ALS-linked mutant superoxide dismutase 1 (SOD1) alters mitochondrial protein composition and decreases protein import Quan Lia,1, Christine Vande Veldeb,c,1, Adrian Israelsonb, Jing Xied, Aaron O. Baileye, Meng-Qui Donge, Seung-Joo Chuna, Tamal Royb, Leah Winera, John R. Yatese, Roderick A. Capaldid, Don W. Clevelandb,2, and Timothy M. Millera,b,2 aDepartment of Neurology, Hope Center for Neurological Disorders, The Washington University School of Medicine, St. Louis, MO 63110; bLudwig Institute and Departments of Cellular and Molecular Medicine and Neuroscience, University of California at San Diego, La Jolla, CA 92093-0670; cDepartment of Medicine, Centre Hospitalier de l’Université de Montréal Research Center, Université de Montréal, Montreal, QC, Canada H2L 4M1; eDepartment of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037; and dMitoSciences, Eugene, OR 97403 Contributed by Don W. Cleveland, October 18, 2010 (sent for review February 4, 2010) Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. system very little SOD1 is found in or on mitochondria (20, 21), Mutant SOD1 preferentially associates with the cytoplasmic face of but mutant SOD1 has been found associated presymptomatically mitochondria from spinal cords of rats and mice expressing SOD1 with the cytoplasmic face of mitochondria from spinal cord in all mutations. Two-dimensional gels and multidimensional liquid chro- rodent models of SOD1 mutant-mediated disease (20, 21). A matography, in combination with tandem mass spectrometry, criticism that apparent association of dismutase inactive mutants fl revealed 33 proteins that were increased and 21 proteins that with mitochondria might re ect cosedimentation of protein G93A aggregates rather than bona fide mitochondrial association (22) were decreased in SOD1 rat spinal cord mitochondria com- fl pared with SOD1WT spinal cord mitochondria. Analysis of this has been refuted by demonstrating that mutant SOD1 oats with group of proteins revealed a higher-than-expected proportion in- mitochondria rather than sedimenting as much more dense pro- tein-only aggregates would under such conditions (21). Indeed, volved in complex I and protein import pathways. Direct import a portion of misfolded mutant SOD1 directly binds to the cyto- assays revealed a 30% decrease in protein import only in spinal plasmic facing surface of the major voltage-dependent anion cord mitochondria, despite an increase in the mitochondrial import NEUROSCIENCE G93A channel (VDAC1), a general diffusion pore for anions and cati- components TOM20, TOM22, and TOM40. Recombinant SOD1 ons that is located on the outer mitochondrial membrane. Mutant G85R WT ’ or SOD1 , but not SOD1 or a Parkinson s disease-causing, mis- SOD1 binding reduces conductance by purified VDAC1 recon- α E46K > folded -synuclein mutant, decreased protein import by 50% stituted in a lipid bilayer, and spinal cord mitochondria have re- in nontransgenic mitochondria from spinal cord, but not from liver. duced ADP uptake (23). Thus, altered mitochondrial protein content accompanied by selec- Three prior efforts have reported use of proteomic tools to in- tive decreases in protein import into spinal cord mitochondria com- vestigate how mutant SOD1 affects mitochondrial protein com- prises part of the mitochondrial damage arising from mutant SOD1. position within whole-spinal-cord extracts of dismutase-active (24) or dismutase-inactive (25) SOD1 mutant mouse models or, more motor neuron disease | proteomic | neurodegenerative disease | selectively, within mitochondria of a cell line retaining some motor amyotrophic neuron-like properties and expressing one dismutase-active, ALS- linked SOD1 mutant (26). We extended these approaches to de- myotrophic lateral sclerosis (ALS) is an adult-onset neuro- termine whether mutant SOD1 affects protein content of spinal Adegenerative disorder caused by loss of the motor neurons cord mitochondria before disease initiation in rodent models that within the spinal cord and dysfunction of the motor pathways in the develop ALS-like disease. Using a combination of mass spectrom- cortex (1, 2). Most cases of ALS (90%) are sporadic. Of the 10% of etry and 2D gels, we now report 21 proteins to be decreased ap- cases that are inherited, about one-fifth are caused by mutation proximately twofold, with another 33 that are increased by a within the superoxide dismutase 1 (SOD1) gene. Rodents expres- comparable amount or more in mitochondria isolated from spinal cords of mutant SOD1 animals, accompanied by diminished pro- sing mutant SOD1 develop an ALS-like disease (1, 2). A consensus fi has emerged that disease arises from acquisition by the mutant tein import activity. By using puri ed components, inhibition of proteins of one or more toxic properties, rather than from loss of mitochondrial protein import is demonstrated to be a direct result dismutase activity (3, 4). At least eight prominent