Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Abstract

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

Fig. 1.

MutSOD1s expressed in motoneuronal cells show diverse properties with respect to superoxide dismutase activity and propensity to monomerize. (A) NSC-34-derived cell lines expressing myc-tagged human wtSOD1 or mutSOD1 were left untreated (−) or treated (+) for 48 h with 1 μg/ml of doxycycline to induce expression of transgenic SOD1. Cell lysates were subjected to reducing SDS/PAGE (Upper) or in-gel SOD1 activity assay (Lower). The anti-SOD1 antibody used for immunodetection recognizes both human (hSOD1-myc) and mouse SOD1 (mSOD1). Position of the bands corresponding to mSOD1 dimers (D) and hSOD1-myc dimers (D) are indicated. Untransfected NSC-34 cells were used as control. (B) NSC-34-derived cell lines were induced as in A, but the extracts were subjected to a mildly denaturing (0.1% SDS), nonreducing PAGE. Western blot analysis was performed with an anti-myc antibody. Position of the bands corresponding to hSOD1-myc dimers (D) and monomers (M) are indicated.

Fig. 2.

A much higher fraction of the mutSOD1s copurify with mitochondria of NSC-34 cells, relative to wtSOD1, and the mitochondrial SOD1 is partially oxidized and thus inaccessible to modification by malPEG. (A) NSC-34-derived cell lines were treated for 48 h with 1 μg/ml of doxycycline. Lysates from mitochondrial (M) and cytosolic (C) fractions were analyzed in Western blot for SOD1 expression with an anti-myc antibody (half of the sample; Upper) or subjected to malPEG modification and then analyzed in Western blot with the same antibody (other half of the sample; Lower). One-half microgram of cytosolic proteins was loaded to obtain levels of SOD1s similar to that from 5 μg of mitochondria proteins in both Upper and Lower, but a short exposure was chosen for Lower to help appreciate the pattern of MP bands. Note that the anti-myc antibody recognizes malPEG-modified SOD1s more efficiently than the unmodified form. (B) Histogram of the mean ratio ± SD of mitochondrial versus cytosolic SOD1s levels (expressed in densitometric arbitrary units) in NSC-34-cells as determined in four independent Western blot experiments. Values significantly different from the relative wtSOD1 (both myc-tagged and untagged) are indicated with an asterisk when P < 0.01. (C) NSC-34 cells expressing the mutSOD1 G93A-myc were treated for 48 h with doxycycline. Lysates from cytosolic (C) and mitochondrial (M) fractions were incubated for 16 h in the absence or in the presence of 2 mM DTT, treated with malPEG, and then subjected to SDS/PAGE and Western blot with an anti-myc antibody.

Fig. 3.

Oxidation of Cys-111 is necessary for C6F mutSOD1 association with mitochondria. NSC-34 cell lines expressing myc-tagged hSOD1s (WT, C6F, C111S, and the double SOD1 mutant C6F/C111S) were treated for 48 h with 1 μg/ml of doxycycline. Lysates from mitochondrial (M) and cytosolic (C) fractions were analyzed in Western blot with an anti-myc antibody (Top) or with an anti-Hsp60 antibody to check for equal loading of mitochondrial fractions (Middle). One-half microgram of cytosolic proteins was loaded to obtain levels of SOD1s similar to that from 5 μg of mitochondria proteins. Histogram of the mean ratio ± SD of mitochondrial versus cytosolic SOD1s levels in NSC-34-cells as determined in three independent Western blot experiments. Values significantly different from wtSOD1 are indicated with an asterisk when P < 0.01 (Bottom).

Fig. 4.

Association of oxidized mutSOD1s with mitochondria decreases reduced/oxidized glutathione ratio (GSH/GSSG). (A) Mitochondrial GSH/GSSG ratio in NSC-34 cell lines uninduced (−) or induced (+) for the expression of the various hSOD1s. (B) Mitochondrial GSH/GSSG ratio in NSC-34 expressing C111S, C6F, or C6F/C111S mutSOD1s. (C) Cell death was measured by trypan blue dye exclusion in induced NSC-34-derived cell lines treated with 50 μM ethacrynic acid for 1 h. Values significantly different from the relative control are indicated with an asterisk when P < 0.01.

Fig. 5.

Oxidized mutSOD1s associated with mitochondria form disulfide cross-linked oligomers and impair mitochondrial function. (A) Cytosolic (C) and mitochondrial (M) fractions from induced NSC-34 cells expressing the indicated SOD1 were lysed and subjected to a denaturing PAGE, either nonreducing (without 2-mercaptoethanol, β-ME; Upper) or reducing (with β-ME; Lower). One-half microgram of cytosolic proteins was loaded to obtain levels of SOD1s similar to that from 5 μg of mitochondria proteins. Western blot analysis was performed with an anti-myc antibody. Positions of the bands corresponding to hSOD1-myc monomers, dimers, and oligomers are indicated. (B) Activities of electron transport chain complexes I, II-III, and IV were measured by spectrophotometric assays on the crude mitochondrial fraction from doxycycline-treated NSC-34-derived cells. Activities are normalized for protein content. (C) ATP content in NSC-34 cells uninduced (−) or induced (+) for the expression of the various hSOD1s. Values significantly different from the relative control are indicated with an asterisk when P < 0.01. (D) Activities of electron transport chain complexes I, II-III, and IV were measured by spectro-photometric assays on the crude mitochondrial fraction from doxycycline-treated NSC-34-derived cells expressing wild type, C6F, C111S SOD1, and the double SOD1 mutant C6F/C111S. Values significantly different from the relative control are indicated with an asterisk when P < 0.01 and with two asterisks when P < 0.05.