Manganese Superoxide Dismutase Levels Are Elevated in a Proportion of Amyotrophic Lateral Sclerosis Patient Cell Lines Gillian McEachern,* Sacha Kassovska-Bratinova,† Sandeep Raha,* , ‡ Mark A. Tarnopolsky,§ John Turnbull,§ Jacqueline Bourgeois, ¶ and Brian Robinson* , ‡ ,1 *Metabolism Research Programme, Research Institute, Hospital for Sick Children, and ‡Department of Biochemistry and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada; †Centre for Cardiovascular Research, Toronto Hospital, Toronto, Ontario, Canada; and §Department of Medicine (Neurology), and ¶ Department of Medicine (Pathology), McMaster University Medical Centre, Hamilton, Ontario, Canada Received May 22, 2000 The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are muta- tions occurring in the gene for copper/zinc superox- ide dismutase (CuZnSOD). The mechanism may in- volve inappropriate formation of hyroxyl radicals, peroxynitrite or malfunctioning of the SOD protein. We hypothesized that undiscovered genetic causes of sporadically occurring amyotrophic lateral scle- rosis might be found in the mechanisms that create and destroy oxygen free radicals within the cell. After determining that there were no CuZnSOD mu- tations present, we measured superoxide produc- tion from mitochondria and manganese superoxide dismutase (MnSOD), glutathione peroxidase, NFB, Bcl-2 and Bax by immunoblot. Of the ten sporadic patients we tested we found three patients with sig- nificantly increased concentrations of MnSOD. These patients also had lower levels of superoxide production from mitochondria and decreased ex- pression of Bcl-2. No mutations were found in the cDNA sequence of either MnSOD in any of the spo- radic patients. A patient with a CuZnSOD mutation (G82R) used as a positive control showed none of these abnormalities. The patients displaying the MnSOD aberrations showed no specific distinguish- ing features. This result suggests that the cause of ALS in a subgroup of ALS patients (30%) is genetic in origin and can be identified by these markers. The alteration in MnSOD and Bcl-2 are likely epiphe- nomena resulting from the primary genetic defect. It suggests also that the oxygen free radicals are part of the cause in this subgroup and that dysregulation of MnSOD or increased endogenous superoxide pro- duction might be responsible. © 2000 Academic Press At least three phenotypically defined forms of famil- ial ALS exist, all inherited in an autosomal dominant fashion (1). Typically there is rapidly progressive loss of motor function, with mainly the anterior horn cells and pyramidal tracts affected. Alternatively there is identical clinical presentation and progression but with the pathological changes including changes in posterior columns and spinocerebellar tracts (2– 4). Variations do seem to occur in the age of onset and duration of the disease and one or both of these param- eters is often consistent within an affected family over a number of generations (5–9). While 10% of ALS is familial, sporadic ALS (SALS) accounts for the other 90%, but this does not mean that the disease does not have a genetic basis. The discovery of mutations in the CuZnSOD gene in the familial group showed that ALS could be a disorder of oxygen free radical processing (10, 11) and that these mutations could operate either in a dominant or recessive manner (12). Enzymes have evolved with the task of detoxifying the primary oxy- gen free radical, superoxide, collectively being named the superoxide dismutases. There are three of them in mammalian systems: a cytosolic CuZn superoxide dis- mutase (SOD1), an intramitochondrial manganese su- peroxide dismutase (MnSOD or SOD2) and an extra- cellular CuZn superoxide dismutase (SOD3) (13). MnSOD, located in a separate compartment from CuZnSOD, is necessary because the respiratory chain assembly of mitochondria which carries out the process of electron transport produces superoxide in substan- tial amounts as an obligatory by-product of its activity. 1 To whom correspondence and reprint requests should be ad- dressed at Metabolism Research Programme, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Fax: (416) 813-8700. E-mail: bhr@sickkids.on.ca. Biochemical and Biophysical Research Communications 273, 359 –363 (2000) doi:10.1006/bbrc.2000.2933, available online at http://www.idealibrary.com on 359 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.