Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis*□S

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93ASOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS. Molecular & Cellular Proteomics 3:1211–1223, 2004. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron death. Approximately 10% of ALS patients are familial cases (fALS), and mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) were linked with a subset of fALS (1, 2). To date, more than 90 mutations in SOD1 are known to be responsible for 25% of fALS (3), most of which are point mutations that are scattered throughout the primary sequence and structure of the protein. There has been intensive research focusing on the etiology of SOD1 mutant-mediated fALS (see reviews in Refs. 4–8). It has been demonstrated that SOD1-null mice did not develop the disease (9). In addition, transgenic mice expressing the ALS-associated mutants G93A-SOD1 (10, 11), G37R-SOD1 (12), and G85R-SOD1 (13, 14) as well as transgenic rats expressing G93A-SOD1 (15, 16) and H46R-SOD1 (16) developed progressive motor neuron disease despite normal or elevated SOD1 activity. Therefore, it is believed that the ALS-linked mutants of SOD1 have acquired unknown toxic properties that eventually lead to the disease. However, the nature of the toxic “gain-of-function” of SOD1 mutations remains incompletely understood. It is neither understood what the downstream target of the toxicity associated with SOD1 mutants is, nor the mechanism by which the toxicity leads to motor neuron degeneration. There are several hypotheses regarding the consequence of the toxic “gain-of-function” of mutant SOD1 and the mechanisms by which motor neurons die. They include mitochondrial dysfunction and activation of apoptosis (17–28), SOD1 mutant-induced protein aggregation and its cytotoxicity (29– 31), glutamate transporter EAAT2-mediated excitotoxicity (15, 32–34), Fas-trigger FADD/caspase-8 apoptotic cascade activation (35), and abnormal axonal transport caused by microfilament accumulation (36–40). This study focuses on the mitochondrial dysfunction/apoptosis hypothesis and searches for mitochondrial proteins that are altered by SOD1 mutants, i.e. the potential targets of the toxicity associated with SOD1 mutants.