Redox regulation of cellular stress response in multiple sclerosis

Redox

regulation

of

cellular

stress

response

in

multiple

sclerosis

G.

Pennisi

a

,

C.

Cornelius

b

,

M.M.

Cavallaro

b

,

A.

Trovato

Salinaro

b

,

M.T.

Cambria

b

,

M.

Pennisi

b

,

R.

Bella

a

,

P.

Milone

c

,

B.

Ventimiglia

d

,

M.R.

Migliore

b

,

L.

Di

Renzo

e

,

A.

De

Lorenzo

e

,

V.

Calabrese

b,

*

a

DepartmentofNeuroscience,UniversityofCatania,I-95125Catania,Italy

b

DepartmentofChemistry,UniversityofCatania,I-95125Catania,Italy

c

DepartmentofRadiologicalSciences,UniversityofCatania,I-95125Catania,Italy

d_{DepartmentofMedicineandSurgery,UniversityofCatania,I-95125Catania,Italy}

e_{DivisionofHumanNutrition,DepartmentofNeuroscience,UniversityofRomeTorVergata,I-00133Rome,Italy}

1. Introduction

Multiplesclerosis(MS)isanautoimmune-mediated neurode-generative disease with characteristic foci of inflammatory demyelinationinthebrain,spinalcord,andopticnerves.Recent studies have demonstrated not only that axonal damage and neuronalloss aresignificant pathologiccomponentsof MS and experimentalautoimmuneencephalomyelitis(EAE),butthatthis neuronaldamageisthoughttocausethepermanentneurologic disabilityoftenseenin MSpatients. Currenttreatments forMS involveimmunomodulation,which can reducetheincidenceof inflammatoryrelapses.However,existingtherapiesareoftennot fullyeffective,andlimitedevidencesuggeststhatthesetherapies preventthelong-termneuronaldamageandphysicaldisabilityof MSpatients[1,2].Newtherapiesthatpreventneurodegeneration

throughnonimmunomodulatorymechanismshaveatremendous potentialtoworksynergisticallywithcurrentMStherapies[3].MS pathologyischaracterizedbyperivenousinfiltrationof lympho-cytesandmacrophageleadingtodamageofmyelinandaxonsin the brain and spinal cord, which underlie the clinical disease courseusuallyoccurringwithrecurrentandreversibleepisodesof neurologicaldysfunctionaffectingoneorseveralsites,duringlate adolescence and earlyadulthood;this form is called relapsing-remitting form (RR) and it is the most prevalent. Usually, approximately20yearslater,thisclinicalpatterntransformsinto a secondaryprogressivephasewithcontinuousandprogressive neurologicaldecline[4].

AlthoughevidenceindicatesthatMSisamultifactorialdisease causedbyacomplexinterplaybetweengeneticandenvironmental factors,itisstillunclearwhicharethecausesorthefactorsthat contributetoitsunpredictablecourse.Itisgenerallyacceptedthat, virus infections of the CNS, vascular factors and/or disturbed immunemechanismsareimplicatedinthepathogenesisofMS[5,6]. Oncetriggered,theimmunesystemattacksanddestroysmyelinand themyelin-formingcell, leadingtothepathological hallmarksof

A R T I C L E I N F O Articlehistory: Received6May2011 Accepted22July2011 Availableonline30July2011

Thispaperisdedicatedtothe memoryofClaudioCavazza, whodiedonJune6th,while thismanuscriptwasinpreparation.

Keywords: Multiplesclerosis Oxidativestress Vitagenes Heatshockproteins Sirtuins

A B S T R A C T

Multiplesclerosis(MS)isanautoimmune-mediatedneurodegenerativediseasewithcharacteristicfociof inflammatorydemyelinationinthebrain,spinalcord,andopticnerves.Recentstudieshavedemonstrated notonlythataxonaldamageandneuronallossaresignificantpathologiccomponentsofMS,butthatthis neuronaldamageisthoughttocausethepermanentneurologicdisabilityoftenseeninMSpatients. Emergingfinding suggests that alteredredox homeostasis and increasedoxidative stress,primarily implicatedinthepathogenesisofMS,areatriggerforactivationofabrainstressresponse.Relevantto maintenanceofredoxhomeostasis,integratedmechanismscontrolledbyvitagenesoperateinbrainin preservingneuronalsurvivalduringstressfulconditions.Vitagenesencodeforheatshockproteins(Hsp) Hsp32,Hsp70,thethioredoxinandthesirtuinproteinsystems.Inthepresentstudyweassessstress responsemechanismsintheCSF,plasmaandlymphocytesofcontrolpatientscomparedtoMSpatients.We foundthatthelevelsofvitagenesHsp72,Hsc70,HO-1,aswellasoxidativestressmarkerscarbonylsand hydroxynonenalsweresignificantlyhigherinthebloodandCSFofMSpatientsthanincontrolpatients.In addition,anincreasedexpressionofTrxandsirtuin1,togetherwithadecreaseintheexpressionofTrxR were observed. Our data strongly support a pivotal role for redox homeostasis disruption in the pathogenesisofMSand,consistentlywiththenotionthatnewtherapiesthatpreventneurodegeneration throughnonimmunomodulatorymechanismscanhaveatremendouspotentialtoworksynergistically withcurrentMStherapies,unravelimportanttargetsfornewcytoprotectivestrategies.

ß2011ElsevierInc.Allrightsreserved.

*Corresponding author at: Department of Chemistry, Faculty of Medicine, UniversityofCatania,Italy.Tel.:+390957384067;fax:+39095580138.

E-mailaddress:calabres@unict.it(V.Calabrese).

ContentslistsavailableatScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

0006-2952/$–seefrontmatterß2011ElsevierInc.Allrightsreserved. doi:10.1016/j.bcp.2011.07.092

MS: the classical actively demyelinating lesions, the cortical demyelination and the diffuse white matter injury, the latter particularlyevidentinthelaterstageofthedisease[6,7].Existing evidenceindicatesthatCNSrespondstotheattackbyimmunecells andtheirsecretedproductsthroughmodulationofitsmetabolism andgeneexpression[8].Inaddition,cytokines,immunoglobulins, andcomplementcomplexesmayelicitasurvivalresponseinthe oligodendrocytes,involvingtheinductionofendogenousheatshock proteinsandotherprotectivemolecules,whichindicatesthatredox systemsandthereforetheoxidant/antioxidantbalanceinthesecells areofgreatimportanceinMS[9–12].

