal cellular functions, such as cell growth, differentiation, signal transduction, membrane excitability regulation, exocytosis and synaptic activity. Cytoplasmic Ca2+ concentration in resting neurons is maintained at low concentrations (~100 nM), 10,000 times lower than extracellular space concentration. To achieve this, neurons possess specialized homeostatic mechanisms, such as regulation of Ca2+ input and output, Ca2+ binding proteins, mitochondrial and endoplasmic reticulum storage, and Ca2+-ATPases. Moreover, neurons not only control intracellular Ca2+ levels, but also its location in the cell by means of complex interactions among Ca2+ input, output, buffering and internal storage. Under physiological conditions, these processes maintain spatial and temporal location of Ca2+, so that multiple Ca2+-regulated signaling pathways can take place independently within the same cell.

Excessive intracellular Ca2+ concentration damages neurons through several mechanisms, including mitochondrial damage, energy metabolism deficit, toxic ROS generation, membrane depolarization, and activation of lytic enzymes such as proteases, lipases, phosphatases and endonucleases. Intracellular Ca2+ accumulation also stimulates ROS production through NOS activation and the conversion of xanthine dehydrogenase to xanthine oxidase through proteases activation. All these events eventually produce membrane destruction and neuronal death (Arundine & Tymianski, 2003; Shaw, 1999).

Intracellular Ca2+ regulation is an expensive process from the energy point of view. Ca2+ is extruded from the cell and sequestered into the endoplasmic reticulum through active transport using Ca2+-ATPases, and it is also removed by secondary active transport using the Na+/Ca2+ exchanger, which activates Na+/K+-ATPases to take out Na+. Mitochondria also play a critical role in the regulation of cytosolic Ca2+ concentration, since they sequester this cation through a Ca2+ uniporter located in the inner mitochondrial membrane and driven by the electric potential (Nicholls, 1985). To prevent a potentially lethal Ca2+ accumulation in mitochondrial matrix, there is an output system that exchanges Na+/Ca2+, besides a mitochondrial Na+/H+ transporter that extrudes Na+, so that ion flux under a constant Ca2+ entrance to mitochondria involves a sequential transfer of Ca2+, Na+ and H+, the latter driven by the respiratory chain (Crompton & Heid, 1978; Nicholls & Budd, 2000). When Ca2+ concentration surpasses a certain critical point, under physiological phosphate concentration an osmotically inactive and rapidly dissociable Ca2+-phosphate complex is formed in the mitochondrial matrix, so that mitochondria work as efficient buffers of extramitochondrial Ca2+ by accumulating this cation (Becker et al., 1980; Nicholls, 1978). Apparently, this organelle acts as a temporary Ca2+ store during high cytoplasmic concentrations peaks, as suggested by the kinetics of mitochondrial Ca2+ transport; because the Ca2+-phosphate complex is rapidly dissociable, mitochondria can release Ca2+ back to the cytoplasm when its concentration decreases below the critical point. As long as mitochondria are polarized, cytosolic Ca2+ accumulates within the mitochondrial matrix through the Ca2+ uniporter. Mitochondrial Ca2+ uptake is driven by m, so it will compete with ATP synthase for proton gradient, in such a way that Ca2+ uptake could dominate due to the fact that ATP synthesis requires a thermodynamic threshold for m, while Ca2+ transport can proceed at much lower m and excessive Ca2+ concentrations reduce <sup>m</sup> dramatically. When Ca2+ concentration does not recover below the critical point, excessive Ca2+ overload in the mitochondrial matrix can lead to mitochondrial swelling, loss of respiratory control, increased mitochondrial ROS generation, m collapse (depolarization) diminished ATP synthesis, and Ca2+ release from the mitochondrial matrix caused by inner mitochondrial membrane permeabilization through the mitochondrial permeability transition pore (MPTP, a large protein complex forming a non-selective pore through the inner mitochondrial membrane) (Al-Nasser & Crompton, 1986; Nicholls & Budd, 2000; Peng & Jou, 2010). When mitochondrion depolarizes, accumulated Ca2+ goes back into the cytoplasm, either through the Ca2+ uniporter, the Na+/Ca2+ exchanger, or through the MPTP. Since p depends mainly on m, its collapse causes p collapse, which results not only in halting ATP synthesis but also in a rapid cytoplasmic ATP hydrolysis because ATP synthase catalytic function reverses in an attempt to restore p.

