Supplementary Materials. Wild-type and mutant SOD1 share an aberrant conformation and

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1 Supplementary Materials Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS Daryl A. Bosco, Gerardo Morfini, Murat Karabacak, Yuyu Song, Francois Gros-Louis, Piera Pasinelli, Holly Goolsby, Benjamin A. Fontaine, Nathan Lemay, Diane McKenna-Yasek, Matthew P. Frosch, Jeffery Agar, Jean-Pierre Julien, Scott Brady, and Robert H. Brown, Jr.

2 Supplementary Figure 1. The hydrogen peroxide-treated AS-SOD1 mutant is not recognized by the C4F6 antibody. The AS-SOD1 mutant, which contains both the C6A and C111S point-mutations, was exposed to hydrogen peroxide (H 2 O 2 ) as described for WT-SOD1 (Methods). (a) In contrast to WT- SOD1 (Fig. 1), there is no evidence of AS-SOD1 oxidation as a result of H 2 O 2 treatment in the mass spectra. The predominate species in the respective mass spectrum for untreated AS-SOD1 and AS-SOD1 exposed to H 2 O 2 (AS-SOD1ox) have similar masses (15,752.5 and 15,753.5 m/z). (b) The indicated recombinant human SOD1 proteins (10 µg/lane) were subjected to a native Western analysis as described in Figure 3. SOD1ox (formed by oxidation of WT- SOD1) and G93A are detected by the C4F6 monoclonal antibody (+C4F6), in agreement with Figure 3, whereas neither AS-SOD1 nor AS-SOD1ox are detected by C4F6 using Western-developing techniques with short (5 seconds) and relatively long (8.5 seconds) exposures. The low molecular weight band in the SOD1 G93A lane is likely a degradation product, which was also detected in the denaturing Western analysis in (d). In the absence of C4F6 antibody (- C4F6), there is no signal detected in the identical duplicate blots, thus demonstrating the lack of non-specific artifacts of the Western blotting techniques employed herein. (c) The native Western analysis described in (b) was performed with a rabbit anti-sod1 polyclonal antibody (Biodesign; 1:500), and demonstrates that the indicated proteins (3.75 µg/lane) are present in similar quantities. (d) The samples analyzed in b-c were diluted identically in SDSloading buffer and subjected to a denaturing Western analysis with a sheep anti- SOD1 polyclonal antibody (Calbiochem; 1:1,000), thus further demonstrating that the indicated proteins (10 ng/lane) are present in similar quantities.

3 Supplementary Figure 2. Extraction of insoluble SOD1 from mouse and human spinal cord tissue lysates. SOD1 G93A transgenic mice, which reportedly form SOD1 G93A aggregates, naïve mice, and human spinal cord (SpC) tissue samples were homogenized in detergent-free lysis buffer. The resulting insoluble-pellet was extensively washed and then extracted with SDS (see Methods). (a) An SDS-Western analysis for the final wash ( W ) of the insoluble pellet before extraction, and the extracted pellet ( Pel ) for SOD1 G93A transgenic (n=3; age=98 days) and naïve (n=3; age=110 days) mice. SOD1 is not detected in the wash samples, demonstrating that the SOD1 detected in the

4 extracted samples is due to the extraction process and not from insufficient washing. SOD1 G93A is extracted from the SpC tissue of the corresponding mouse model, whereas insoluble SOD is not detected from the extracted pellets derived from the naïve mice, indicating that this extraction procedure is capable of extracting insoluble SOD1 from animal tissues. (b) The same extraction protocol described in (a) was applied to insoluble pellets derived from frozen, end-stage human tissues from the indicated control and ALS cases. All cases contain some insoluble SOD1. Because the end-stage human tissues were frozen prior to the homogenation, and the resulting lysate pellets were frozen prior to the extraction, we cannot exclude that some level of insoluble protein may have resulted from the multiple freeze-thaw cycles. In contrast, the mouse tissue was isolated and immediately homogenized (Methods). (c) An independent experiment that is the same as (b) for the indicated cases. Note that the SALS1 and SALS4 cases are represented in both (a) and (b). (d) Densitometry of the insoluble SOD1 band is shown for the extractions performed in panels (b; solid bars) and (c; hatched bars). The results for SALS1 and SALS4 illustrate that the extraction protocol is reproducible between independent experiments. Although SALS1 and FALS2 cases contain the highest levels of insoluble SOD1, there is not a significant difference in the levels of insoluble SOD1 for the ALS versus control cases (n=4 cases for controls (C3,4,5,6) and SALS(SALS1,2,3,4); P=0.5 by the T-test, two-tailed).