Theadaptationandsurvivalofcellsandorganismsrequiresthe abilitytosenseproteotoxicinsultsandtocoordinateprotective cellularstressresponsepathwaysandchaperonenetworksrelated to protein quality control [13]. Despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, brain cells appears poorlyadaptedforchronicproteotoxicstresswhichincreasesin neurodegenerativediseasessuchasMS[14].Intheseconditions,a declineinbiosyntheticandrepairactivitiesthatcompromisesthe integrityoftheproteomeisstronglyinfluencedbyprotectivegenes called ‘‘vitagenes’’ that control aging, thus linking stress and proteinhomeostasiswiththehealthantidegenerativemechanisms

[15].Vitagenesencodeforheatshockproteins(Hsp)Hsp32,Hsp70, thethioredoxinandthesirtuinproteinsystems.IntheCNS,Hsps synthesisoccursafterhyperthermia,alterationsintheintracellular redoxenvironment,exposuretoheavymetals,aminoacidanalogs orcytotoxicdrugs[15].Whileprolongedexposuretoconditionsof extreme stress is detrimental and can induce cell death, Hsps synthesis can result in stress tolerance and cytoprotection in variousmetabolicdiseasesandinjuries,includinghypoxia,stroke, epilepsy,celland tissuetrauma,neurodegenerativediseaseand aging [14]. Sirtuins (SIRTs) represent a highly conserved gene familyencodingNAD+-dependentdeacetylases,originallyfoundto deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals.Membersofthesirtuinfamilyhavesincebeenfoundto functionasdeacetylasesfornumerousproteintargetsinvolvedin many cellular pathways, including cellular stress responses, apoptosis,andaxonal degeneration. Theactivityof sirtuinscan beregulatedbyothercompoundsthatcanserveasactivators(e.g., plantpolyphenols)thatincreaseenzymeactivitybyloweringthe Kmofitssubstratesorthatcanserveasinhibitors(e.g.,sirtinol)

[15,16].NeuroprotectionmediatedbySIRT1activationhasbeen demonstratedinaxotomizeddorsalrootganglionneuronsandin neuronscontainingtheHuntingtingenemutationresponsiblefor neurodegenerationinHuntingtondisease.Thus,pharmacological modulationofcellularstresspathwayshasemergingimplications for the treatment of human diseases, including cardiovascular disease,cancerandneurodegenerativedisorders[17,18].

The present study was undertaken in order to investigate systemic stress response and the associated oxidative stress measuredthrough the determination of markers ofprotein and lipidoxidationinplasma, lymphocytesandCSFofpatientswith activeMS,ascomparedtoage-matchedcontrols,inordertogaina betterinsightintothemolecularmechanismsregulatingthecellular stressresponseduringtheprogressionofthediseaseand,assuch, provide a potential target for novel cytoprotective strategies impactingtheclinicalsettingsofthisdegenerativedisease.

2. Materialsandmethods

2.1. Ethicalpermission

ThestudywasconductedaccordingtoguidelinesoflocalEthics Committee,andinformedconsentwasobtainedfromallpatients.

2.2. Patients

2.2.1. Multiplesclerosispatients

Twenty-six patients withanage rangeof 20–60years were usedasthe‘‘test’’groupforthisstudy.Themeandiseaseduration was 3.92.0 years. All these patients had a confirmed clinical diagnosisofMSaccordingtothediagnosticcriteriaofMcDonaldetal.

[19].Furthermore,allthesubjectswereclinicallyclassifiedhavingthe relapsingremittingformofMSaccordingtothecriteriadescribedin Lublin andReingold [20]. The patients had not been undergoing corticosteroidorimmunosuppressivetreatmentforatleast2months beforetheCSFsampleswerecollected.

2.2.2. Controlpatients

Twenty control patients, withan age range of 30–60 years, underwentlumbarpuncturebecauseofsuspectedsubarachnoid hemorrage, pseudotumor cerebri, oculomotor palsies, or other indications in the usual neurological survey. Laboratory and neuroimagingtestswerenormal.Thereforethefinaldiagnosiswas mainlytensionheadacheorconversiondisorder.

2.3. Sampling

Bloodwascollectedfromcontrolsandpatientsbyvenipuncture fromanantecubitalveinintotubescontaining500mMEDTAasan anticoagulant. Immediatelyafter sampling,1ml the blood was centrifugedat10,000gfor10minat48Ctoseparateserumfrom redbloodcellsand4mLwereutilizedforlymphocytes purifica-tion.Lymphocytesfromperipheralbloodwerepurifiedusingthe Ficoll Paque System following the procedure provided by the manufacturer (GE Healthcare, Piscataway, NJ, USA). CSF was obtained(onice)fromallsubjectsbylumbarspinaltap.TheCSF sampleswereimmediatelycentrifugedat10,000gfor3minat 48Ctoremoveanycontaminatingcellsandkeptoniceuntilthe biochemicalassayswereperformed.

ChemicalanalysisofCSFshowednohemepresentintheCSFin anyofthecontrolorMSCSFsamples.CSFlymphocytescountwas 1.051.17inthefirstMSCSFsampleand1.041.15insecondones. Thecontrolgrouplymphocytescountwas10.12.

2.4. Westernblotanalysis

Carbonyls (DPNH),HNE, Hsp60, Hsc70, Hsp-72,HO-1, HO-2, Trx,TrxR-1,Sirt-1 wereevaluatedby Westernblotanalyses.In brief,proteins(40

m

g)foreachsamplewereseparatedbya12.5% SDS-PAGE and electroblotted (1h at 100V) to nitrocellulose membranes(Bio-Rad)using25mMTris,192mMglycineand20% (v/v)methanol.Equalproteinloadingwasconfirmedbystaining with0.2%(v/v)PonceauSin 7%acetic acid.Blottedmembranes were blocked with3% non fat dry milk in phosphate-buffered saline and challenged with appropriate primary antibodies, namely anti-Hsc70, monoclonal antibody (Santa Cruz Biotech. Inc.,SantaCruz,CA,USA),thatrecognizestheconstitutiveformof Hsp 70, orwitha monoclonal anti-Hsp72 antibody(RPN 1197, Amersham)that recognizesonlytheinducibleform,anti-DPNH (V0401 DAKO, Glostrup, Denmark), anti-HNE (HNE11-S Alpha DiagnosticInternational,SanAntonio, TX,USA),anti-HO-1, anti-HO-2(Stressgen,Victoria,BC,Canada)anti-Trx,antiTrxR1andanti Sirt-1rabbitpolyclonalantibody(SantaCruzBiotech.Inc.,Santa Cruz,CA,USA),for1hatroomtemperature.Immunodetectionof Hsp60wasperformed usinga polyclonalgoatantibodysc-1052 (SantaCruzBiotech.Inc.,SantaCruz,CA,USA).Unboundantibodies wereremovedbywashingTBS-Tfor5min.Themembraneswere thenincubatedwithhorseradishperoxidase-conjugated second-aryantibodydiluted1:10,000.Proteinbandswerevisualizedwith ECLPlusTM_{(Amersham,NJ,USA)accordingtothemanufacturer’s}

protocol. Immunoreactive bands were quantified by scanning Western blot-imaged films with a laser densitometer (LKB-Ultrascan,XLmodel,Pharmacia,AmericanInstruments,Haverhill, MA, USA). Molecular weights of the proteins detected were determinedusingastandardcurveobtainedwithproteinsofknow molecularweight.

2.5. Proteinmeasurements

Samples protein concentrations were determined by the bicinchoninicacidmethod[21].

2.6. Statisticalanalysis

All resultsare expressedas meansstandarderrorof mean. Each experiment was performed, unless otherwise specified, in triplicate. Data were analyzed by one-way analysis of variance, followedbyinspectionofalldifferencesbyDuncan’snew multiple-rangetest.DifferenceswereconsideredsignificantatP<0.05.