In motor neurons, the damage produced by these alterations may be enhanced because they do not have sufficient mitochondrial Ca2+-buffering capacity, due in part to a lower mitochondrial density per volume compared to non-motor neurons (Grosskreutz et al., 2007). In addition, other buffering mechanisms are deficient in spinal and cortical motor neurons because they lack the Ca2+-binding proteins calbindin D-28K and parvalbumin. This may explain why other motor neurons that express these proteins, such as those located in oculomotor and Onuf's nuclei, are not usually affected in ALS (Alexianu et al., 1994; Celio, 1990; Ince et al., 1993; Palecek et al., 1999). For all these reasons, mitochondrial Ca2+ overload plays a key role in glutamatergic excitotoxicity (Nicholls et al., 2003), given that overactivation of Ca2+-permeable AMPA receptors, which are abundant in spinal motor neurons, confers to these cells a special vulnerability to AMPA receptor-mediated excitotoxicity (Corona & Tapia, 2007; Grosskreutz et al., 2010). AMPA exposure to spinal motor neuron cultures results in an intracellular Ca2+ concentration increase that triggers mitochondrial Ca2+ overload, depolarization and ROS generation (Carriedo et al., 2000). So, there is abundant evidence that suggest that mitochondrial damage, probably related to Ca2+ homeostasis disturbances, is involved in SALS and FALS (Manfredi & Xu, 2005; Menzies et al., 2002a; Swerdlow et al., 1998; von Lewinski & Keller, 2005).

### **3.2 Energy deficits**

204 Amyotrophic Lateral Sclerosis

rats perfused only with Krebs-Ringer medium (Santa-Cruz and Tapia, in preparation). These results suggest that mitochondrial dysfunction plays a crucial role in spinal motoneuron degeneration induced by overactivation of AMPA receptors in vivo. These

Under physiological conditions, Ca2+ participates as intracellular messenger in many normal cellular functions, such as cell growth, differentiation, signal transduction, membrane excitability regulation, exocytosis and synaptic activity. Cytoplasmic Ca2+ concentration in resting neurons is maintained at low concentrations (~100 nM), 10,000 times lower than extracellular space concentration. To achieve this, neurons possess specialized homeostatic mechanisms, such as regulation of Ca2+ input and output, Ca2+ binding proteins, mitochondrial and endoplasmic reticulum storage, and Ca2+-ATPases. Moreover, neurons not only control intracellular Ca2+ levels, but also its location in the cell by means of complex interactions among Ca2+ input, output, buffering and internal storage. Under physiological conditions, these processes maintain spatial and temporal location of Ca2+, so that multiple Ca2+-regulated signaling pathways can take place independently within the same cell. Excessive intracellular Ca2+ concentration damages neurons through several mechanisms, including mitochondrial damage, energy metabolism deficit, toxic ROS generation, membrane depolarization, and activation of lytic enzymes such as proteases, lipases, phosphatases and endonucleases. Intracellular Ca2+ accumulation also stimulates ROS production through NOS activation and the conversion of xanthine dehydrogenase to xanthine oxidase through proteases activation. All these events eventually produce membrane destruction and neuronal death (Arundine & Tymianski, 2003; Shaw, 1999). Intracellular Ca2+ regulation is an expensive process from the energy point of view. Ca2+ is extruded from the cell and sequestered into the endoplasmic reticulum through active transport using Ca2+-ATPases, and it is also removed by secondary active transport using the Na+/Ca2+ exchanger, which activates Na+/K+-ATPases to take out Na+. Mitochondria also play a critical role in the regulation of cytosolic Ca2+ concentration, since they sequester this cation through a Ca2+ uniporter located in the inner mitochondrial membrane and driven by the electric potential (Nicholls, 1985). To prevent a potentially lethal Ca2+ accumulation in mitochondrial matrix, there is an output system that exchanges Na+/Ca2+, besides a mitochondrial Na+/H+ transporter that extrudes Na+, so that ion flux under a constant Ca2+ entrance to mitochondria involves a sequential transfer of Ca2+, Na+ and H+, the latter driven by the respiratory chain (Crompton & Heid, 1978; Nicholls & Budd, 2000). When Ca2+ concentration surpasses a certain critical point, under physiological phosphate concentration an osmotically inactive and rapidly dissociable Ca2+-phosphate complex is formed in the mitochondrial matrix, so that mitochondria work as efficient buffers of extramitochondrial Ca2+ by accumulating this cation (Becker et al., 1980; Nicholls, 1978). Apparently, this organelle acts as a temporary Ca2+ store during high cytoplasmic concentrations peaks, as suggested by the kinetics of mitochondrial Ca2+ transport; because the Ca2+-phosphate complex is rapidly dissociable, mitochondria can release Ca2+ back to the cytoplasm when its concentration decreases below the critical point. As long as mitochondria are polarized, cytosolic Ca2+ accumulates within the mitochondrial matrix through the Ca2+ uniporter. Mitochondrial Ca2+ uptake is driven by m, so it will compete with ATP synthase for proton gradient, in such a way that Ca2+ uptake could dominate due

