The Fourier-transform mass spectrometry (FT-MS) spectra for (a) untreated WT-SOD1 and (b) oxidized WT-SOD1 (SOD1ox). Data shown have been automatically deconvoluted and reconstructed into a mass domain. (a) The conditions under which the FT-MS analysis was performed reduced the integrity of the SOD1 dimer interface and the SOD1 metal binding capacity. Therefore, the apo form of WT-SOD1 (15,844 Da – average nominal mass) is the predominate species in the mass spectrum of unmodified WT-SOD. (a-b) Peaks representing SOD1 adducts containing sodium, potassium and phosphate ions from the buffers employed during the purification of SOD1 are indicated. (b) The predominant species in the SOD1ox spectrum has a mass increase of 48 Da (15,892 Da) relative to apo-SOD1, which corresponds to the incorporation of 3 oxygens (+3ox; +48 Da). (c) SOD1 proteins were subjected to gas-phase isolation followed by electron capture dissociation (ECD, shown for SOD1ox). MS/MS fragments were assigned using monoisotopic masses with a 5 ppm cutoff and superimposed upon the SOD1 primary sequence (top), where indicates unmodified, c-type fragment ions that include the N-terminus, indicates unmodified z-type fragment ions that include the C-terminus, and indicates +48 Da modified z-type fragment ions corresponding to the conversion of the sulfhydryl group at Cys 111 into sulfonic acid (+3ox). Inset shows raw data for c₇₂⁹⁺ fragment. The SOD1ox peptides resulting from EDC that were used to deduce the Cys111 site of oxidation are shown in Supplementary Table 1.

The X-ray crystallographic structure of WT-SOD1 (pdb2C9V) 48 modeled in PyMOL. WT-SOD1 residues G93 and C111 within exon 2 are highlighted and labeled in purple. The zinc and copper atoms are shown in light cyan and orange, respectively. SOD1 conformation specific antibodies epitope map to the following regions: C4F6 to exon 4 (residues H80-V118 highlighted in red); A9G3 (Fig. 4c) to exons 1 and 2 (comprised of β-strands 1-4); SEDI 38 to β-strand 8; and USOD 33 to β-strand 4.

(a) Recombinant SOD1ox and WT-SOD1 (6 μg/lane) were subjected to a Western analysis using native (non-denaturing) gels with the C4F6 and SDG6 monoclonal antibodies. Native SOD1ox, but not WT-SOD1, is detected by C4F6, whereas SDG6 is reactive for both proteins. The samples were diluted (1 ng/lane) and subjected to an SDS (denaturing) Western analysis with a polyclonal anti-SOD1 antibody (Binding Site) to demonstrate equal gel loading. (b) The native Western shows that C4F6 is reactive only for native SOD1 G93A, whereas SDG6 is reactive only for native WT-SOD1 in lysates (30 μg total protein/lane) that were derived from the respective transgenic mouse. The SDS (denaturing) Western analysis, performed as in (a), demonstrates equal gel loading. (c) C4F6 is reactive for recombinant SOD1 G93A (55 ng/ lane), but not SODox (55 ng/ lane), whereas a polyclonal anti-SOD1 antibody (Calbiochem) detects both proteins. (d) Under denaturing conditions, C4F6 is reactive only for SOD1 G93A but not the other indicated SOD1 mutants. (e) C4F6 eptitope maps to exon 4. Lysates (30 μg total protein) from HEK 293 mammalian cells transfected with the indicated GST-tagged construct (Δ1-5 denote the respective exon deleted construct, FL = full length) were probed with C4F6 or a polyclonal anti-SOD1 antibody (Binding Site).

Vesicle motility assays in isolated squid axoplasm. Individual fast axonal transport (FAT) velocity (μm/sec) measurements (arrowheads) are plotted as a function of time (minutes). Dark arrowheads and line represent anterograde, conventional kinesin-dependent FAT rates. Grey arrows and line represent retrograde, dynein-dependent FAT rates. (a) Perfusion of 5 μM of the FALS-linked H46R mutant into squid axoplasm caused a marked reduction in the rate of anterograde FAT (n=4). (b) In contrast to (a), perfusion of 5 μM WT-SOD1 in the squid axoplasm had no effect on anterograde or retrograde FAT rates (n=4). (c) Perfusion of 5 μM SOD1ox mimicked the inhibitory effect of SOD1 H46R on anterograde FAT (n=4).

(a) Immunoblotting analysis using activation-specific phosphoantibodies reveals a marked activation of p38 (p-p38) in axoplasms perfused with recombinant oxidized SOD1 (SOD1ox), compared to those perfused with recombinant unmodified WT-SOD1 (WT). In contrast, no changes were found in the activities of ERK (pERK) and GSK3 (pGSK3) in association with a specific SOD1 species. A monoclonal antibody against SOD1 (D3H5) 22 confirmed similar levels of SOD1 perfusion, and antibodies against kinesin-1 (KHC) provided a loading control for total levels of axoplasmic protein. Results from three independent experiments are shown (Squid 1-3). (b) Quantitation of results in (a) reveals an approximately 4-fold increase in the phosphorylation of p38 kinase (indicative of p38 activation) in SOD1ox-perfused axoplasms, compared to unmodified WT-SOD1-perfused axoplasms (n=6, P<0.05 (*) by the pooled t-test of μ1-μ2). Error bars reflect the standard error of multiple measurements. Co-perfusion of the highly specific p38 inhibitors SB203580 (c) and MW01-2-069SRM (d) blocked the inhibitory effect of SOD1ox on anterograde FAT (compare to Fig. 4c). Similarly, FALS-linked mutant SOD1 polypeptides inhibit anterograde FAT through a mechanism involving activation of p38 kinase (Gerardo Morfini and Scott Brady, submitted and 10).

(a,d,e,f) C4F6 positive staining is shown for 4 SALS cases (SALS1-4). The positive staining observed for SALS1 (shown in panel a) is lost when C4F6 is excluded from the staining protocol (b) or when the alternative SOD1-mutant specific A9G3 antibody is employed (c). Representative control cases (g-h) and an SOD1-negative FALS case (i) illustrate the lack of positive C4F6 reactivity for such cases. In total, in 4/9 SALS cases exhibited positive C4F6 staining, whereas 0/17 control cases exhibited positive staining. For clinical and demographic information on these cases, see Supplementary Tables 2 and 3.

hSOD1 immunopurified from spinal cords of SALS (SALS hSOD1) and control (Ctrl hSOD1) were perfused into isolated squid axoplasm, and the effects on FAT evaluated as in Fig. 4. (a) Perfusion of SALS-derived hSOD1 (1 μM) selectively inhibits anterograde FAT (dark lines, right arrowheads) while retrograde FAT (gray lines, left arrowheads) remains unchanged (n=5 motility plots, from 2 independent immunopurifications of hSOD1). The inhibitory effect of SALS-derived hSOD1 on FAT mimics that of FALS-SOD1 H46R and SOD1ox (Fig. 4). (b) Perfusion of control-derived hSOD1 has no effect on FAT (n=3 motility plots, from 2 independent immunopurifications). (c) Co-perfusion of the C4F6 monoclonal antibody (22.5 ng) with SALS-derived hSOD1 blocked the inhibitory effect of SOD1 on anterograde FAT (n=3 axoplasms), demonstrating that the C4F6-reactive SOD1 species mediate the inhibitory effect on FAT.