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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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p38 mediates the inhibition of anterograde FAT induced by SOD1ox(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).
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Figure 5: p38 mediates the inhibition of anterograde FAT induced by SOD1ox(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).

Mentions: Phosphorylation of the molecular motor conventional kinesin is known to regulate FAT in vivo10. Further, FALS-linked mutant SOD1 inhibits FAT by a mechanism involving the activation of a kinase pathway (Gerardo Morfini and Scott Brady, submitted and 10). To evaluate the possibility that SOD1ox-mediated inhibition of anterograde FAT similarly involves the activation of axonal kinases, we screened for changes in the activity of various protein kinases in axoplasms perfused with either WT-SOD1 or SOD1ox by immunoblotting with activation-specific phosphoantibodies. No changes in the activities of GSK3 and ERK were observed between WT-SOD1 and SOD1ox-perfused axoplasms (Fig. 5a, b). In contrast, antibodies against phosphorylated, catalytically active p38 (pp38) revealed a dramatic increase in p38 activation in axoplasms perfused with SOD1ox, by comparison with those perfused with WT-SOD1 (Fig. 5a). Quantitative analysis of immunoblots indicated that SOD1ox induced an approximately 4-fold increase in p38 activation, compared to WT-SOD1 (n=6; P<0.05, Fig. 5b). Consistent with these data, co-perfusion of the highly specific p38 MAPK inhibitors SB203580 31 (5μM; Fig. 5c) or MW01-2-069SRM 32 (10μM; Fig. 5d) with SOD1ox prevented the inhibitory effect of SOD1ox on anterograde FAT. Thus, inhibition of anterograde FAT by SOD1ox requires activation of p38 kinase. Taken together, these data indicate that an aberrantly modified form of WT-SOD1, which share conformational motifs with FALS-linked SOD1 mutant proteins, inhibit conventional kinesin-based FAT through a mechanism involving p38 activation.


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)

p38 mediates the inhibition of anterograde FAT induced by SOD1ox(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).
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Figure 5: p38 mediates the inhibition of anterograde FAT induced by SOD1ox(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).
Mentions: Phosphorylation of the molecular motor conventional kinesin is known to regulate FAT in vivo10. Further, FALS-linked mutant SOD1 inhibits FAT by a mechanism involving the activation of a kinase pathway (Gerardo Morfini and Scott Brady, submitted and 10). To evaluate the possibility that SOD1ox-mediated inhibition of anterograde FAT similarly involves the activation of axonal kinases, we screened for changes in the activity of various protein kinases in axoplasms perfused with either WT-SOD1 or SOD1ox by immunoblotting with activation-specific phosphoantibodies. No changes in the activities of GSK3 and ERK were observed between WT-SOD1 and SOD1ox-perfused axoplasms (Fig. 5a, b). In contrast, antibodies against phosphorylated, catalytically active p38 (pp38) revealed a dramatic increase in p38 activation in axoplasms perfused with SOD1ox, by comparison with those perfused with WT-SOD1 (Fig. 5a). Quantitative analysis of immunoblots indicated that SOD1ox induced an approximately 4-fold increase in p38 activation, compared to WT-SOD1 (n=6; P<0.05, Fig. 5b). Consistent with these data, co-perfusion of the highly specific p38 MAPK inhibitors SB203580 31 (5μM; Fig. 5c) or MW01-2-069SRM 32 (10μM; Fig. 5d) with SOD1ox prevented the inhibitory effect of SOD1ox on anterograde FAT. Thus, inhibition of anterograde FAT by SOD1ox requires activation of p38 kinase. Taken together, these data indicate that an aberrantly modified form of WT-SOD1, which share conformational motifs with FALS-linked SOD1 mutant proteins, inhibit conventional kinesin-based FAT through a mechanism involving p38 activation.

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