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Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS.

Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, Frosch MP, Agar JN, Julien JP, Brady ST, Brown RH - Nat. Neurosci. (2010)

Bottom Line: Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1.In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present.Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts, USA. daryl.bosco@umassmed.edu

ABSTRACT
Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

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WT-SOD1 purified from SALS tissues inhibits anterograde FAThSOD1 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.
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Figure 7: WT-SOD1 purified from SALS tissues inhibits anterograde FAThSOD1 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.

Mentions: The IHC analysis described above suggested that misfolded, C4F6-positive WT-SOD1 is significantly associated with many SALS cases. We evaluated the possibility that endogenous misfolded WT-SOD1 from SpC tissue inhibits FAT as observed with SOD1ox (Fig. 4c), To this end, WT-SOD1 was immunopurified from both SALS and control SpC tissues under detergent-free conditions (Methods) and perfused in isolated squid axoplasm. Mass spectrometry confirmed that the purified SOD1 preparations were 99% free of contaminating proteins. Perfusion of WT-SOD1 (1 μM) immunopurified from SALS tissues selectively inhibited anterograde, but not retrograde FAT, a pattern of FAT inhibition consistent with that induced by both FALS-linked mutant SOD1 (Fig. 4a; Gerardo Morfini and Scott Brady, submitted and 10) and SOD1ox (Fig. 4c). By contrast, WT-SOD1 immunopurified from control tissues had no effect on FAT (Fig. 7b). Co-perfusion of the conformation specific C4F6 monoclonal antibody (22.5 ng) blocked the inhibitory effect of SALS-derived SOD1 (Fig. 7c), demonstrating that C4F6-reactive SOD1 species mediate the inhibitory effect on FAT. Results shown in Figure 7 are representative of 3-5 independent squid axoplasm assays, each performed with human-derived WT-SOD1 proteins from two separate immunopurification preparations (SALS1, 2 and 4, and control 6). The fact that mild, detergent-free conditions (Methods) were employed to immunopurify WT-SOD1 proteins from human SpC lysates further suggested that the toxic, SALS-derived WT-SOD1 species are relatively soluble.


Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS.

Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, Frosch MP, Agar JN, Julien JP, Brady ST, Brown RH - Nat. Neurosci. (2010)

WT-SOD1 purified from SALS tissues inhibits anterograde FAThSOD1 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.
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Related In: Results  -  Collection

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Figure 7: WT-SOD1 purified from SALS tissues inhibits anterograde FAThSOD1 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.
Mentions: The IHC analysis described above suggested that misfolded, C4F6-positive WT-SOD1 is significantly associated with many SALS cases. We evaluated the possibility that endogenous misfolded WT-SOD1 from SpC tissue inhibits FAT as observed with SOD1ox (Fig. 4c), To this end, WT-SOD1 was immunopurified from both SALS and control SpC tissues under detergent-free conditions (Methods) and perfused in isolated squid axoplasm. Mass spectrometry confirmed that the purified SOD1 preparations were 99% free of contaminating proteins. Perfusion of WT-SOD1 (1 μM) immunopurified from SALS tissues selectively inhibited anterograde, but not retrograde FAT, a pattern of FAT inhibition consistent with that induced by both FALS-linked mutant SOD1 (Fig. 4a; Gerardo Morfini and Scott Brady, submitted and 10) and SOD1ox (Fig. 4c). By contrast, WT-SOD1 immunopurified from control tissues had no effect on FAT (Fig. 7b). Co-perfusion of the conformation specific C4F6 monoclonal antibody (22.5 ng) blocked the inhibitory effect of SALS-derived SOD1 (Fig. 7c), demonstrating that C4F6-reactive SOD1 species mediate the inhibitory effect on FAT. Results shown in Figure 7 are representative of 3-5 independent squid axoplasm assays, each performed with human-derived WT-SOD1 proteins from two separate immunopurification preparations (SALS1, 2 and 4, and control 6). The fact that mild, detergent-free conditions (Methods) were employed to immunopurify WT-SOD1 proteins from human SpC lysates further suggested that the toxic, SALS-derived WT-SOD1 species are relatively soluble.

Bottom Line: Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1.In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present.Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts, USA. daryl.bosco@umassmed.edu

ABSTRACT
Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

Show MeSH
Related in: MedlinePlus