SHARE AN ABERRANT CONFORMATION AND A COMMON PATHOGENIC PATHWAY IN - - PowerPoint PPT Presentation

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 H2O2 to generate

• x-SOD1

 Fourier-transform mass

spectrometry (FT-MS) was employed to confirm the oxidation

48 Da

 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

• bserved

Results Oxidized and mutant SOD1 are conformationally similar

conversion of the sulfhydryl group at Cys 111 into sulfonic acid (+3 O2)

Results Oxidized and mutant SOD1 are conformationally similar

 WB analyses were performed to

address the specificity and reactivity

• f 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;

 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!!!

Denaturing

Results Oxidized and mutant SOD1 are conformationally similar

 under native conditions C4F6 is

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!!!

Denaturing

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

 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



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!!!

MAPK Addition prevent the inhibitory effect of ox-SOD1

Results ox-SOD1 inhibits kinesin-based fast axonal transport

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

 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

Results Misfolded SOD1 is present in SALS spinal cord tissues

 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

• f 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’ model of motor neuron degeneration

Discussion

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

THANK YOU...

End of Presentation