**3. Proposed mechanisms for mutant SOD1 toxicity in mitochondria**

Mitochondrial abnormalities and degeneration of motor neurons are early signs of ALS disease (Wong et al., 1995; Dal Canto & Gurney, 1997; Kong & Xu, 1998). They also represent pathological hallmarks in mutant SOD1 transgenic animal models for FALS as well as in patients with sporadic ALS (Kong & Xu, 1998; Mattiazzi et al., 2002; Manfredi & Xu, 2005). Mitochondrial toxicity may thus be an important factor in the degeneration of motor neurons. The pathology, demonstrated in sporadic ALS cases includes mitochondrial aggregates, mitochondrion swelling and increased calcium levels in mitochondria (Atsumi, 1981; Siklós et al., 1996). In G93A-SOD1 transgenic mice the disease onset is associated with a remarkable increase of vacuolated mitochondria in motor neurons (Kong & Xu, 1998). It

Mutant Cu/Zn-Superoxide Dismutase Induced

(Panov et al., n.d.)

pathology in ALS.

Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis 253

Despite of rather different mechanisms proposed for the toxic properties of mutant SOD1 in mitochondria, most of the recent studies document that mitochondrial dysfunction results in increased ROS production (Beretta et al., 2003). Mitochondria isolated from the neural tissue (brain, spinal cord) have distinct metabolic properties regarding the extent of ROS produced upon oxidation of respiratory substrates (Panov et al., 2011). Especially in G93A-SOD1 transgenic rats, brain and spinal cord mitochondria generate 5–7 fold more ROS than mitochondria of corresponding wild-type tissues. Particularly, the spinal cord mitochondria produce two times more hydroperoxide than brain mitochondria of the same animals

Analysis of mitochondrial morphology in G37R and G85R-SOD1 transgenic mice has revealed that somal mitochondria become shorter and rounder in both dismutase active and inactive mutant SOD1 mouse lines. In contrast, axonal mitochondria in G37R-SOD1 animals shift from elongated tubular mitochondria to punctate mitochondria, while in G85R-SOD1 mice the mitochondria have been reported to show an increase in length (Vande Velde et al., 2011). These changes in mitochondrial shape and distribution were characteristic prior to ALS disease onset and support the notion of early mitochondrial

The growing body of evidence provides support to the concept that superoxide dismutation in IMS may cause an increased hydroperoxide production with toxic consequences. Mitochondria are the major intracellular source of superoxide, the primary ROS, where

The two major pathways of superoxide production in mitochondria are autooxidation or complex III catalyzed oxidation of ubisemiquinone (Muller et al., 2004) and complex I catalyzed reduction of oxygen through reversed electron flow in the respiratory chain (Fig. 2.) (Liu et al., 2002). The produced superoxide anion radical has ability to actively react with a number of cellular targets leading to the loss of their proper function. The main detoxifying mechanism for superoxide instead of reverse oxidation of superoxide to oxygen,

Besides SOD1, there are other dedicated enzymes catalyzing this dismutation reaction. In mitochondria Mn-superoxide dismutase (SOD2), which is found in the mitochondrial matrix, scavenges superoxide in this compartment. Extracellular superoxide dismutase (SOD3) is secreted into the extracellular space and protects tissues against excess of superoxide (Zelko et al., 2002). In the IMS superoxide is produced presumably by complex III (Fig. 2.) (Muller et al., 2004). Unlike hydroperoxide, which freely diffuses through the membranes, superoxide cannot cross the mitochondrial inner membrane. In the matrix SOD2 converts superoxide to hydroperoxide, which in turn is reduced to water by the matrix glutathione peroxidase (Inoue et al., 2003). Homozygous SOD2 knockout mice are neonatally lethal (Li et al., 1995), whereas deletion of SOD1 gene does not have apparent

 In IMS the fate of superoxide is determined by SOD1 and cytochrome c, which is present there in millimolar concentrations (Forman & Azzi, 1997; van Beek-Harmsen & van der Laarse, 2005). Cytochrome c is a heme containing protein, which functions as an electron carrier between complex III and cytochrome oxidase in the respiratory chain. Cytocrome c can also efficiently oxidize superoxide to oxygen. In this respect, cytochrome c can function

**4. SOD1 catalyzes increased hydroperoxide production in IMS** 

superoxide anion radical is generated by one electron reduction of oxygen.

includes dismutation to hydroperoxide and oxygen.

motor neuron disease phenotype (Maier & Chan, 2002).

has been proposed that formation of vacuoles originates from the expansion of mitochondrial IMS and degeneration of mitochondrial matrix (Jaarsma et al., 2001; Bendotti et al., 2001; Higgins et al., 2003; Xu et al., 2004).

Currently, there is no consensus on how mutant SOD1 causes mitochondrial pathology. The proposed mechanisms for mitochondrial toxicity of mutant SOD1 are summarized in Table 1. Among other toxic mechanisms reduced activities of respiratory complexes (Browne et al., 1998), mitochondrial depolarization and impaired calcium homeostasis (Kruman et al., 1999) have been demonstrated in the spinal cord of G93A-SOD1 mice. The observed dysfunctions of mitochondria might be caused by the recruitment of mutant SOD1, which has been shown to be selective to spinal cord mitochondria (Stathopulos et al., 2003; Liu et al., 2004; Pasinelli et al., 2004).


Table 1. Proposed mechanisms for mitochondrial toxicity of mutant SOD1

Among the proposed mechanisms, impairment of mitochondrial calcium buffering capacity has been shown in motor neurons of transgenic ALS mice (Damiano et al., 2006). On the other hand, ATP levels have been reported to be diminished in spinal cords of mutant SOD1 mouse model (Mattiazzi et al., 2002). Another view to the mitochondrial toxicity of mutant SOD1 was brought up by Vande Velde et al., who demonstrated that misfolded mutant SOD1 damages mitochondria by its deposition onto the cytoplasmic side of the outer membrane of spinal cord mitochondria (Vande Velde et al., 2008).

