Forum Original Research Communication Oligomerization of Mutant SOD1 in Mitochondria of Motoneuronal Cells Drives Mitochondrial Damage and Cell Toxicity Mauro Cozzolino, 1 Maria Grazia Pesaresi, 1 Ilaria Amori, 1 Claudia Crosio, 2 Alberto Ferri, 1,3 Monica Nencini, 1 and Maria Teresa Carrı ` 1,4 Abstract Increasing evidence indicates that the accumulation and aggregation of mutant Cu,Zn superoxide dismutase (mutSOD1) in spinal cord mitochondria is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (FALS). Although the mechanisms underlying this effect are only partially understood, a deficit in the import mechanism of mutSOD1 and=or in its folding and maturation inside mitochondria is likely involved. To in- vestigate this issue, we overexpressed mitochondria-targeted wild-type and mutSOD1s in neuronal cell lines. Mitochondria-targeted G93A mutSOD1 induces a significant impairment of mitochondrial morphology and metabolism, resulting in caspase-3 activation and cell death. These effects are paralleled by the formation of disulfide-linked, insoluble oligomers of mutSOD1 inside mitochondria. Overexpression of the copper chaperone for SOD1 (CCS) improves the solubility of cytosolic mutSOD1s, but has no effect or even worsens the insolubility of mitochondria-targeted G93A mutSOD1, indicating that CCS may increase the availability of an aggregating form of mutSOD1. Interestingly, prevention of the formation of such aggregates by removal of disulfide-bonded cysteines counteracts the effects produced by mutSOD1 accumulated inside mitochondria. Overall, our results demonstrate for the first time that aggregation of mutSOD1s into mitochondria is important for mutSOD1 to induce damage, although other forms of misfolded SOD1s might be involved. Antioxid. Redox Signal. 11, 1547–1558. Introduction A myotrophic lateral sclerosis (ALS) is a lethal disease which is sporadic in *90% of cases (SALS), while the remaining 10% are associated to familial forms (FALS). In particular, >100 different mutations in the gene encoding Cu=Zn superoxide dismutase (SOD1) have been causally linked to FALS. A gained toxic function is probably shared by all the mutants (mutSOD1s), which are able to produce an ALS-like disease when introduced in the genome of rodents, yet the nature of this toxic property is still unknown (6). Ab- normal aggregation of the mutant protein is an obvious can- didate, since mutSOD1-containing aggregates have been observed in cellular and animal models of the disease (3, 10, 34), and extensive disulfide crosslinking between cysteine residues that are present in SOD1 may have a pivotal role in this phenomenon (2, 5, 19, 23). Although evidence has been provided showing that the removal of such aggregates by the elimination of reactive cysteines from mutSOD1s is able to rescue the viability of transfected cells (5, 23), transgenic mice that overexpress both the G93A mutSOD1 and its copper chaperone (CCS) die from an ALS-like disease at a time when cytosolic aggregates are not present, thus questioning about a causal role of such aggregates in the pathogenesis of the dis- ease (28). Interestingly, these mice have a clearly enhanced mitochondrial damage (28, 29), and mitochondrial abnor- malities appear to be shared by models and patients suffering from ALS (reviewed in Ref. 17). Compelling evidence has 1 Laboratory of Neurochemistry, Fondazione S.Lucia IRCCS, Rome, Italy. 2 Department of Physiological, Biochemical and Cell Science, University of Sassari, Sassari, Italy. 3 Institute of Neuroscience, Department of Psychobiology and Psycopharmacology, CNR, Rome, Italy. 4 Department of Biology, University of Rome ‘‘Tor Vergata’’, Rome, Italy. ANTIOXIDANTS & REDOX SIGNALING Volume 11, Number 7, 2009 ª Mary Ann Liebert, Inc. DOI:10.1089=ars.2009.2545 1547