3. Results

Multiplesclerosis(MS)isachronicinflammatory demyelinat-ingdiseaseofthecentralnervoussystem(CNS)ofunclearetiology, characterized by perivenous infiltration of lymphocytes and macrophageleading damage of myelin and axons in thebrain andspinalcord[1].Clinicaldiseaseusuallyoccurswithrecurrent andreversibleepisodesofneurologicaldysfunctionaffectingone orseveralsites,duringlateadolescenceandearlyadulthood;this formbeingthemostprevalentiscalledrelapsing-remittingform (RR)andwaschoseninthisstudy.Table1summarizesclinicaldata ofpatientsandcontrolsubjects.Asshown,themeanageatdisease onsetwas31.49.2yearswithadiseasedurationof3.922.08 years,arelativelyshortdurationtypicaloftherelapsingremitting formnotyetchangedintothesecondaryprogressiveform.Themean valueofExpandedDisabilityStatusScale(EDSS),whichthemostused methodofquantifyingdisabilityinmultiplesclerosis,was1.51.18 andthisindicatesthatpatientshavealowdegreeofdisabilitylinked totheshortperiodofdisease.BrainMRIfromMSpatientspresenting twohypointenseareasdistributedbilaterallyonthewhitematterof thesemioval center (A), enhanced bygadolinium injection (B) is showninFig.1AandB.

Theheatshockresponsecontributestoestablishinga cytopro-tective state in a wide variety of human diseases, including inflammation,cancer,agingandneurodegenerativedisorders.The vitagenefamilyiscomposedoftheheatshockproteinsHO-1(also calledHsp32), Hsp70, by the thioredoxinsystem and bysirtuin proteins[22–24].Wethereforeevaluatedtheexpressionlevelsof cellularstressresponseproteinsintheCSF,plasmaandlymphocytes incontrolandinMSpatients.WesternblotanalysisofCSFprobedfor heme oxygenase-1 is reported in Fig. 2. HO-1 expression is significantly(P<0.05)increasedintheCSFofMSpatients,compared tocontrols. This findingwas associated withincreased levelsof proteinoxidation(Fig.3A)aswellaslipidoxidation(Fig.3C).Protein oxidation was evaluated by measuring the amount of protein carbonyls(DNPH).Proteincarbonylsmostoftenaredetectedbytwo methods, i.e., derivatization with 2,4-dinitrophenylhydrazine

Table1

ClinicalanddemographicdataofMSpatientsandcontrolsubjects.

Numberofsubjects Age(meanSD) Gender(F/M) Ageatdiseaseonset(meanSD) Diseaseduration(meanSD) EDSS(meanSD)

Patients 26 35.39.9 16/10 31.49.2 3.922.08 1.51.18

Controls 20 36.910.9 12/8

EDSS:ExpandedDisabilityStatusScale.

Fig.1.BrainMRIfromMSpatients:axialT1WIshowstwohypointenseareasonthe whitematterofthesemiovalcenterbilaterally(A)thatpresentenhancementafter gadoliniuminjection(B).

(DNPH),followedbyimmunochemicaldetectionwithanantibody againsttheresultingproteinhydrazone,orformationoftheSchiff basebybiotinhydrazidefollowedbydetectionoftheprotein-bound iminewithenzyme-orfluorophore-linkedavidinorstreptavidin. HerewedescribetheDNPH-baseddetectionmethodthatisroutinely usedfordetectionofcarbonylatedproteins.IntheDNPHmethod, samplesare derivatizedwith DNPH.DNPH reactswith carbonyl groupstoformprotein-resident2,4-dinitrophenylhydrazone(DNP), which is then detected using commercially available anti-DNP antibodies(Fig.3B).Asshown,proteincarbonylswerefoundata significantly(P<0.05)higherlevelintheCSFofMSpatientsthanin

controlsubjects.Furthermore,onemeasureofoxidativemarkeris lipidoxidation,indexedbyHNE[25,26],whichcanalsooccurinbrain underoxidativestress[27].HNE,formedfromarachidonicacidor otherunsaturatedfattyacidsfollowingfreeradicalattack,bindsby Michaeladditiontoproteins,particularlyatcysteine,histidine,or lysineresidues[27].ExaminationofHNElevelsintheCSFofMS patientsshowedasignificant(P<0.05)elevationofprotein-bound HNEascomparedtocontrolgroup(Fig.3D).

The 70kDa family of stress proteins is one of the most extensivelystudiedcytoprotectivesystem.Includedinthisfamily aretheconstitutiveisoform,Hsc70(heatshockcognate),andthe inducibleisoform,Hsp70(alsoreferredtoasHsp72).Figs.4and5

showa significant(P<0.05)increasein theexpressionlevel of

Fig.2.(A)Hemeoxygenase-1(HO-1)levelsintheCSFofMSpatients.CSFsampleswereassayedforHO-1byWesternblotasdescribedinSection2.(B)Arepresentative immunoblotisshown.b-Actinhasbeenusedasloadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesareexpressedasmeanstandarderrorofmean of3independentanalysesontwenty-two/twenty-sixpatientspergrouppergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiplesclerosis;CTRL,control;St, standard.

Fig.3.ProteincarbonylsandHNE(4-hydroxy-2-nonenals)levelsintheCSFofMS patients.CSFsampleswereassayedforproteincarbonyls(A,B),andHNE(C,D)by WesternblotasdescribedinSection2.Representativeimmunoblotsareshown(B, D).Thebargraphshowsthedensitometricevaluationandvaluesareexpressedas meanstandarderrorofmeanof3independentanalysesontwenty-two/twenty-six patientspergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiple sclerosis;CTRL,control.

Fig.4.(A)Heatshockprotein70(Hsc70)levelsinMSlymphocytes.Lymphocytes were assayed for Hsc-70 by Western blot asdescribed in Section 2. (B) A representativeimmunoblotisshown.b-Actinhasbeenusedasloadingcontrol.The bargraphshowsthedensitometricevaluationandvaluesareexpressedasmean standard errorofmeanof 3independentanalysesontwenty-two/twenty-six patientspergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiple sclerosis;CTRL,control.

Hsc70andHsp70,respectively,inlymphocytesofMSpatientswith respecttocontrols(Figs.4Aand5A).Representativeimmunoblots arealsoshown(Figs.4Band5B).

Hemeoxygenase-1(HO-1),alsoreferredtoasHsp32,belongsto theHsp familyandprotectsbraincells fromoxidativestressby degrading toxic heme into free iron, carbon monoxide and biliverdin [17,24]. This latter is then reduced by biliverdin reductase (BVR) into bilirubin (BR), a linear tetrapyrrole with antioxidant properties; very recently, BR has been shown to effectivelycounteractalsonitrosativestress,duetoitsabilityto bindandinactivateNOandRNS[14,17].Theconstitutiveisoform ofhemeoxygenaseisHO-2[28,29].Weevaluatedtheexpressionof bothisoformsinlymphocytesofMSpatientsandincontrols.As showninFig.6,HO-1proteinexpressionwashigherinMSsamples compared tocontrol. Notably,increase in HO-1wasassociated withasignificant(P<0.05)decreasein HO-2expressioninthe sameexperimentalgroup(Fig.7).AnalysisoflymphocytesinMS patients, comparedtocontrol group, revealedalsoa significant (P<0.05)increaseinthioredoxinexpression,whereasexpression of the related enzyme thioredoxin reductase-1 was decreased (Figs. 8A, B and 9A, B). These changes were associated with significantly(P<0.05)increasedformationofproteincarbonyls (Fig.10AandB)andHNE(Fig. 10CandD). Consistentlytothe observedchangesinMS lymphocytes,analysisofplasmain MS patientsshowedhigherandsignificant(P<0.05)expressionlevels ofHO-1,Hsp60thanincontrolsubjects(Figs.11and12),whileno difference was found to a significant extent between the two experimentalgroupsinHsp70proteinlevels(Fig.13). Interesting-ly,wefoundintheplasmasignificantly(P<0.05)higherlevelsof sirtuin-1proteininMSpatientsthanincontrolgroup(Fig.14).As farasweareconcernedthisisthefirstevidenceofchangesin SIRT-1expressioninMS,althoughatthemomentwecannotexclude that this might not be a specific alteration of this progressive inflammatoryneurodegenerativedisease.Asummaryofchanges ofallstressresponseproteinsinvestigatedinCSF,lymphocytesand plasmaarereportedinTable2.