Other studies have demonstrated that the increased dismutase activity in rodent ALS models expressing mutant SOD1 paradoxically boosts the production of toxic ROS in the IMS (Goldsteins et al., 2008). It was shown that in a G93A-SOD1 rat model of ALS, the stability and quaternary structure of mutant SOD1 are lost most prominently in the spinal cord already several weeks before the onset of the disease (Ahtoniemi et al., 2008). These results suggest that destabilization of mutant SOD1 is associated with its increased binding to the inner mitochondrial membrane and elevated ROS production in the IMS. (Liu et al., 2004; Kirkinezos et al., 2005; Ahtoniemi et al., 2008).

Importantly, it was also recently demonstrated, that disulphide-reduced apo-SOD1 can rapidly initiate SOD1 fibrillation upon physiological conditions, suggesting that such disulphide-reduced apo-SOD1 may act as a seed for the amyloid like aggregates originating from the destabilized and folding intermediates of mutant SOD1 (Chattopadhyay et al., 2008).

has been proposed that formation of vacuoles originates from the expansion of mitochondrial IMS and degeneration of mitochondrial matrix (Jaarsma et al., 2001; Bendotti

Currently, there is no consensus on how mutant SOD1 causes mitochondrial pathology. The proposed mechanisms for mitochondrial toxicity of mutant SOD1 are summarized in Table 1. Among other toxic mechanisms reduced activities of respiratory complexes (Browne et al., 1998), mitochondrial depolarization and impaired calcium homeostasis (Kruman et al., 1999) have been demonstrated in the spinal cord of G93A-SOD1 mice. The observed dysfunctions of mitochondria might be caused by the recruitment of mutant SOD1, which has been shown to be selective to spinal cord mitochondria (Stathopulos et al., 2003; Liu et al., 2004;

Aggregate accumulation in mitochondria (Higgins et al., 2002; Vande Velde et

causing ROS production (Estévez et al., 1999; Elliott, 2001) Impaired energy metabolism (Siciliano et al., 2001; Mattiazzi et

Impaired Ca2+ buffering (Jaiswal & Keller, 2009; Grosskreutz

Among the proposed mechanisms, impairment of mitochondrial calcium buffering capacity has been shown in motor neurons of transgenic ALS mice (Damiano et al., 2006). On the other hand, ATP levels have been reported to be diminished in spinal cords of mutant SOD1 mouse model (Mattiazzi et al., 2002). Another view to the mitochondrial toxicity of mutant SOD1 was brought up by Vande Velde et al., who demonstrated that misfolded mutant SOD1 damages mitochondria by its deposition onto the cytoplasmic side of the outer

Other studies have demonstrated that the increased dismutase activity in rodent ALS models expressing mutant SOD1 paradoxically boosts the production of toxic ROS in the IMS (Goldsteins et al., 2008). It was shown that in a G93A-SOD1 rat model of ALS, the stability and quaternary structure of mutant SOD1 are lost most prominently in the spinal cord already several weeks before the onset of the disease (Ahtoniemi et al., 2008). These results suggest that destabilization of mutant SOD1 is associated with its increased binding to the inner mitochondrial membrane and elevated ROS production in the IMS. (Liu et al.,

Importantly, it was also recently demonstrated, that disulphide-reduced apo-SOD1 can rapidly initiate SOD1 fibrillation upon physiological conditions, suggesting that such disulphide-reduced apo-SOD1 may act as a seed for the amyloid like aggregates originating from the destabilized and folding intermediates of mutant SOD1 (Chattopadhyay et al.,

Gain in pro-apoptotic function (Pasinelli et al., 2004) Interfering with mitochondrial protein import (Liu et al., 2004)

Increased hydroperoxide production in IMS (Goldsteins et al., 2008)

Table 1. Proposed mechanisms for mitochondrial toxicity of mutant SOD1

membrane of spinal cord mitochondria (Vande Velde et al., 2008).

2004; Kirkinezos et al., 2005; Ahtoniemi et al., 2008).

2008).

al., 2008)

al., 2002)

et al., 2010)

TOXIC MECHANISM REFERENCES

et al., 2001; Higgins et al., 2003; Xu et al., 2004).

Aberrant mutant SOD1 enzymatic activities,

Pasinelli et al., 2004).

Despite of rather different mechanisms proposed for the toxic properties of mutant SOD1 in mitochondria, most of the recent studies document that mitochondrial dysfunction results in increased ROS production (Beretta et al., 2003). Mitochondria isolated from the neural tissue (brain, spinal cord) have distinct metabolic properties regarding the extent of ROS produced upon oxidation of respiratory substrates (Panov et al., 2011). Especially in G93A-SOD1 transgenic rats, brain and spinal cord mitochondria generate 5–7 fold more ROS than mitochondria of corresponding wild-type tissues. Particularly, the spinal cord mitochondria produce two times more hydroperoxide than brain mitochondria of the same animals (Panov et al., n.d.)

Analysis of mitochondrial morphology in G37R and G85R-SOD1 transgenic mice has revealed that somal mitochondria become shorter and rounder in both dismutase active and inactive mutant SOD1 mouse lines. In contrast, axonal mitochondria in G37R-SOD1 animals shift from elongated tubular mitochondria to punctate mitochondria, while in G85R-SOD1 mice the mitochondria have been reported to show an increase in length (Vande Velde et al., 2011). These changes in mitochondrial shape and distribution were characteristic prior to ALS disease onset and support the notion of early mitochondrial pathology in ALS.