mechanisms could be involved in ALS motoneuron degeneration.

**3.1 Ca2+, mitochondria and motor neuron degeneration** 

Due to the large size of motor neurons and their long processes reaching muscles, they have an expensive energy cost and this renders them very vulnerable to energy deficits. Much of the ATP demand in neurons is used in the ion pumping through plasma membrane to maintain membrane potential. Thus, Na+/K+-ATPase is the most demanding ATP process in neurons (Scott & Nicholls, 1980) in order to expel Na+ excess resulting from excitation. Intracellular Ca2+ regulation by Ca-ATPases is also highly energy consuming, as previously discussed.

There is abundant evidence both in vitro and in vivo that any restriction in the ability of the cell to generate ATP can exacerbate or even induce glutamatergic excitotoxicity. The energylinked excitotoxic hypothesis (Beal et al., 1993; Greene & Greenamyre, 1996; Novelli et al., 1988) proposes that the correlation between excitotoxic damage and energy restriction is due to plasma membrane depolarization. Diminished ATP levels cause a decrease in Na+/K+- ATPase and Ca2+-ATPase functions, lessening Na+ and Ca2+ removal. This triggers plasma

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 207

ROS production, triggering or increasing cellular injure. Among them, mitochondrial Ca2+ overload resulting from NMDA, AMPA or kainate receptor overactivation (Carriedo et al., 1998; Carriedo et al., 2000; Dugan et al., 1995) increases ROS production (Dykens, 1994; Peng & Jou, 2010); thus, an initial excitotoxic event might also contribute to increased oxidative

In addition, it is important to consider that mitochondria are not only ROS producers but also that they are a susceptible target of them. Thereby, in a pathologic situation where an increased ROS production occurs initially, oxidative damage to mitochondrial lipids, nucleic acids and proteins can reduce mitochondrial respiration, disturb normal function and seriously damage this organelle (Lenaz et al., 2002). Furthermore, mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA, due to its close location next to an important ROS production site, to the lack of protective histones and to less effective repair mechanisms, as compared to the nuclear DNA (Richter et al., 1988). Mitochondrial redox status also influences the opening of the MPTP, since it is enhanced by oxidative stress in

**4. Mitochondrial structural damage in ALS and experimental motor neuron** 

The death process involved in the motor neuron loss characteristic of ALS is not yet fully understood. Several functional alterations present in both human disease and experimental models have been reviewed in the previous sections, but several studies have shown also morphological and ultrastructural changes in motor neurons that may be associated with

Postmortem examination of ALS patients tissues has revealed morphological and ultrastructural abnormalities in mitochondria. Atypical mitochondrial aggregates were found in skeletal muscle subsarcolemmal region and in intramuscular axons (Afifi et al., 1966; Atsumi, 1981), and morphological abnormalities were also detected in proximal axons, as well as dense clusters of mitochondria in the ventral horn of spinal cord SALS patients (Hirano et al., 1984a; b; Sasaki & Iwata, 1996). Giant mitochondria with intramitochondrial inclusions were observed in the liver of some ALS patients and these alterations were disease specific (Nakano et al., 1987). Further, mitochondria with increased volume and with high Ca2+ concentration were found in motor nerve terminals in muscle biopsies of alive ALS patients, which were not observed in patients with other neuropathies or in control subjects (Siklos et al., 1996). Ultrastructural damage of mitochondria, characterized by swelling and rounding, was recently described in platelets of ALS patients (Shrivastava