4. Discussion

Multiple sclerosis is a relatively common and etiologically unknown disease with no cure. It is the leading cause of neurologicaldisabilityinyoungadults,affectingovertwomillion people worldwide. Traditionally, MS has been considered a chronic, inflammatory disorder of the central white matter in which ensuingdemyelinationresultsinphysical disability[30].

Fig.5.(A)Heatshockprotein72(Hsp72)levelsinmultiplesclerosispatients. LymphocytessampleswereassayedforHsc-72byWesternblotasdescribedin Section2.(B)Arepresentativeimmunoblotisshown.b-Actinhasbeenusedas loadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesare expressedasmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits; MS,multiplesclerosis;CTRL,control.

Fig.6. (A) Heme oxygenase-1(HO-1) levels in lymphocytes ofMS patients. LymphocytessampleswereassayedforHO-1byWesternblotasdescribedin Section2.(B)Arepresentativeimmunoblotisshown.b-Actinhasbeenusedas loadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesare expressedasmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits; MS,multiplesclerosis;CTRL,control.

Fig.7. (A) Heme oxygenase-2(HO-2) levels in lymphocytes ofMS patients. LymphocytessampleswereassayedforHO-2byWesternblotasdescribedin Section2.(B)Arepresentativeimmunoblotisshown.b-Actinhasbeenusedas loadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesare expressedasmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits; MS,multiplesclerosis;CTRL,control.

Fig.8.(A)Thioredoxin(Trx)levelsinlymphocytesfrommultiplesclerosispatients. LymphocytessampleswereassayedforTrxbyWesternblotasdescribedinSection

2.(B)Arepresentativeimmunoblotisshown.b-Actinhasbeenusedasloading control.Thebargraphshowsthedensitometricevaluationandvaluesareexpressed asmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiple sclerosis;CTRL,control.

Recently, MS has become increasingly viewed not only an inflammatory disease, but alsoa neurodegenerativedisorderin which axonal injury, neuronal loss and atrophy of the central nervous system lead to permanent neurological and clinical disability [31]. There are three main types of MS, defined as: relapsing-remitting(RR),secondaryprogressive(SP)and primary-progressive(PP) withprogressive-relapsing(PR)recently distin-guishedasanadditionalsubtype[31,32].ThereisevidencethatMS is characterized not only by immune mediated inflammatory reactions but also by neurodegenerative processes [33,34]. A varietyof studies, supportinga role foroxidative stressin MS, indicatethat endogenousantioxidantsaredecreasedinMS,and damagetomitochondriainducedbylipidperoxidationcanleadto further ROS generation [35–39]. However, up-regulation of antioxidantsinastrocytesandmacrophagesinactiveMSlesions

[30]aswellasinleukocytessuggeststhatendogenousprotection fromoxidativedamagecouldlimitthedeleteriousconsequencesof the oxidative stress per se [40]. Interestingly, exogenously administrated antioxidantshave shown somebeneficial effects inanimalstudiesofMSbutnoclinicalstudiessupporttheuseof suchtherapy[41–44].Furthermore,thepossiblelinksbetweenMS and redox balance perturbation is indicated by increased malonaldehydeand F2-iPlevels inMS patientsas comparedto controlindividualswithotherneurologicaldisorders[11]and,in addition,aninappropriateexpressionofHspsonoligodendrocytes

[36,45].Thislasteventcouldrepresentapossibleinitiatingfactor atthelevelofMSlesions,capableofmodulatingthesubsequent susceptibilityorresistanceofcellstooxidativestress.Inaprevious reportwehavedemonstratedhighlevelsofNO,peroxynitrite,and superoxideinspinalfluidfrompatientswithMS[5,9].Activated glia secrete RNS products of NO metabolism with superoxide radicals (O2 ) to form peroxynitrite anion (ONOO ). RNS can

causenitrosativestress,whichresultsinthedestructionofmyelin and(myelin-forming)oligodendrocytecells[46].Furthermore,the formationofS-nitrosoglutathione(GSNO)cancauseGSH deple-tion, and hence trigger redox dependent changes in cellular signalingaswellasmodificationofkeyintracellularenzymes,such aschainrespiratorycomplexactivities[47].

Inthisstudy,significantincreasesinoxidativestressmarkers, such as carbonyls and HNE were foundin theCSF, peripheral lymphocytesandplasmaofMSpatients,ascomparedtocontrol subjects.Thesechangeswereassociatedwithinductionofstress responsiveproteins,suchasHO-1,Hsp72,Hsp60andthioredoxin (TRX), measured in CSF, lymphocytes and in plasma. Heme oxygenase is the rate-limiting enzyme in the production of bilirubin.In thepast decadethe heme oxygenase(HO) system has been focused for its potential significance in maintaining cellularhomeostasis.Itislocatedintheendoplasmicreticulumina complexwithNADPHcytochromecP450reductase.Thereaction catalyzedbyHOistheoxidativecleavageofthehememoleculeto formbiliverdinandcarbonmonoxide(CO).Ironisreducedtoits ferrous statethrough the action of NADPHcytochrome cP450

Fig.9.(A)Thioredoxinreductase-1(TrxR-1)levelsinlymphocytesfrompatients withmultiplesclerosis.LymphocytessampleswereassayedforTrxR-1byWestern blotasdescribedinSection2.(B)Arepresentativeimmunoblotisshown.b-Actin hasbeenusedasloadingcontrol.Thebargraphshowsthedensitometricevaluation andvaluesareexpressedasmeanstandarderrorofmeanof3independentanalyses on twenty-two/twenty-six patients per group. P0.05 vs control. D.U., densitometricunits;MS,multiplesclerosis;CTRL,control.

Fig.10.(A)Proteincarbonyls(DPNH)and(C)HNElevelsinlymphocytesfrommultiplesclerosisandcontrolpatients.Lymphocytessampleswereassayedforcarbonylsand HNEbyWesternblotasdescribedinSection2.Representativeimmunoblotsareshown(B,D).Thebargraphshowsthedensitometricevaluationandvaluesareexpressedas meanstandarderrorofmeanof3independentanalysesontwenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits,MS,multiplesclerosis; CTRL,control;St,standard.

reductase. Further degradationof biliverdin to bilirubinoccurs throughtheactionofacytosolicenzyme,biliverdinreductase[48]. HOispresentinvarioustissueswiththehighestactivityin the brain,liver,spleen,andtestes.Therearethreeisoformsofheme oxygenase, HO-1 or inducible isoform [13,16,22], HO-2 or constitutive isoform [49], and the recently discovered HO-3, clonedonlyinrattodate[50].ElevationofHO-1expressionand activityin MSisprobablyduetoelevatedoxidative stress.This findingisconsistentwithevidencesuggestingthattheHO-1gene isredoxregulatedand,similartootherantioxidantenzymes[50], this occurs because it contains in its promoter region the antioxidantresponsiveelement(ARE).Therefore,theHO-1gene undergoesaredoxsensitivemodulationbytranscriptionfactors recognizingspecificbindingsiteswithinthepromoteranddistal enhancer regions of the HO-1 gene [14,15]. In addition, heme oxygenase-1israpidlyupregulatedbyoxidativeand nitrosative stresses,aswellasbyglutathionedepletion[51].

