**Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS**

Alexander Panov1, 3, Nury Steuerwald1, Valentin Vavilin2, Svetlana Dambinova3 and Herbert L. Bonkovsky1 *1Cannon Research Center, Carolinas Medical Center, Charlotte, NC, 2Institute of Molecular Biology and Biophysics, Novosibirsk, 3Laboratory of Brain Biomarkers, WellStar College of Health & Human Services, Kennesaw State University, Kennesaw, GA, 1,3USA 2Russia* 

### **1. Introduction**

224 Amyotrophic Lateral Sclerosis

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Amyotrophic lateral sclerosis (ALS) is one of the group of diseases of the central nerve system (CNS), which are characterized by progressive loss of structure and function of neurons in different regions of brain or spinal cord. Therefore, these diseases are collectively designated as "Neurodegenerative Diseases" (NDDs). Usually the loss of specific functions precedes the death of affected neurons, and the related clinical features depend on localization and degree of neurodegeneration. NDDs include such diseases as Alzheimer's, Parkinson's, Huntington's, spinocerebellar ataxias, and ALS. In spite of differences in predominant localization of neurodegeneration and clinical features, there are many parallels among different neurodegenerative disorders. These include involvement of mitochondrial dysfunctions, increased oxidative stress, and atypical protein assemblies (Backman et al., 2006).

Amyotrophic lateral sclerosis (ALS) refers to several adult-onset conditions characterized by progressive degeneration of motor neurons. "Amyotrophic" refers to the muscle atrophy, weakness, and fasciculation (spontaneous contraction affecting a small number of muscle fibers) that signify disease of the lower motor neurons. There are two forms of this fatal disease: sporadic, with no known genetic component, and familial, which make up about 10% of all ALS cases (Rowland & Schneider, 2001; Martin et al., 2009). Among the familial cases, approximately 20% are caused by dominantly inherited mutations in the Cu/Zn superoxide dismutase (*SOD1*) gene, with more than 100 known mutations (reviewed in Bruijn et al., 2004). So far, there is very little information that links familial and sporadic cases of the disease. One established fact, based on studies of patients and transgenic animals, is that mitochondria dysfunction is an early manifestation. However, it is unclear whether mitochondrial dysfunctions are the primary pathogenic mechanism, or the result of some other proximate pathogenic mechanism.

Although the cases associated with mutations in the *SOD1* gene comprise only about 2% of all ALS cases, understandably, transgenic animals bearing mutated *SOD1* gene

Role of Neuronal Mitochondrial Metabolic Phenotype in Pathogenesis of ALS 227

We suggest a hypothesis, which links together pathogenic mechanisms of sporadic and familial forms of ALS. Our hypothesis is based on results obtained in experiments on animals, which showed that between species and even within one species there may exist different phenotypes of mitochondrial metabolism. Neuronal mitochondria of some phenotypes may produce very large amounts of superoxide radical, which is dismutated to hydrogen peroxide (H2O2). High levels of H2O2 may damage the extramitochondrial isoform of superoxide dismutase (SOD1). The oxidatively damaged proteins lose Cu and Zn from the hem centers, and the demetallated protein enters mitochondrial membranes, further increases ROS production by a mechanism similar to that described for cholesterol-β-D-glucoside and fitosterol glucosides (Panov et al., 2010b), causes mitochondrial dysfunctions, and finally the death of neurons. These are sporadic cases of ALS associated with disorders of mitochondrial metabolism. The familial cases with mutations in the *SOD1* gene have basically the same mechanism of motor neuron death, but, because mutated SOD1 protein is exceptionally sensitive to oxidative damage, the loss of Cu and Zn may occur at relatively low levels of H2O2 formation by neuronal mitochondria. The predominant involvement of spinal cord can be accounted for by metabolic and structural features of spinal cord and SCM, which were published

Clinical features of ALS, a severe neuromuscular degenerative disease, were described by Charcot in 1874 (Rowland & Shneider, 2001). Since that time clinical definitions (Shook and Pioro, 2009) and pathological features of the disease have been greatly expanded, aided in no small measure by advances in genetics of the disease (Rowland & Shneider, 2001) and development of transgenic animal models of ALS (Bendotti & Carri, 2004; Matsumoto et al., 2006; Howland et al., 2002). However, the etiology and pathogenesis of the disease remain