The main problem with pathological studies in human ALS is the difficulty in determining whether the alterations observed are a cause or a consequence of the disease. This highlights the importance of developing experimental models of motor neuron death to study the temporal progress of the morphological changes, including the alterations of mitochondrial structure. With this objective, we have recently studied the ultrastructural changes of mitochondria in both our acute and chronic models of spinal motor neuron death described above. In the acute model we observed motor neurons with mitochondrial swelling as soon as 2 h after AMPA perfusion, followed in a few hours by the rupture of mitochondrial, nuclear and plasma membranes, which led to total neuronal disruption. These ultrastructural alterations are characteristic of a necrotic process. In contrast, in the chronic

stress.

**degeneration** 

apoptosis and/or necrosis.

isolated mitochondria (Saxena et al., 1995).

& Vivekanandhan, 2011; Shrivastava et al., 2011a,b).

membrane depolarization and as a consequence Ca2+ enters the cell through voltagedependent Ca2+channels and glutamate is released to the extracellular space by exocytosis. This in turn activates Ca2+ influx through the NMDA receptor, which is also voltagedependent. Further, under energetic failure conditions, glutamate transporters operate in reverse because Na+/K+ electrochemical gradient collapse due to ATP decrease, resulting in diminished glutamate uptake and non-vesicular glutamate release into extracellular space (Jabaudon et al., 2000; Longuemare & Swanson, 1995).

The observation that inhibition of mitochondrial respiratory chain complexes activity can induce pathological changes similar to those observed in some neurodegenerative diseases in specific CNS regions has generated great interest. Association among glutamatergic excitotoxicity and bioenergetic limitation has been proposed for Alzheimer, Parkinson, Huntington's disease and ALS (Beal, 1998), and in many cases specific respiratory chain complexes are involved. In organotypic spinal cord cultures, motor neurons are selectively vulnerable to chronic mitochondrial blockade by inhibitors of mitochondrial respiratory chain complex II and complex IV and this motor neuron degeneration displays structural changes similar to those seen following excitotoxicity (Brunet et al., 2009; Kaal et al., 2000).

In our acute model of excitotoxic motor neuron degeneration previously described (Corona & Tapia, 2004, 2007) we have demonstrated the importance of Ca2+-permeable AMPA receptors and of intracellular Ca2+ overload in motor neuron death process. Using this model, we aimed to study the importance of energy deficits and oxidative stress in AMPAinduced degeneration. With this purpose, we assessed the potential neuroprotection of various energy substrates and antioxidants at different concentrations, co-perfusing them with AMPA in the rat lumbar spinal cord. We observed protection at different degrees depending on the concentration of each compound, but in general antioxidants only partially protected, while various energy substrates prevented the AMPA-induced motor impairment and the spinal motor neuron loss (Santa-Cruz and Tapia, in preparation). These findings suggest that intracellular Ca2+ overload in vivo disrupts mitochondrial energy metabolism. On the other hand, energy substrates can directly prevent m collapse and thus prevent mitochondrial dysfunction. Because one of the factors that control m is substrate availability, excess mitochondrial substrates administered exogenously can stimulate respiratory chain and increase oxidative phosphorylation, maintaining the electrochemical proton gradient and thus preventing the collapse of ATP synthesis.

### **3.3 Oxidative stress**

Since mitochondria are the organelles where oxidative phosphorylation is accomplished, they consume about 98 % of the cell oxygen requirement and constitute a major site for intracellular ROS production. Some steps along mitochondrial oxygen reduction pathway have the potential to produce, and indeed generate free radicals, due to the fact that electron flux along respiratory chain may have leakage of electrons to oxygen. The intermediate radical ubisemiquinone, involved in the transfer of electrons through respiratory complexes III and I, can grant an electron to oxygen, forming the superoxide radical O2•-, a powerful oxidant and a very reactive intermediate (Turrens et al., 1985) that must be rapidly removed by antioxidant enzymes to avoid its lethal effects. About 0.1-4% of the O2 used by actively respiring mitochondria is converted to O2•-. Nevertheless, respiratory chain enzymes defects or other mitochondrial perturbations could be responsible of an excessive mitochondrial

membrane depolarization and as a consequence Ca2+ enters the cell through voltagedependent Ca2+channels and glutamate is released to the extracellular space by exocytosis. This in turn activates Ca2+ influx through the NMDA receptor, which is also voltagedependent. Further, under energetic failure conditions, glutamate transporters operate in reverse because Na+/K+ electrochemical gradient collapse due to ATP decrease, resulting in diminished glutamate uptake and non-vesicular glutamate release into extracellular space

