Supporting information Materials and Methods ALS cases The human cases included in this study were 2 SOD1-ALS cases (A4V, ΔG27/P28) and 10 sporadic ALS cases (negative for SOD1 mutations), 1 case with Alzheimer’s disease (AD), and 1 case with corticobasal degeneration (CBD). See Table S1 for detailed case information. ALS cases were diagnosed in accordance with the revised criteria of El Escorial [1]. Patient consents were obtained from the legal representative in accordance with the respective Ethical Review Boards at Sunnybrook Health Sciences Centre (Toronto, Canada). Synthesis of multiple antigenic peptide (MAP) We carried out peptide synthesis using standard Fmoc-based chemistry on a PS3 Peptide synthesizer (Protein Technologies Inc.). The MAP was eluted from the resin with cleavage mixture (95% trifluoroacetic acid, 2% thioanisole, 2% anisole, 1% triisopropylsilane, all from Aldrich), precipitated with ice-cold ether, and partially purified using ether washes to remove protecting groups and scavengers.

Antibody purification We synthesized a linear peptide with identical sequence to the antigen on a (noncleavable) TentaGel-SH resin (Advanced ChemTech). After deprotection and acetylation, this resin was packed into disposable columns (Evergreen Scientific) and equilibrated in PBS-T (phosphate-buffered saline containing 0.05% Tween-20) before antiserum purification. We precleared antiserum (1 mL) by centrifugation and added an equal volume of ice-cold saturated ammonium sulfate. After incubation at 4 °C for one hour, precipitated antibody was recovered by centrifugation and washed several times with 50% saturated ammonium sulfate. The pellets were dissolved in PBS-T, combined with the equilibrated TentaGel resin, and incubated overnight at 4 C with end-over-end rotation. The resin was then transferred to a plastic column and washed extensively with PBS-T at room temperature. The bound antibody was eluted with 0.1 M glycine (pH 2.8) in 1 mL fractions that were each neutralized with 250 uL 1.5 M Tris-HCl (pH 8.8). We determined the concentration of the antibody in the fractions using UV-vis absorption spectroscopy, with the IgG extinction coefficient ε280 = 1.35 (mg/ml)-1 and an IgG molecular weight of 150,000 Da. We used serum only from the third bleed or later. The purified antibody was stored at 4 °C and was generally stable for at least 1 month.

In the case of the SEDI antibody, ammonium sulfate precipitations were prepared as described above for USOD. The pellets were dissolved in PBS-T and used in ELISAs without further purification. Preparation of folded (holo)SOD1 Lyophilized wild-type human erythrocyte SOD1 (Sigma-Aldrich, St. Louis, MO) was dissolved in 20 mM HEPES buffer, pH 7.5. SOD1 from human tissues often bears a persulfide modification at C111 detectable by a 325 nm absorbance band [2]. We treated SOD1 overnight at room temperature with 1 mM TCEP (tris(2-carboxyethyl)phosphine) to remove the persulfide. TCEP was then removed by extensive dialysis against 20 mM HEPES, pH 7.5, at 4° C. Removal of the persulfide modification was confirmed by the disappearance of the 325 nm absorbance band. SOD1