Clinical and experimental evidence showed that ALS is a systemic disease, with particular vulnerability of motor neurons due to some unique properties (Martin et al., 2007; von Lewinski & Keller, 2005; Panov et al., 2011a). Many ALS patients are hypermetabolic, an early and persistent phenomenon (Bouteloup et al., 2009, Desport et al., 2005). Muscular mitochondrial function in amyotrophic lateral sclerosis is progressively altered in ALS patients (Echaniz-Laguna et al., 2006, Krasnianski et al., 2005), and subtle ultrastructural changes of hepatocytes and liver dysfunction have also been described in biopsy specimens from ALS patients (Nakano et al., 1987). Significant changes were also found in skeletal muscle mitochondria of transgenic SOD1 (tgSOD1) animals (Dupuis et al., 2009, Krasnianski et al., 2005). It was suggested that increased ROS generation by skeletal muscle mitochondria (Muller et al., 2007) or mitochondrial uncoupling (Dupuis et al., 2009) may be primarily responsible for the loss of neuromuscular junctions and secondary distal degeneration of motor neurons in SOD1 mice. This is an interesting and important alternative hypothesis, which requires a more

The paper by Muller et al. (2007) presents data on generation of ROS by skeletal muscle mitochondria (SMM) from two lines of transgenic mice bearing different mutations in SOD1 gene, SOD1 knockout mice, and mice with denervated muscle. The authors

elsewhere (Panov et al., 2011a, 2011b).

poorly understood.

detailed and critical discussion.

**2. General characterization of mitochondria in ALS** 

became the major targets for research on pathogenesis of ALS (Bendotti and Carri, 2004; Bruijn et al., 2004; Matsumoto et al., 2006). Of these, a transgenic mouse carrying the G93A (Gly-93 Ala) mutant human *SOD1* gene was the first described (Gurney et al., 1994). This animal model of ALS was used all over the world because it closely recapitulates clinical and histopathological features of the human disease (Matsumoto et al., 2006).

Researchers from Wyeth, John Hopkins and Harvard in collaboration with ALS Association as part of its Lou Gehrig Challenge Initiative established the SOD1G93A mutant rat line. Taconic (Germantown, NY) established a production colony in 2002, which was sponsored by grant funding from ALS Association. The original publication on this strain (tgSOD1) reported disease onset around 115 days of age, with rapid disease progression thereafter (Howland et al., 2002). Taconic has maintained a colony since 2002. In our recent work (Panov et al., 2011b) we presented data obtained in 2007 on 45 tgSOD1 animals obtained from 9 separate simultaneous isolations of BM and SCM. We studied respiration and reactive oxygen species production by mitochondria isolated from the brains (tgBM) and spinal cords (tgSCM), of such rats and the results obtained were highly reproducible (Panov et al., 2011b). Beginning late in 2008 and in 2009 we encountered difficulties in reproducing the results obtained in 2007. Moreover, we have found that the transgenic rat line did not develop symptoms of the disease at age of more than 200 days even though the mutated gene was evident in DNA samples from tails and ears. In 2010 we received information from Taconic (Germantown, NY) that changes in features can be attributed to a variable phenotype in this model, "which may be due to in part to both the outbred nature of the background strain as well as to possible copy number variation of the transgene" (Information Letter from Taconic). Whether these phenotypic changes were associated with the decline of the level to which the transgene was expressed is impossible to conclude because, in the rats studied in 2007, neither Taconic company, nor our lab quantified the expression of the mutated SOD1 gene or protein. Nevertheless, our comparative studies of metabolic properties of BM and SCM suggest that potentially the loss of morbidity could be explained by different patterns of substrates metabolism and associated ROS generation in BM and SCM studied in 2007 and 2010.

In 2008 we found that BM and SCM isolated from the wild type Sprague Dawley rats began to show metabolic features that were different from those described earlier for the same strain of the wild type and tgSOD1 rats (Panov et al., 2009, 2011a). In this work we present a comparison of metabolic phenotypes and the substrate–dependent ROS generation in the wild type and transgenic rats with mutant G93A Cu/Zn-superoxide dismutase gene isolated in 2007 and 2010. We conclude that the shift in mitochondrial metabolic phenotype, which resulted in a dramatic decrease in ROS production by tgBM and tgSCM, may have contributed to the significant loss of morbidity of the established tgSOD1 rat line. This could result from the breeding method of the transgenic animals, when males bearing the dominant mutated *SOD1* gene were mated with the wild type females. Because mitochondrial DNA has maternal inheritance (Wallace, 2001), the resulting progeny has the mitochondrial phenotype of females, and thus the mutated SOD1 protein was acting on mitochondria with different metabolism that resulted in much lower rates of ROS production. We conclude that mitochondria are the key players in the pathogenesis of ALS.