Ithasbeenalsosuggestedthatinappropriatestressresponse withintheCNScouldinfluenceboththepermeabilityoftheblood– brainbarrierandtheexpressionofHsps,therebyinitiatingtheMS lesion [34,52,53]. In addition, cytokines, immunoglobulins, and complementcomplexesmayelicita survivalresponseinvolving theinductionofendogenousHspsandotherprotectivemolecules, whichindicatesthatredoxdependentmechanismsinthesecells areofgreatimportanceinMS[54–56].Theheatshockresponse

contributes to establish a cytoprotective state in a variety of metabolicdisturbancesandinjuries,includingstroke,epilepsy,cell andtissuetrauma,neurodegenerativedisease,andaging[15,24]. In mammalian cells, the induction of theheat shock response requirestheactivationandtranslocationtothenucleusofoneor moreheatshocktranscription factors(HSFs),whichcontrolthe expressionofaspecificsetofgenesencodingcytoprotectiveHsps. Production of Hsps is essential for the folding and repair of damagedproteins,servingthustopromotecellsurvival[34,52]. Hspsserveaschaperonesthatbindtootherproteinsandregulate theirconformation,regulateproteinmovementacrossmembranes orthroughorganelles,orregulatetheavailabilityofareceptoror activityofanenzyme.The70-kDafamilyofstressproteinsisoneof themostextensivelystudied.ThisfamilycomprisesHsc70 (heat-shockcognate,theconstitutiveform),Hsp70(theinducibleformor Hsp72),andGRP75(aconstitutivelyexpressedglucose-regulated proteinfoundintheendoplasmicreticulum).AfteraCNSinjury, Hsp70issynthesizedathighlevelsandispresentinthecytosol, nucleus, and endoplasmic reticulum [52]. Hsp60 chaperone proteinswithinmitochondriaand,also,protectbraincellsagainst

Fig.11.(A)Hemeoxygenase-1(HO-1)levelsinplasmafrommultiplesclerosis patients.PlasmasampleswereassayedforHO-1byWesternblotasdescribedin Section2.(B)Arepresentativeimmunoblotisshown.b-Actinhasbeenusedas loadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesare expressedasmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits; MS,multiplesclerosis;CTRL,control.

Fig.12.(A)Heatshockprotein60(Hsp60)levelsinplasmafrommultiplesclerosis patients.Plasmasamplesfrommultiplesclerosispatientsandcontrolsubjectswere assayedforHsp60byWesternblotasdescribedinSection2.Arepresentative immunoblotisshownin(B).b-Actinhasbeenusedasloadingcontrol.Thebar graphshows thedensitometricevaluationand valuesare expressedasmean standarderrorof meanof3independentanalyses ontwenty-two/twenty-six patientspergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiple sclerosis;CTRL,control.

Fig.13.(A)Heatshockprotein72(Hsp72)levelsinplasmafrommultiplesclerosis patients.PlasmasampleswereassayedforHsp72byWesternblotasdescribedin Section2.Arepresentativeimmunoblotisshownin(B).b-Actinhasbeenusedas loadingcontrol.Thebargraphshowsthedensitometricevaluationandvaluesare expressedasmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits; MS,multiplesclerosis;CTRL,control.

Fig.14.(A)Sirtuin-1(Sirt-1)levelsinplasmafrommultiplesclerosispatients. PlasmasampleswereassayedforSirt-1byWesternblotasdescribedinSection2.A representativeimmunoblotisshownin(B).b-Actinhasbeenusedasloading control.Thebargraphshowsthedensitometricevaluationandvaluesareexpressed asmeanstandarderrorofmeanof3independentanalyseson twenty-two/twenty-sixpatientspergroup.P0.05vscontrol.D.U.,densitometricunits;MS,multiple sclerosis;CTRL,control.

ischemiaandseizuresinvivo,afterviral-inducedoverexpression

[18].Hsp60isencodedinthenucleusandresidesmainlyinthe mitochondria[53].Hsp60formsthechaperonincomplex,whichis implicatedinproteinfoldingandassemblywithinthe mitochon-dria under normal conditions [53]. Hsp60 together with its cochaperonin Hsp10 produce a large, efficient protein-editing machinery that facilitates proper folding and assembly of mitochondrial-importedproteinsandcorrectsmisfolded polypep-tidesgeneratedundermitochondrialoxidativestress[57]. Muta-tionsintheHSPD1geneencodingHsp60haverecentlybeenfound to underlie spastic paraplegia 13 (SPG13 [MIM 605280]), an autosomal-dominant spinal-cord neurodegenerativedisorder of lateonset,characterizedbyprogressiveweaknessandspasticityof the lower limbs, and, more recently, a homozygous missense mutation, D29G, in HSPD1, encoding the mitochondrial Hsp60 chaperonin, causing a mitochondrial Hsp60 chaperonopathy linkedtobrainhypomyelinationandleukodystrophy.Thisfinding providesevidenceforthepivotalroleofthemitochondrialHsp60 chaperoninintheprocessofnormalbrainmyelinationandinthe pathogenesisofhypomyelinatingneurodegenerativedisease[58]. Notably,wefoundincreasedexpressionofthioredoxininMSas compared to control subjects. To date this is the first report demonstrating changes in thioredoxin/thioredoxin reductase protein levelsin MS patients. Thioredoxin (TRX) represents an intracellularredoxregulatorthathasbeenshowntobeimportant fortheregulationofredox-sensitivetranscriptionfactors[18].In itsreducedstateTRXcanoxidativelyreactivateinactive transcrip-tionfactorssuchasJun,Fos,AP-1,redoxfactor-1(ref-1),andNrf-2

[59].Thioredoxinreductase(TRXr)isaflavoproteinthatcatalyzes theNADPH-dependentreductionofoxidizedthioredoxinprotein. Itis usually located in thecytosol, but it translocates into the nucleusinresponsetovariousstimuliassociatedwithoxidative stress.TRXr playsa pivotalrole in protecting againstoxidative stressandinregulatingcellgrowthandcelldeath. Constitutive TRXrexpressionhas beenobservedin several cell typesof the mammalians,includingneuronalcellsafternitrosativestress.Both invivoandinvitrostudiesdemonstratedthatTRXandTRXrhave protectiverolesagainstcytotoxicitymediatedbythegenerationof ROS[59].Notably,inourstudywedemonstratethatthioredoxin reductaseisdecreasedinMSlymphocytes.Thismayberelevantto thepathogenesisofMS,aswehavepreviouslydemonstratedinCSF andplasmaofMSpatientsasignificantdecreaseofGSH/GSSGratio