mutant-derived of mutant but not wild-type SOD1. toxicities have been proposed, including mitochondrial dysfunc- Results tion, excitotoxicity, endoplasmic reticulum stress, toxic extracel- lular SOD1, superoxide production from microglia or astrocytes, Altered Spinal Cord Mitochondrial Protein Content in SOD1 Mutant and disruption of the blood–brain barrier (5). Furthermore, Rats. To identify possible differences in protein composition be- pathogenesis is noncell autonomous with disease progression tween mitochondria isolated from spinal cords of age-matched driven by mutant synthesis in astrocytes (6–8) and microglia (3, 9). SOD1WT and SOD1G93A mutant rats, we used 2D gel electro- Dysfunction of mitochondria can clearly cause specific damage phoresis as well as liquid chromatography followed by tandem to neurons or muscle because deletion/mutation in mitochondrial mass spectrometry [multidimensional protein identification tech- genes causes specific nerve and muscle diseases (10). Accompa- nying mutant SOD1-mediated disease in some mice is evidence for altered mitochondrial calcium-buffering capacity (11) and Author contributions: Q.L., C.V.V., R.A.C., D.W.C., and T.M.M. designed research; Q.L., C.V.V., changes in the activity of complexes of the electron transport chain A.I., J.X., A.O.B., M.-Q.D., S.-J.C., T.R., L.W., and T.M.M. performed research; Q.L., C.V.V., A.I., (12). Deletion of the mitochondrial BCL-2–related proteins BAX J.X., A.O.B., M.-Q.D., S.-J.C., T.R., L.W., J.R.Y., R.A.C., D.W.C., and T.M.M. analyzed data; and and BAK delays disease onset in SOD1G93A mice (13), perhaps Q.L., C.V.V., D.W.C., and T.M.M. wrote the paper. by modulating effects of SOD1 conformational changes to BCL-2 The authors declare no conflict of interest. (14). Apparent damage to mitochondria has been reported in 1Q.L. and C.V.V. contributed equally to this work. autopsy material from ALS patients (15, 16). 2To whom correspondence may be addressed. E-mail: [email protected] or Although SOD1 is a primarily cytosolic enzyme, wild-type [email protected]. SOD1 is also found in the intermembrane space of mitochondria This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in liver (17) and in yeast (18, 19). However, in the normal nervous 1073/pnas.1014862107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1014862107 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 G93A nology (MudPIT)] (27). Initially, a presymptomatic age (11 wk) Decreased Complex I Activity in Mitochondria from SOD1 Rats. was chosen because it was known to correspond to a time of Six (NDUFS1, ND5, NDUFB8, NDUFC2, NDUFA5, NDUFB9) very active denervation of lower motor neurons (28). Although of the 26 down-regulated proteins in mitochondria from mutant a change in staining on 2D gels may reflect altered abundance, spinal cord are components of the 42-subunit complex I, also size, or isoelectric point, the overall patterns of major proteins known as NADH dehydrogenase. Complex I is located on the (visible in Coomassie (Fig. 1) or silver (Fig. S1) staining were inner membrane of mitochondria, catalyzes the shuttling of elec- highly similar. Twelve spots (Fig. 1 B and C compared with Fig. 1 trons from NADH to coenzyme Q, and helps to build the proton E and F and Fig. S1 B and C compared with Fig. S1 E and F) were gradient required for ATP generation. Immunoprecipitation of clearly different between the two samples, with five (spots 1, 6, 7, mitochondrial extracts with a monoclonal antibody raised against 8, and 9) increased in SOD1G93A mitochondria and seven (spots complex I, which had been crosslinked to protein G agarose beads (32), confirmed that at least six complex I polypeptides were 2–5 and 10–12) decreased. Each was excised, digested with tryp- changed in relative abundance: two were increased [(V1, S2) and sin, and identified via tandem mass spectrometry (Table S1). (A10, ND5)] and four were decreased [S1, (A9, D1), S3, and (B10, Three spots contained more than one protein, resulting in a total fi V2; B7, A5, B4, B5, B6, S5, B16, 6, A7)] (some stained bands of 15 proteins identi ed. represent multiple polypeptides) (Fig. 3A). Of those decreased in Using MudPIT mass spectrometry, protein differences were G93A fi SOD1 mitochondria, a polypeptide migrating at 75 kDa (Fig. also identi ed from three independent preparations of mitochon- 3A) most likely represents S1 (also known as NDUFS1) (32–34), dria from 11-wk-old SOD1WT and age-matched presymptomatic G93A which was also seen to be decreased by mass spectrometry (Table SOD1 rat spinal cords, using normalized spectral counts as an 1) and 2D gels (Fig. 3B, arrows). A prominent complex I poly- indication of the relative amount of any given protein (29, 30). peptide migrating at 35 kDa, most likely representing ND5 (32– We identified 299 mitochondrial proteins present in all of the 34), was increased in immunoprecipitates. ND5 is a mitochond- mitochondrial samples.