The observation that inhibition of mitochondrial respiratory chain complexes activity can induce pathological changes similar to those observed in some neurodegenerative diseases in specific CNS regions has generated great interest. Association among glutamatergic excitotoxicity and bioenergetic limitation has been proposed for Alzheimer, Parkinson, Huntington's disease and ALS (Beal, 1998), and in many cases specific respiratory chain complexes are involved. In organotypic spinal cord cultures, motor neurons are selectively vulnerable to chronic mitochondrial blockade by inhibitors of mitochondrial respiratory chain complex II and complex IV and this motor neuron degeneration displays structural changes similar to those seen following excitotoxicity

In our acute model of excitotoxic motor neuron degeneration previously described (Corona & Tapia, 2004, 2007) we have demonstrated the importance of Ca2+-permeable AMPA receptors and of intracellular Ca2+ overload in motor neuron death process. Using this model, we aimed to study the importance of energy deficits and oxidative stress in AMPAinduced degeneration. With this purpose, we assessed the potential neuroprotection of various energy substrates and antioxidants at different concentrations, co-perfusing them with AMPA in the rat lumbar spinal cord. We observed protection at different degrees depending on the concentration of each compound, but in general antioxidants only partially protected, while various energy substrates prevented the AMPA-induced motor impairment and the spinal motor neuron loss (Santa-Cruz and Tapia, in preparation). These findings suggest that intracellular Ca2+ overload in vivo disrupts mitochondrial energy metabolism. On the other hand, energy substrates can directly prevent m collapse and thus prevent mitochondrial dysfunction. Because one of the factors that control m is substrate availability, excess mitochondrial substrates administered exogenously can stimulate respiratory chain and increase oxidative phosphorylation, maintaining the

electrochemical proton gradient and thus preventing the collapse of ATP synthesis.

Since mitochondria are the organelles where oxidative phosphorylation is accomplished, they consume about 98 % of the cell oxygen requirement and constitute a major site for intracellular ROS production. Some steps along mitochondrial oxygen reduction pathway have the potential to produce, and indeed generate free radicals, due to the fact that electron flux along respiratory chain may have leakage of electrons to oxygen. The intermediate radical ubisemiquinone, involved in the transfer of electrons through respiratory complexes III and I, can grant an electron to oxygen, forming the superoxide radical O2•-, a powerful oxidant and a very reactive intermediate (Turrens et al., 1985) that must be rapidly removed by antioxidant enzymes to avoid its lethal effects. About 0.1-4% of the O2 used by actively

or other mitochondrial perturbations could be responsible of an excessive mitochondrial

•-. Nevertheless, respiratory chain enzymes defects

(Jabaudon et al., 2000; Longuemare & Swanson, 1995).

(Brunet et al., 2009; Kaal et al., 2000).

**3.3 Oxidative stress** 

respiring mitochondria is converted to O2

ROS production, triggering or increasing cellular injure. Among them, mitochondrial Ca2+ overload resulting from NMDA, AMPA or kainate receptor overactivation (Carriedo et al., 1998; Carriedo et al., 2000; Dugan et al., 1995) increases ROS production (Dykens, 1994; Peng & Jou, 2010); thus, an initial excitotoxic event might also contribute to increased oxidative stress.

In addition, it is important to consider that mitochondria are not only ROS producers but also that they are a susceptible target of them. Thereby, in a pathologic situation where an increased ROS production occurs initially, oxidative damage to mitochondrial lipids, nucleic acids and proteins can reduce mitochondrial respiration, disturb normal function and seriously damage this organelle (Lenaz et al., 2002). Furthermore, mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA, due to its close location next to an important ROS production site, to the lack of protective histones and to less effective repair mechanisms, as compared to the nuclear DNA (Richter et al., 1988). Mitochondrial redox status also influences the opening of the MPTP, since it is enhanced by oxidative stress in isolated mitochondria (Saxena et al., 1995).