5 Supplementary Figure 3. Immunohistochemistry of human spinal cord tissues with a commercial, polyclonal anti-sod1 antibody. Human paraffin spinal cord tissues (see Supplementary tables 2 and 3 for clinical and demographic information) from Figure 6 were stained with a commercial polyclonal antibody (Calbiochem; Methods). Dark arrows denote SOD1-positive cells.

6 Supplementary Table 1: Mass spectrometry analysis of SOD1 fragments resulting from electron capture dissociation (EDC) SOD1 amino acid sequence Modification status SOD1 residue at EDC cleaveage site Theoretical +1 mass of the peptide (z+1 ion) (monoisotopic) 1 Theoretical +1 mass +48Da (z+1 ion) (monoisotopic) 2 Observed mass (monoisotopic) Deviation of observedtheoretical ox N ox T ox A ox D ox D ox G ox A ox D ox S ox S * +3 ox C * none L none V none V none E none K The theoretical mass of the indicated SOD1 peptide fragment, assuming there is no oxidation of the SOD1 amino acid side chains. 2 The theoretical mass of the indicated SOD1 peptide fragment, taking into account a potential 48Da increase in mass, which is observed in the FT-MS spectrum of SOD1ox (Fig. 1b). 1,2 Those masses in bold italics correspond to the actual observed mass detected in the experiment, within 1-2 Da. *The overlapping sequence in these peptides is 111-CIIGRTL-117, in which only C111 can undergo oxidation of +48 Da, thus forming sulfonic acid.

7 Supplementary Table 2: Clinical information pertaining to ALS patient tissues used in this study. Case number Sex Diagnosis Age at onset (years) Age at death (years) Duration of disease (months) Site of onset 1 Tissue source (analysis) 3 SALS1 M SALS LE Paraffin (IHC); frozen (pellet extraction, motility assay) SALS2 M SALS LE Paraffin (IHC); frozen (pellet extraction, motility assay) SALS3 M SALS LE Paraffin (IHC); frozen (pellet extraction) SALS4 F SALS NA 2 65 NA NA Paraffin (IHC); frozen (pellet extraction, motility assay) SALS5 F SALS UE Paraffin (IHC) SALS6 M SALS UE Paraffin (IHC) SALS7 M SALS UE Paraffin (IHC) SALS8 F SALS LE Paraffin (IHC) SALS9 M SALS UE Paraffin (IHC) FALS1 F FALS/ (SOD1 negative) FALS2 M FALS/ (SOD A4V) NA 54 NA NA Paraffin (IHC) B & LE frozen (pellet extraction) 1 LE=lower extremity; UE=upper extremity; B=bulbar. 2 NA=information not available. 3 The indicated cases were used for immunohistochemistry analysis (IHC; Figure 6), extraction of insoluble SOD1 from tissue pellets (Supplementary Figure 2), and/or the squid motility assay (Figure 7).

8 Supplementary Table 3: Clinical information for control tissues used in this study. Case number Sex Age at death (years) Cause of death Control 1 F 60 End-stage renal disease; systemic lupus erythematosus (SLE) Tissue source (analysis) 3 Paraffin (IHC) Control 2 M 52 Hepatocellular carcinoma Paraffin (IHC) Control 3 M 93 NA 1 frozen (pellet extraction) Control 4 M 34 NA frozen (pellet extraction) Control 5 F 76 NA frozen (pellet extraction) Control 6 M 65 NA frozen (pellet extraction, motility assay) Control 7 M 24 NA Paraffin (IHC) Control 8 NA 42 Spinal cord tumor, Paraffin (IHC) pulmonary illness Control 9 NA 63 Heart illness Paraffin (IHC) Control 10 M 75 Gastrointestinal tumor Paraffin (IHC) Control 11 M 56 Heart illness Paraffin (IHC) Control 12 M 66 Heart illness Paraffin (IHC) Control 13 F 86 Pulmonary infection Paraffin (IHC) Control 14 M 68 Pulmonary infection, Paraffin (IHC) cancer, heart illness Control 15 F 64 Post-transplant Paraffin (IHC) lymphoproliferative disorder Control 16 F 44 Chronic obstructive Paraffin (IHC) pulmonary isease Control 17 M 9 months Krabbe s disease Paraffin (IHC) Control 18 F 50 Pneumonia Paraffin (IHC) Control 19 NA 49 Pneumonia Paraffin (IHC) Control 20 NA 56 Sarcoma of unknown Paraffin (IHC) origin Control 21 NA NA NA Paraffin (IHC) 1 NA=information not available. 2 The indicated cases were used for immunohistochemistry analysis (IHC; Figure 6), extraction of insoluble SOD1 from tissue pellets (Supplementary Figure 2), and/or the squid motility assay (Figure 7).