[5,9].Consistentwiththisnotion,itiswellestablishedthatcellular redoxregulationofmanyprocessesisprovidedbythecooperation betweentheTrxandglutathionesystems[60].Infact,TrxandGSH systemsareinvolvedin avarietyofredox-dependentreactions, suchassupplyingreducingequivalentsforribonucleotide reduc-tase,andpeptidemethioninesulfoxidereductase,thelatterbeing involvedinantioxidantdefenseandregulationofthecellularredox state [59]. Therefore, Trx and GSH form a powerful system controllingredoxregulationofgeneexpression,signal transduc-tion,cell proliferation,protection against oxidative stress, anti-apoptoticfunctions,growthfactorandco-cytokineeffects,aswell asregulationoftheredoxstateoftheextracellularenvironment

[61,62].Inaddition,increasedTrx-1expressionhasbeendescribed inmanyhumanprimarycancersandtumorcelllines[63].Changes inMSredoxstatusobservedinthisstudywerereflectedalsobythe

interestingfindingthatSirtuin-1ispresentintheplasmaofMS patientsatsignificantlyhigherlevelscomparedtocontrolsubjects. Asfarasweareconcernedthisisthefirstevidenceofincreased Sirtuin-1 expressioninthe bloodof MSpatients. Sirtuins area familyofhistonedeacetylase, that,inhumans,includesatleast sevenmembers,withdifferentcellularandsubcellularlocalization andsubstratebindingactivity[15,61][15,64].Themoststudiedis theSIRT1,aNAD+dependentenzyme,involvedindeacetylationof differentproteinsandintheregulationofenergymetabolismand redoxstate.Sirtuinsareinvolved intheregulationofimportant biologicalprocesses,suchasapoptosis,celldifferentiation,energy transduction,glucosehomeostasis[18,65],aswellasinmediating enhancementofsynapticplasticityandneurogenesisinresponse to exercise, dietary energy restriction, and other hormetic environmental factors[15,66]. Becauseoftheir rolesin cellular stressresponses,sirtuinswouldbeexpectedtoplayparticularly importantrolesinadaptiveresponsesofneuralcellstostress,such as thatassociated withtheMS pathogenesis.Insupportofthis conceivablepossibility, SIRT1hasbeenshowntointeracteither directlyorindirectlywithseveralpathwaysknowntobeinvolved inadaptiveneuralplasticity,includingtheCa2+_{-calmodulin-cyclic}

adenosine monophosphate (AMP) response element binding proteinandNF-kBsystems[15].Thus,ourfindingmayberelevant totheMSpathogenesis,inviewofthefactthatSIRT1areemerging asa promisingcandidatetargetfortherapeuticinterventions in metabolicandneurodegenerativedisorders[67].

MS is a progressiveinflammatory neurodegenerative disease exhibitingavariableclinicalpresentationwhichhasno‘diagnostic’ laboratorytest;thisoftenresultsindelaystodefinitediagnosis.In confronting the disease, early diagnosis and appropriate,timely therapeuticinterventionarecriticalfactorsinensuringfavorable long-termoutcomes.Theavailabilityofreliablebiomarkerscould radicallyalterourmanagementofMSatcriticalphasesofthedisease spectrum.Identificationofmarkersthatcouldpredictthe develop-mentofMSinhigh-riskpopulationswouldallowforintervention strategiesthatmaypreventevolutiontodefinitedisease.Ongoing workwithbiomarkersthatreflectdrugbioavailabilityandfactors thatdistinguishbetweenmedicationrespondersand nonrespon-dersarealsounderinvestigation.Pharmacologicalmodulationof cellularstresspathwayshasemergingimplicationsinthetreatment of several human diseases [15]. Consistent with this notion, inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), has been recently reported to be associated with increased susceptibilitytoMS,suggestingapossiblerolefortheNQO1genetic backgroundinthe developmentofprimary progressiveMS[68]. Basedontheoverlappinginducibilityofthecytoprotectivephase2 responseandthevitagenenetwork,thereisnowprovenexplanation forsensingmechanismoftheinitialchemicalsignalthatultimately leadstoenhancedgeneexpression.Thus,modulationofendogenous cellular defense mechanisms via the stress response signaling representsaninnovativeapproachtotherapeuticinterventionin diseasescausingtissuedamage,suchasneurodegeneration.Thisis accomplished by a complex networkof the so-called longevity assuranceprocesses,whicharecomposedofseveralgenestermed vitagenes[15].Maintainingorrecoveringtheactivityofvitagenes throughnutritionalinterventionsorspecificallyactivedrugswill helpuncovernovelcluesforneuroprotectivetherapeuticstrategies withrelevanceforclinicalapplicationswellbeyondthefieldofMS alone.

Acknowledgments

Workfromtheauthors’laboratorieswassupportedbygrants fromMIUR, FIRB RBRN07BMCT,I.N.B.B.,and by ‘‘FondiAteneo’’ 2008and2009.

Table2

SummaryofdifferentstressresponseproteinsexpressedintheCSF,plasmaand lymphocytesofMSpatientsandcontrolsubjects.

MSpatients HO-1 HO-2 Hsp60 Hsc70 Hsp72 Trx TrxR-1 Sirt-1

Lymphocytes ++ ++ ++ ++

Plasma ++ ++ ++

CSF ++

CVwouldliketothankGiorgioFedericiforhelpfuldiscussions duringthegenesisofthisarticle.

References

[1]CompstonA,ColesA.Multiplesclerosis.Lancet2008;372:1502–17. [2]NesslerS,Bru¨ckW.Advancesinmultiplesclerosisresearchin2009.JNeurol

2010;257:1590–3.

[3]DerwenskusJ.Currentdisease-modifyingtreatmentofmultiplesclerosis.Mt SinaiJMed2011;78:161–75.

[4]TumaniH,HartungHP,HemmerB,TeunissenC,DeisenhammerF,Giovannoni. etal.Cerebrospinalfluid biomarkersin multiplesclerosis. NeurobiolDis 2009;35:117–27.

[5]CalabreseV,ScapagniniG,RavagnaA,BellaR,ForestiR,BatesTE,etal.Nitric oxidesynthaseispresentinthecerebrospinalfluidofpatientswithactive multiplesclerosisand isassociated with increasesin cerebrospinalfluid proteinnitrotyrosineandS-nitrosothiols andwithchangesinglutathione levels.JNeurosciRes2002;70:580–7.

[6]WitherickJ,WilkinsA,ScoldingN,KempK.Mechanismsofoxidativedamage inmultiplesclerosisandacelltherapyapproachtotreatment.Autoimmune Dis2010;2011:164608.

[7]ZiemannU,WahlM,HattingenE,TumaniH.Developmentofbiomarkersfor multiplesclerosisasaneurodegenerativedisorder.ProgNeurobiol2011[Epub April16].

[8]VanNoortJM,vandenElsenPJ,vanHorssenJ,GeurtsJJ,vanderValkP,AmorS. Preactivemultiple sclerosis lesionsoffer novel clues for neuroprotective therapeuticstrategies.CNSNeurolDisordDrugTargets2011;10:68–81. [9]CalabreseV,ScapagniniG,RavagnaA,BellaR,ButterfieldDA,CalvaniM,etal.

Disruptionofthiolhomeostasisandnitrosativestressinthecerebrospinal fluidofpatientswithactivemultiplesclerosis:evidenceforaprotectiveroleof acetylcarnitine.NeurochemRes2003;28:1321–8.