disulfide status was confirmed by denaturing SOD1 in 6 M guanidine and 5 mM iodoacetamide. The mass of the product was measured by MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry, and was found to correspond to SOD1 with two of the four cysteine residues modified to carboxy-methyl-cysteine, which indicates that there are two free cysteines and two cysteines linked by a disulfide bond. A single peak corresponding to the holoSOD1 dimer was observed in size-exclusion chromatography, confirming SOD1 purity and homogeneity. The zinc and copper content of SOD1 was measured as described previously [3], using a final SOD1 concentration of 4.25 μM. Standard curves were prepared using 0 to 14 μM CuCl2 and ZnCl2 solutions. All holo-SOD1 solutions were found to contain between 0.95 and 1.00 moles of copper and between 0.90 and 1.00 moles of zinc per mole of SOD1 monomers. Determination of IC50s from competition ELISA data IC50s were estimated by fitting the data from the competition ELISAs to the following equation: A450 = Amax * IC50 IC50 + [unfolded SOD1] Where A450 is the observed absorbance at 450 nm, Amax is the maximum absorbance, and IC50 is the concentration of competing antigen (unfolded SOD1) which gives 50% competition. Immunohistochemistry For labeling with USOD, tissue sections were pretreated with Tris-EDTA (pH 9.0) and heating (120 C for 2 minutes) to retrieve antigen. For labeling with SEDI, sections were pretreated with citrate and heating. For the SOD100 antibody (Stressgen), antigen retrieval was performed as described above for USOD. Immunostaining was visualized on a Leica DM6000 microscope and images captured using a Micropublisher 3.3 RTV color digital camera and Openlab software (Improvision). Western blotting Eluates from the immunoprecipitation experiments were separated by 12% SDS-PAGE. After transferring to PVDF membrane, the blot was blocked in 5% milk-TBST (Tris-buffered saline, 0.1% Tween) and then incubated with sheep polyclonal SOD1 antibody (1:1000, Calbiochem) followed by secondary anti-sheep IgG-horse-radish peroxidase (1:5000, Biodesign). Immunoreactivity was visualized using chemiluminescence reagent plus (PerkinElmer). A separate blot loaded with 0.1% of the supernatant input was probed with an antibody to SOD1 (1:1000, Calbiochem) to show equalization of samples used for the immunoprecipitations. References 1. Brooks, B.R. et al. (2000) El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders 1:293-299. 2. de Beus, M.D., Chung, J., Colón, W. (2004) Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties. Protein Sci. 13(5):1347-55. 3. Mulligan, V.K., Kerman, A., Ho, S., Chakrabartty, A. (2008) Denaturational stress induces formation of zinc-deficient monomers of Cu,Zn superoxide dismutase: implications for pathogenesis in amyotrophic lateral sclerosis. J Mol Biol. 383:424-36.

Table S1: Information for patients included in this study Case Diagnosis Sex Age at

Death (years)

Duration of Disease (years)

Onset* Motor neuron counts**

A4V SOD1-ALS M 46 0.4 Lumbar 11 ΔG27/P28 SOD1-ALS F 55 4 Lumbar 8 sALS#1 sALS F

63 2.9 Lumbar 16

sALS#2 sALS M

66 2.1 Bulbar 29

sALS#3 sALS F

71 0.7 Lumbar 37

sALS#4 sALS F

57 2.8 Bulbar 41

sALS#5 sALS M

64 1.4 Lumbar 25

sALS#6 sALS F

73 2.1 Bulbar 50

sALS#7 sALS M

70 1 Bulbar 23

sALS#8 sALS M

58 2.2 Bulbar 31

sALS#9 sALS M

51 3 Lumbar 33

sALS#10 sALS F

59 1.4 Lumbar 26

AD Alzheimer’s disease

F 91 >10 N/A N/A

CBD Corticobasal degeneration

F 79 5.8 N/A N/A

M: male, F: female, SOD1-ALS: amyotrophic lateral sclerosis due to SOD1 mutation, sALS: sporadic ALS, AD: Alzheimer’s disease, CBD, corticobasal degeneration. N/A: not applicable. * From symptom onset. ** Average number of motor neurons present in the transverse section of lumbar spinal cord. Minimal three sections per case counted.

Supplementary Figure Legends

Figure S1. Immunohistochemistry of G93A mouse spinal cord tissue with the USOD antibody (top-left and bottom-left panels), USOD incubated with antigenic peptide (middle panels) to show specificity of labeling, and a commercial anti-SOD1 antibody (top-right and bottom-right panels) to show the presence of SOD1 throughout the spinal cord. Scale bar: 70 μm. Figure S2: USOD/SEDI immunohistochemistry of ALS cases. a-b: Two consecutive serial sections from the A4V case, showing labeling of the same motor neuron/inclusion by both USOD (a) and SEDI (b). c-g: Absence of USOD labeling of motor neurons from five additional sALS cases. Some motor neurons are highlighted with arrows. Scale bar: a: 35 μm. c-f: 100 μm. g: 70 μm. Figure S3: Absence of amyloid in TDP-43-positive and ubiquitin-positive inclusions. In the first three rows, each row shows the results of staining of three consecutive serial sections from a sALS case. The first section was stained with either anti-TDP-43 (a) or anti-ubiquitin (e,i) antibody; the second section was stained with Congo Red (b,f,j) and viewed through crossed polarizers (c,g,k); the third section was stained with ThS (d,h,l). m-n: The same tissue section from Figure 4q (i.e. stained with ThS), viewed through a 488 nm filter (m) and a 594 nm filter (n). The presence of strong fluorescence intensity at both wavelengths indicates that the fluorescence is due to lipofuscin and not ThS. Positions of immunopositive inclusions (or lipofuscin in panels d,h,l) are indicated with arrows. Scale bar: 35 μm.

Figure S1

Figure S2

Figure S3