Approximately 150 mutations dispersed throughout the SOD1 sequence have been linked to FALS (http://alsod.iop.kcl.ac.uk/
). In many cases, these represent subtle, conservative (e.g. Gly→Ala) amino acid substitutions 3
; nonetheless, all these mutations lead to an ALS phenotype. While pathogenic mechanisms underlying FALS-linked SOD1-mediated toxicity have not been definitively elucidated, a prevailing hypothesis is that FALS-linked mutations induce an altered or misfolded conformation in SOD1 4, 5, 7-9
that modifies its interactions with other proteins and perturbs its cellular localization 39-41
Given the common pathological effects of diverse FALS-linked SOD1 mutations, it seems plausible that alterations to the non-Mendelian, post-translational modifications of SOD1 may similarly lead to an ALS phenotype 13-15
. For example, disruptions of the normal SOD1 post-translational modifications (), such as subunit dimerization, the intrasubunit disulfide bond between residues Cys57 and Cys146, and the coordination of copper and zinc, have all been shown to cause WT-SOD1 to aggregate 16, 17, 25, 38, 42
. Moreover, aberrant post-translational modifications of SOD1, such as oxidation, have adverse effects on WT-SOD1 protein conformation 11, 15, 18
. By employing the C4F6 monoclonal antibody 20
, we found that both oxidation of Cys111 () and mutagenesis of G93→A induce the formation of a conformational epitope that includes elements of exon 4 () and that is not normally exposed by WT-SOD1 (). In addition to this conformational component of the C4F6 epitope that is shared by SOD1ox and SOD1 G93A, there is an aminoacid sequence component that includes G93A, which could be expected based on the fact that the SOD1 G93A antigen was used to raise the C4F6 antibody 20
, and which likely explains the stronger reactivity of C4F6 for G93A relative to SOD1ox under native conditions (Supplementary Fig. 1
). Under denaturing conditions, the conformational epitope is lost and only the G93A sequence element remains to confer reactivity with C4F6, thus explaining a lack of C4F6 reactivity for SODox and other G93 variants under denaturing conditions ().
Studies of human spinal cord tissues with C4F6 revealed the presence of aberrantly folded WT-SOD1 species in approximately half of the available SALS cases, but not in control cases (). These results indicate that at least a subset of SALS cases contain WT-SOD1 proteins that are structurally similar to FALS-linked SOD1 mutant proteins. The lack of C4F6 reactivity in the remaining SALS cases may indicate that misfolded SOD1 is not associated with ALS pathogenesis for these cases, suggesting that modified WT-SOD1 plays a role in a subset of SALS in an analogous manner to the role that mutant-SOD1 plays in a subset of FALS. However, we cannot confirm that the C4F6 antibody is reactive for all possible misfolded forms of WT-SOD1.
Our finding that misfolded SOD1 is associated with SALS is consistent with the report that an aberrant 32-kDa, SOD1-containing species is indirectly detected through a biotinylation cross-linking reaction with homogenized tissue lysates from both SALS and FALS 12
. Here, we probe for a misfolded SOD1 conformation in SALS using an IHC approach on fixed tissues with C4F6, a conformation-specific monoclonal antibody. Moreover, we demonstrate that these misfolded WT-SOD1 species derived from C4F6-positive SALS cases recapitulate the toxic effect of FALS-linked mutant SOD1 on FAT (). Further, we show that activation of p38 MAPK is a common feature of both SALS- and FALS-linked SOD1 inhibition of FAT ( and 10
While we have shown that C4F6 is reactive for an oxidized form of WT-SOD1 (), it is possible that other modifications to WT-SOD1, including alterations in normal post-translational modifications 16, 17, 25, 38
, might also induce an altered conformation that confers C4F6 reactivity. That different modifications to SOD1 can induce similar structural consequences is supported by observations that C4F6 reacts with different FALS-linked SOD1 mutants 20
, and by a recent hydrogen/deuterium exchange study that revealed enhanced flexibility within the same SOD1 electrostatic loop region (residues 133-144, located between β-strands 7 and 8; ) for a panel of 13 different FALS-linked SOD1 mutants 7
. Additional studies with alternate misfolded forms of WT-SOD1 and different SOD1 conformation specific antibodies will provide greater insight into the conformational similarities of these proteins and their prevalence in SALS.