[10]MattssonN,YaongM,RosengrenL,BlennowK,Ma˚nssonJE,AndersenO,etal. ElevatedcerebrospinalfluidlevelsofprostaglandinE2and 15-(S)-hydroxyei-cosatetraenoicacidinmultiplesclerosis.JInternMed2009;265:459–64. [11]MattssonN,HaghighiS,AndersenO,YaoY,RosengrenL,BlennowK,etal.

ElevatedcerebrospinalfluidF2-isoprostanelevelsindicatingoxidativestress inhealthysiblingsofmultiplesclerosispatients.NeurosciLett2007;414: 233–6.

[12]CalabreseV,BellaR,TestaD,SpadaroF,ScrofaniA,RizzaV,etal.Increased cerebrospinalfluidandplasmalevelsofultraweakchemiluminescenceare associatedwithchangesinthethiolpoolandlipid-solublefluorescencein multiplesclerosis:thepathogenicroleofoxidativestress.DrugsExpClinRes 1998;24:125–31.

[13]CalabreseV,CorneliusC,Dinkova-KostovaAT,CalabreseEJ.Vitagenes,cellular stress response and acetylcarnitine: relevance to hormesis. Biofactors 2009;35:146–60.

[14]CalabreseV,CorneliusC,RizzarelliE,OwenJB,Dinkova-KostovaAT, Butter-fieldDA.Nitricoxideincellsurvival:aJanusmolecule.AntioxidRedoxSignal 2009;11:2717–39.

[15]CalabreseV,Cornelius C,Dinkova-KostovaAT, CalabreseEJ,MattsonMP. Cellularstressresponses,thehormesisparadigm,andvitagenes:noveltargets fortherapeuticinterventioninneurodegenerativedisorders.AntioxidRedox Signal2010;13:1763–811.

[16]CalabreseV,CorneliusC,StellaAM,CalabreseEJ.Cellularstressresponses, mitostressandcarnitineinsufficienciesascriticaldeterminantsinagingand neurodegenerativedisorders:roleofhormesisandvitagenes.NeurochemRes 2010;35:1880–915.

[17]CalabreseV,MancusoC,CalvaniM,RizzarelliE,ButterfieldDA,GiuffridaStella AM.NitricoxideintheCNS:neuroprotectionversusneurotoxicity.Nat Neu-rosci2007;8:766–75.

[18]CalabreseV,CorneliusC,MancusoC,PennisiG,CalafatoS,BelliaF,etal. Cellularstressresponse:anoveltargetforchemopreventionandnutritional neuroprotectioninaging,neurodegenerativedisordersandlongevity. Neu-rochemRes2008;33:2444–71.

[19]McDonaldWI,CompstonA,EdanG,GoodkinD,HartungHP,LublinFD,etal. Recommendeddiagnosticcriteriaformultiplesclerosis:guidelinesfromthe International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50(1):121–7.

[20]LublinFD,ReingoldSC.Definingtheclinicalcourseofmultiplesclerosis: resultsofaninternationalsurveyNationalMultipleSclerosisSociety(USA) AdvisorCommitteeonClinicalTrialsofNewAgentsinMultipleSclerosis. Neurology1996;46:907–11.

[21]SmithP,KrohnR,HermansonG,MalliaA,GartnerF,ProvenzanoM,etal. Measurementofproteinsusingbicinchoninicacid.AnnBiochem1985;150: 76–85.

[22]CalabreseV,GuaglianoE,SapienzaM,PanebiancoM,CalafatoS,PuleoE,etal. Redoxregulationofcellularstressresponseinagingandneurodegenerative disorders:roleofvitagenes.NeurochemRes2007;32:757–73.

[23]CalabreseV,Boyd-KimballD,ScapagniniG,ButterfieldDA.Nitricoxideand cellularstressresponseinbrainagingandneurodegenerativedisorders:the roleofvitagenes.InVivo2004;18:245–67.

[24]MancusoC,ScapagniniG,CurroD,GiuffridaStellaAM,DeMarcoC,Butterfield DA,etal.Mitochondrialdysfunction,freeradicalgenerationandcellularstress responseinneurodegenerativedisorders.FrontBiosci2007;12:1107–23.

[25]ButterfieldDA,StadtmanER.Proteinoxidationprocessesinagingbrain.Adv CellAgingGerontol1997;2:161–91.

[26]LauderbackCM,HackettJM,HuangFF,KellerJN,SzwedaLI,MarkesberyWR, etal.Theglialglutamatetransporter,GLT-1,isoxidativelymodifiedby 4-hydroxy-2-nonenalintheAlzheimer’sdiseasebrain:roleofA-beta1-42.J Neurochem2001;78:413–6.

[27]ButterfieldDA,HowardBJ,YatinS,AllenKL,CarneyJM.Freeradicaloxidation ofbrainproteinsinacceleratedsenescenceanditsmodulationby N-tert-butyl-phenylnitrone.ProcNatlAcadSciUSA1997;94:674–8.

[28]TudorC,Lerner-MarmaroshN,EngelborghsY,GibbsPE,MainesMD.Biliverdin reductaseis a transporterofhaemintothe nucleusandis essential for regulationofHO-1geneexpressionbyhaematin.BiochemJ2008;413:405–16. [29]Yoneyama-SarneckyT,OlivasAD,AzariS,FerrieroDM,ManvelyanHM, Noble-HaeussleinLJ.Hemeoxygenase-2modulatesearlypathogenesisafter trau-maticinjurytotheimmaturebrain.DevNeurosci2010;32:81–90. [30]MaoP,ReddyPH.Ismultiplesclerosisa mitochondrialdisease?Biochim

BiophysActa2010;1802:66–79.

[31]FrohmanEM,RackeMK,RaineCS.Multiplesclerosis—theplaqueandits pathogenesis.NEnglJMed2006;354:942–55.

[32]Pokryszko-DraganA,GruszkaE,Bilins´kaM.Secondaryprogressivemultiple sclerosis-clinicalcourseandpotentialpredictivefactors.NeurolNeurochirPol 2008;42:6–11.

[33]GonsetteRE.Neurodegenerationinmultiplesclerosis:theroleofoxidative stressandexcitotoxicity.JNeurolSci2008;274:48–53.

[34]TurturiciG,SconzoG,GeraciF.Hsp70anditsmolecularroleinnervoussystem diseases.BiochemResInt2011;2011:618127.

[35]GonsetteRE.Oxidativestressandexcitotoxicity:atherapeuticissuein multi-plesclerosis?MultScler2008;14:22–34.

[36]LindseyJW,AgarwalSK,TanFK.Geneexpressionchangesinmultiplesclerosis relapsesuggestactivationofTandnon-Tcells.MolMed2011;17:95–102. [37]GonsetteRE.Endogenousneuroprotectioninmultiplesclerosis.ActaNeurol

Belg2010;110:26–35.

[38]CalabreseV,RaffaeleR,CosentinoE,RizzaV.Changesincerebrospinalfluid levelsofmalondialdehydeandglutathionereductase activityin multiple sclerosis.IntJClinPharmacolRes1994;14:119–23.

[39]MillerE,MrowickaM,Saluk-JuszczakJ,IreneuszM.Thelevelofisoprostanes asanon-invasivemarkerforinvivolipidperoxidationinsecondary progres-sivemultiplesclerosis.NeurochemRes2011;36(6):1012–6.