became the major targets for research on pathogenesis of ALS (Bendotti and Carri, 2004; Bruijn et al., 2004; Matsumoto et al., 2006). Of these, a transgenic mouse carrying the G93A (Gly-93 Ala) mutant human *SOD1* gene was the first described (Gurney et al., 1994). This animal model of ALS was used all over the world because it closely recapitulates clinical and histopathological features of the human disease (Matsumoto et

Researchers from Wyeth, John Hopkins and Harvard in collaboration with ALS Association as part of its Lou Gehrig Challenge Initiative established the SOD1G93A mutant rat line. Taconic (Germantown, NY) established a production colony in 2002, which was sponsored by grant funding from ALS Association. The original publication on this strain (tgSOD1) reported disease onset around 115 days of age, with rapid disease progression thereafter (Howland et al., 2002). Taconic has maintained a colony since 2002. In our recent work (Panov et al., 2011b) we presented data obtained in 2007 on 45 tgSOD1 animals obtained from 9 separate simultaneous isolations of BM and SCM. We studied respiration and reactive oxygen species production by mitochondria isolated from the brains (tgBM) and spinal cords (tgSCM), of such rats and the results obtained were highly reproducible (Panov et al., 2011b). Beginning late in 2008 and in 2009 we encountered difficulties in reproducing the results obtained in 2007. Moreover, we have found that the transgenic rat line did not develop symptoms of the disease at age of more than 200 days even though the mutated gene was evident in DNA samples from tails and ears. In 2010 we received information from Taconic (Germantown, NY) that changes in features can be attributed to a variable phenotype in this model, "which may be due to in part to both the outbred nature of the background strain as well as to possible copy number variation of the transgene" (Information Letter from Taconic). Whether these phenotypic changes were associated with the decline of the level to which the transgene was expressed is impossible to conclude because, in the rats studied in 2007, neither Taconic company, nor our lab quantified the expression of the mutated SOD1 gene or protein. Nevertheless, our comparative studies of metabolic properties of BM and SCM suggest that potentially the loss of morbidity could be explained by different patterns of substrates metabolism and associated ROS generation in BM and SCM studied in 2007

In 2008 we found that BM and SCM isolated from the wild type Sprague Dawley rats began to show metabolic features that were different from those described earlier for the same strain of the wild type and tgSOD1 rats (Panov et al., 2009, 2011a). In this work we present a comparison of metabolic phenotypes and the substrate–dependent ROS generation in the wild type and transgenic rats with mutant G93A Cu/Zn-superoxide dismutase gene isolated in 2007 and 2010. We conclude that the shift in mitochondrial metabolic phenotype, which resulted in a dramatic decrease in ROS production by tgBM and tgSCM, may have contributed to the significant loss of morbidity of the established tgSOD1 rat line. This could result from the breeding method of the transgenic animals, when males bearing the dominant mutated *SOD1* gene were mated with the wild type females. Because mitochondrial DNA has maternal inheritance (Wallace, 2001), the resulting progeny has the mitochondrial phenotype of females, and thus the mutated SOD1 protein was acting on mitochondria with different metabolism that resulted in much lower rates of ROS production. We conclude that mitochondria are the key players

al., 2006).

and 2010.

in the pathogenesis of ALS.

We suggest a hypothesis, which links together pathogenic mechanisms of sporadic and familial forms of ALS. Our hypothesis is based on results obtained in experiments on animals, which showed that between species and even within one species there may exist different phenotypes of mitochondrial metabolism. Neuronal mitochondria of some phenotypes may produce very large amounts of superoxide radical, which is dismutated to hydrogen peroxide (H2O2). High levels of H2O2 may damage the extramitochondrial isoform of superoxide dismutase (SOD1). The oxidatively damaged proteins lose Cu and Zn from the hem centers, and the demetallated protein enters mitochondrial membranes, further increases ROS production by a mechanism similar to that described for cholesterol-β-D-glucoside and fitosterol glucosides (Panov et al., 2010b), causes mitochondrial dysfunctions, and finally the death of neurons. These are sporadic cases of ALS associated with disorders of mitochondrial metabolism. The familial cases with mutations in the *SOD1* gene have basically the same mechanism of motor neuron death, but, because mutated SOD1 protein is exceptionally sensitive to oxidative damage, the loss of Cu and Zn may occur at relatively low levels of H2O2 formation by neuronal mitochondria. The predominant involvement of spinal cord can be accounted for by metabolic and structural features of spinal cord and SCM, which were published elsewhere (Panov et al., 2011a, 2011b).