Although the C4F6 antibody recognizes a conformation-dependent epitope common to both mutant and WT-SOD1 proteins associated with ALS pathology, the critical epitope is not detected by all antibodies that recognize other misfolded SOD1 species. For example, our A9G3 antibody detects a subset of FALS-linked mutant SOD1 species, but does not react with spinal cord sections from SALS patients that are immunoreactive for C4F6 (). Similarly, both the SEDI (S
nterface) and USOD (U
1) antibodies failed to detect WT-SOD1 in SALS cases 33, 34
. There are several significant distinctions between the epitopes recognized by C4F6 and the SEDI and USOD antibodies. C4F6 is reactive for a conformational epitope that includes G93 encoded within exon 4 (), which is distal to those epitopes recognized by SEDI and USOD (). Moreover, both SEDI and USOD are reactive for linear sequences that can become exposed in aggregated SOD1 inclusions in FALS cases, whereas the misfolded WT-SOD1 species in our SALS cases is relatively soluble as evidenced by the diffuse C4F6 staining pattern (), the low levels of insoluble WT-SOD1 detected in our SALS cases (Supplementary Fig. 2
), and the ability to purify these species under detergent-free conditions while maintaining their inhibitory effect on anterograde FAT (). Importantly, our IHC methods do not require the harsh conditions used for antigen-retrieval. Such treatments may disrupt the C4F6-like conformational-epitope, but enhance the detection of epitopes like those for the SEDI and USOD antibodies that are otherwise buried 33, 34
. Thus, the differing results obtained with the C4F6, SEDI and USOD antibodies are likely due to the different epitopes recognized by these antibodies.
The foregoing analyses of immunoreactivity patterns for the C4F6 antibody reveal that genetic variants transmitted as Mendelian traits and non-inherited modifications to SOD1 can both induce similar structural perturbations within the protein, and that non-inherited modifications of SOD1 can be associated with SALS. A critical question that follows directly from these observations is whether these SALS-linked modifications confer upon WT-SOD1 the same toxic properties that are elicited by FALS-linked SOD1 mutations, including activation of p38 and inhibition of FAT. Both recombinant SOD1ox () and WT-SOD1 derived from C4F6-postive SALS spinal cord tissues () recapitulate this pattern of mutant-SOD1 mediated FAT inhibition, whereas untreated recombinant WT-SOD1 and WT-SOD1 derived from control spinal cord tissues had no effect ( and , respectively). The effect of SOD1 from C4F6-postive SALS spinal cord on anterograde FAT was abolished by incubation with the C4F6 antibody prior to perfusion (). C4F6 also abolished the ability of FALS mutant SOD1 to inhibit FAT (data not shown). Biochemical and pharmacological experiments further indicated that the inhibition of anterograde FAT induced by SOD1ox involved activation of p38 kinase (). FALS-linked mutant SOD1 mediated defects in axonal transport have been reported previously 10, 43, 44
, and were thought to represent an early pathogenic event in mutant-SOD1 transgenic mice that contributes to a “dying back” mode of motor neuron degeneration 45, 46, 47
. That the inhibition of FAT is selectively in the anterograde direction demonstrates that this effect of mutant SOD1 and SOD1ox shows specificity, as this effect is not universally observed for all toxic, neurodegenerative disease associated proteins 10
. The pattern of FAT inhibition is largely determined by the nature of the regulatory kinases that become activated by the toxic protein 10
. Both modified WT-SOD1 () and several mutant-SOD1 proteins (Gerardo Morfini and Scott Brady, submitted and 10
) inhibit FAT through a mechanism involving specific activation of p38 MAPK.
Concurrent studies showed that p38 directly phosphorylates kinesin-1 subunits of conventional kinesin and dramatically inhibited the translocation of this motor protein along axonal microtubules, thereby providing a common molecular basis for the effects of activated p38 kinase activity on anterograde FAT 10
(Morfini and Brady, submitted). Thus, SALS-associated WT-SOD1 species induce the same defects on conventional kinesin-based FAT as FALS-linked SOD1 mutants, and by the same molecular mechanism.
The concept that proteins can become pathogenic via both inheritable and non-heritable modifications has precedence in the context of other neurodenegerative diseases, as exemplified by α-synuclein in Parkinson's disease, Aβ or tau in Alzheimer's disease and frontotemporal dementia (FTDP). Although our data do not rule out potential toxic effects of aggregated WT-SOD1 species in ALS pathogenesis, they reveal toxic effects associated with relatively soluble, misfolded WT-SOD1 species in SALS. While aberrantly modified WT-SOD1 is aggregation-prone in vitro
16, 17, 25, 38
, the toxic species in vivo
may in fact be a pre-aggregated, conformationally mis-folded form of the protein.
In conclusion, our investigations indicate that misfolded, SALS-linked WT-SOD1 proteins activate the same neurotoxic mechanism that is invoked by FALS-linked SOD1 mutants, strongly suggesting that conformational abnormalities and post-translational modifications in WT-SOD1 can contribute to SALS pathogenesis. These studies identify a novel pathogenic mechanism for ALS common to both mutant-SOD1-mediated FALS and many cases of SALS.