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WILD-TYPE AND MUTANT SOD1 SHARE AN ABERRANT CONFORMATION AND A COMMON PATHOGENIC PATHWAY IN ALS DARYL A BOSCO, GERARDO MORFINI, N MURAT KARABACAK, YUYU SONG, FRANCOIS GROS-LOUIS, PIERA PASINELLI, HOLLY GOOLSBY, BENJAMIN A FONTAINE, NATHAN LEMAY, DIANE MCKENNA-YASEK, MATTHEW P FROSCH, JEFFREY N AGAR, JEAN-PIERRE JULIEN, SCOTT T BRADY & ROBERT H BROWN JR PRESENTED BY SUNA LAHUT VOLUME 13 | NUMBER 11 | NOVEMBER 2010 Nature Neuroscience

Abbreviation  wt-SOD1 : wild type SOD1  mut-SOD1 : mutant SOD1  ox-SOD1 : Recombinant, oxidized SOD1  SALS-SOD1 : SOD1 derived from sALS patients (abarrent wt-SOD1)

Abstract  Using C4F6 antibody that detects misfolded SOD1  ox- and mut- SOD1 share a common conformational epitope that is not present in wt-SOD1  In sALS cases, motor neurons in the lumbosacral spinal cord were immunoreactive for C4F6  indicating that an aberrant wt-SOD1 species was present  ox-SOD1 and SALS-SOD1 inhibited kinesin- based fast axonal transport as in the case of FALS-linked mut-SOD1

Introduction  C4F6 antibody  Monoclonal  Generated for SOD1-G93A mutant protein  Binds to several fALS-linked mut-SOD1  Does not bind to wt-SOD1  Appears to be specific for misfolded SOD1

Introduction  fALS and sALS are clinically and neuropathologically similar  the pathogenesis of these diseases share a common pathogenic pathway  such factors are yet unknown  A hypothesis;  defects in the post-translational modifications  aberrant covalent modifications inducing conformational changes in wt-SOD1 or mimic structural features of mut-SOD1 responsible of fALS

If so...  If aberrant modifications to wt-SOD1induce the protein to adopt a mutant-like conformation  new modifications (oxidation of wt-SOD1) would acquire the properties of mut-SOD1 C4F6  SALS-SOD1  If wt-SOD1 has a pathogenic role in sALS, these aberrant wt-SOD1 species would recapitulate the toxic effect(s) of fALS-linked mut-SOD1  functional experiments in isolated squid axoplasm (FAT)

Results Oxidized and mutant SOD1 are conformationally similar  They exposed wt-SOD1 to H 2 O 2 to generate ox-SOD1  Fourier-transform mass spectrometry (FT-MS) was employed to confirm the oxidation 48 Da

Results Oxidized and mutant SOD1 are conformationally similar  ox-SOD1 was subjected to electron capture dissociation  the sulfhydryl group of Cys 111 encoded in exon 4 was fully and irreversibly oxidized to sulfonic acid through the addition of 3oxygen molecules  no other oxidated forms of wild-type SOD1 were observed conversion of the sulfhydryl group at Cys 111 into sulfonic acid (+3 O 2 )

Results Oxidized and mutant SOD1 are conformationally similar  WB analyses were performed to address the specificity and reactivity of the C4F6 antibody  SDG6 antibody is reactive for the native form of SOD1  The migration patterns of ox-SOD1  a loss of hydrodynamic volume as a result of misfolding (slower)  loss or exchange of metals (faster)  a shift in the monomer-dimer equilibrium toward the monomer (faster) Tissue lysates of transgenic mice

Results Oxidized and mutant SOD1 are conformationally similar  Under denaturing conditions; Denaturing  C4F6 reacted to SOD1 G93A  no longer reactive for ox-SOD There is a conformational epitope in ox-SOD1, rather than the sulfonic part at Cys111, that is recognized by C4F6!!!

Results Oxidized and mutant SOD1 are conformationally similar  under native conditions C4F6 is Denaturing reactive for other FALS-linked mut- SOD1 proteins  under denaturing conditions only SOD1 G93A and not others C4F6 recognizes an epitope in SOD1G93A that contains both a conformational component and the G93A sequence component !!!