[40]ChoiIY,LeeSP,DenneyDR,LynchSG.Lowerlevelsofglutathioneinthebrains ofsecondaryprogressivemultiplesclerosispatientsmeasuredby1Hmagnetic resonancechemicalshiftimagingat3T.MultScler2011;17:289–96. [41]LiuY,LiuJ,TetzlaffW,PatyDW,CynaderMS.Biliverdinreductase,amajor

physiologiccytoprotectant,suppressesexperimentalautoimmune encepha-lomyelitis.FreeRadicBiolMed2006;40:960–7.

[42]LeungG,SunW,ZhengL,BrookesS,TullyM,ShiR.Anti-acroleintreatment improvesbehavioraloutcomeandalleviatesmyelindamageinexperimental autoimmuneencephalomyelitismouse.Neuroscience2011;173:150–5. [43]EspositoE,CuzzocreaS.Antiinflammatoryactivityofmelatoninincentral

nervoussystem.CurrNeuropharmacol2010;8(September(3)):228–42. [44]HultqvistM,OlofssonP,HolmbergJ,BackstromBT,TordssonJ,HolmdahlR.

Enhancedautoimmunity,arthritis, andencephalomyelitisin micewitha reducedoxidativeburstduetoamutationintheNcf1gene.ProcNatlAcad SciUSA2004;101:12646–51.

[45]DelloRussoC,PolakPE,MercadoPR,SpagnoloA,SharpA,MurphyP,etal.The heat-shockprotein90inhibitor17-allylamino-17-demethoxygeldanamycin suppressesglialinflammatoryresponsesandamelioratesexperimental auto-immuneencephalomyelitis.JNeurochem2006;99:1351–62.

[46]SahaRN,PahanK.Regulationofinduciblenitricoxidesynthasegeneinglial cells.AntioxidRedoxSignal2006;8:929–47.

[47]Bolan˜ osJP,HealesSJ.Persistentmitochondrialdamagebynitricoxideandits derivatives:neuropathologicalimplications.FrontNeuroenergetics2010;2 (February):1.

[48]GibbsPE,MainesMD.BiliverdininhibitsactivationofNF-kappaB:reversalof inhibitionbyhumanbiliverdinreductase.IntJCancer2007;121:2567–74. [49]MainesMD.Thehemeoxygenasesystem:past,present,andfuture.Antioxid

RedoxSignal2004;6:797–801.

[50]CalabreseV,ButterfieldDA, ScapagniniG, Stella AM,MainesMD.Redox regulationofheatshockproteinexpressionbysignalinginvolvingnitricoxide andcarbonmonoxide:relevancetobrainaging,neurodegenerativedisorders, andlongevity.AntioxidRedoxSignal2006;8:444–77.

[51]MotterliniR,ForestiR,BassiR,CalabreseV,ClarkJE,GreenCJ.Endothelial hemeoxygenase-1inductionbyhypoxia.Modulationbyinducible nitric-oxidesynthaseandS-nitrosothiols.JBiolChem2000;275:13613–20. [52]CalabreseV,ButterfieldDA,StellaAM.Agingandoxidativestressresponsein

theCNS. In:AbelL,ReginoP-P,SteffenR,editors.DevelopmentandAging ChangesintheNervousSystem.HandbookofNeurochemistryandMolecular Neurobiology.2008.p.128–234.

[53]CalabreseV,ScapagniniG,ColombritaC,RavagnaA,PennisiG,GiuffridaStella AM,etal.Redoxregulationofheatshockproteinexpressioninagingand neurodegenerativedisordersassociatedwithoxidativestress:anutritional approach.AminoAcids2003;25:437–44.

[54]CidC,AlcazarA.Protectionofoligodendrocyteprecursorcellsbylowdosesof HSP90inhibitorsincellculture.ExpNeurol2010;225:29–33.

[55]Mha´illeAN,McQuaidS,WindebankA,CunneaP,McMahonJ,SamaliA,etal. Increasedexpressionofendoplasmicreticulumstress-relatedsignalingpathway moleculesinmultiplesclerosislesions.JNeuropatholExpNeurol2008;67:200–11.

[56]StahnkeT,StadelmannC,NetzlerA,Bru¨ckW,Richter-LandsbergC. Differ-ential upregulation of heme oxygenase-1 (HSP32) in glial cells after oxidativestressandindemyelinatingdisorders.JMolNeurosci2007;32: 25–37.

[57]BenderT,LewrenzI,FrankenS,BaitzelC,VoosW.Mitochondrialenzymesare protectedfromstress-inducedaggregationbymitochondrialchaperonesand thePim1/LONprotease.MolBiolCell2011;22(March(5)):541–54. [58]MagenD,GeorgopoulosC,BrossP,AngD,SegevY,GoldsherD,etal.

Mito-chondrialhsp60chaperonopathycausesanautosomal-recessive neurodegen-erativedisorderlinkedtobrainhypomyelinationandleukodystrophy.AmJ HumGenet2008;83:30–42.

[59]CalabreseV,CorneliusC,MancusoC,BaroneE,CalafatoS,BatesT,etal. Vitagenes,dietaryantioxidantsandneuroprotectioninneurodegenerative diseases.FrontBiosci2009;14:376–97.

[60]MandalPK,SeilerA,PerisicT,Ko¨lleP,BanjacCanakA,Fo¨rsterH,etal.System x(c)-andthioredoxinreductase1cooperativelyrescueglutathionedeficiency. JBiolChem2010;285:22244–53.

[61]LowesDA,GalleyHF.Mitochondrialprotectionbythethioredoxin-2and glutathionesystemsinaninvitroendothelialmodelofsepsis.BiochemJ 2011;436:123–32.

[62]BellKF,HardinghamGE.CNSperoxiredoxinsandtheirregulationinhealthand disease.AntioxidRedoxSignal2011;14:1467–77.

[63]QuY,WangJ,RayPS,GuoH,HuangJ,Shin-SimM,etal.Thioredoxin-like2 regulateshumancancercellgrowthandmetastasisviaredoxhomeostasisand NF-kBsignaling.JClinInvest2011;121:212–25.

[64]Dali-Youcef N,LagougeM,FroelichS, Koehl C, SchoonjansK,AuwerxJ. Sirtuins:the‘magnificentseven’,function,metabolismandlongevity.Ann Med2007;39:335–45.

[65]YiJ,LuoJ.SIRT1andp53,effectoncancer,senescenceandbeyond.Biochim BiophysActa2010;1804:1684–9.

[66]MinSW,ChoSH,ZhouY,SchroederS,HaroutunianV,SeeleyWW,etal. Acetylation oftauinhibitsits degradationandcontributes to tauopathy. Neuron2010;67:953–66.

[67]HerranzD,SerranoM.SIRT1:recentlessonsfrommousemodels.NatRev Cancer2010;10:819–23.

[68]StavropoulouC,ZachakiS,AlexoudiA,ChatziI,GeorgakakosVN,TerzoudiGI, etal.TheC(609)TinbornpolymorphisminNAD(P)H:quinoneoxidoreductase 1isassociatedwithsusceptibilitytomultiplesclerosisandaffectstheriskof developmentoftheprimaryprogressiveformofthedisease.FreeRadicBiol Med2011;51:713–8.