Results Oxidized and mutant SOD1 are conformationally similar  The C4F6 reactive epitope in both SOD1-G93A and ox-SOD1 was further investigated in an epitope mapping analysis.  GST-tagged constructs encoding  full-length SOD1-G93A gene  SOD1-G93A lacking one of the five SOD1 exons were transfected into the HEK-293 cells  C4F6 reactivity required the presence of exon 4, which harbors the G93 → A mutation

Results Oxidized and mutant SOD1 are conformationally similar

Results Oxidized and mutant SOD1 are conformationally similar  The formation of this epitope was induced by both the G93 → A mutation and the Cys111 sulfonic acid moiety  both of which are in exon 4  However, the conformational component of the epitope was lost when denaturated,  leaving only the G93A sequence element of the epitope to confer C4F6 reactivity

Results ox-SOD1 inhibits kinesin-based fast axonal transport  The common conformational change shared by fALS-linked mut-SOD1 and ox-SOD1led to compare  the effects of ox-SOD1 with FALS-linked mut-SOD1 in an ALS-relevant biological assay:  Vesicle motility assay in isolated squid axoplasm and found that the fALS-linked SOD1-H46R selectively inhibited kinesin-based FAT in the anterograde direction  Whereas the wild-type SOD1 protein did not affect FAT

Results ox-SOD1 inhibits kinesin-based fast axonal transport  They perfused ox-SOD1into the axoplasm and it selectively inhibited anterograde FAT  to a similar extent as the SOD1 H46R mutant.  Thus, ox-SOD1mimics the toxic effect of the FALS- linked mut-SOD1 in these assays.

Results ox-SOD1 inhibits kinesin-based fast axonal transport  Phosphorylation of the molecular motor conventional kinesin is known to regulate FAT in vivo  Furthermore, fALS-linked mut-SOD1 inhibits FAT by a mechanism involving the activation of a kinase pathway  To evaluate the possibility that ox-SOD1mediated inhibition of FAT also involves the activation of axonal kinases, changes in the activity of various protein kinases perfused with either wt- or ox- SOD1 were screened

Results ox-SOD1 inhibits kinesin-based fast axonal transport MAPK Addition prevent the inhibitory effect of ox-SOD1 p38: kinase that phosphorylate (inactivate) kinesin  MAPK: inhibitor of kinesin phosphorylation  Aberrantly modified form of wt-SOD1shares conformational motifs with mut-SOD1 inhibits anterograde FAT through a mechanism involving p38 activation!!!

Results Misfolded SOD1 is present in SALS spinal cord tissues  To address the hypothesis that aberrantly modified forms of wt-SOD1 are associated with SALS in vivo, they performed immunohistochemistry on human spinal cord tissue, using the C4F6 antibody  Positive C4F6 staining was observed in four of nine SALS cases  extensive degeneration in the motor regions  motor neurons could not be detedted

Results Misfolded SOD1 is present in SALS spinal cord tissues  a,d,e,f are positive for C4F6  17 controls are negative for C4F6  Lack of C4F6 immunoreactivity in a fALS case without SOD1 mutations confirmed the specificity of staining  The A9G3 monoclonal antibody did not react to SALS-SOD1  a specific epitope

Results Misfolded SOD1 is present in SALS spinal cord tissues  To assess the levels of aggregated SALS-SOD1 in the C4F6-positive SALS cases  insoluble SOD1 derived from postmortem human spinal cord tissue  The levels of insoluble SOD1 were not substantially different between human ALS cases and controls Misfolded SALS-SOD1 are relatively soluble!!!

Results Misfolded SOD1 is present in SALS spinal cord tissues  They evaluated the possibility SALS-SOD1 also inhibits FAT as ox-SOD1  immunopurified wild-type SOD1 from both SALS and control human cases and perfused them into the squid axoplasm  SALS-SOD1 inhibited anterograde FAT  wt-SOD1 had no effect on FAT  Co-perfusion of the C4F6 antibody blocked the inhibitory effect of SALS-SOD1  indicating that C4F6-reactive SOD1 species mediate the inhibitory effect on FAT

Discussion  similar structural perturbations within the protein  genetic variants  non-inherited modifications  non-inherited modifications of SOD1 can be associated with sALS  Do these sALS-linked modifications confer on wt- SOD1 have the same toxic properties with fALS- linked mut-SOD1 including activation???

Discussion  Both ox- and SALS-SOD1 recapitulated the pattern of mut-SOD1-mediated FAT inhibition  whereas wt-SOD1 had no effect  The effect of SALS-SOD1 on anterograde FAT was abolished by incubation with the C4F6 antibody

Discussion  FALS-linked mut-SOD1-mediated defects in anterograde FAT have been reported previously  an early pathogenic event in mutant SOD1 transgenic mice that contributes to a ‘ dying back ’ mode l of motor neuron degeneration

Conclusion  They found that SALS-SOD1 proteins activate the same neurotoxic mechanism that is invoked by FALS- linked mut-SOD1  strongly suggesting that conformational abnormalities and post-translational modifications in wt-SOD1 can contribute to SALS pathogenesis.  The results identify a pathogenic mechanism for ALS that is common to both  Mut-SOD1 – mediated fALS  many cases of sALS

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