Leigh’s syndromeZassociated neurodegeneration: NDUFS4Z mutations and oxidative phosphorylation dysfunction

Literature(Review(

(

(

T.A.O.(Muntslag(

MSc(Brain(and(Cognitive(Sciences(

University(of(Amsterdam(

UvA(ID#(10436928(

(

September(18

th

,(2015(

10(EC(

July(–(September(2015(

(

(

Supervisor:(Werner(J.H.(Koopman,(Ph.D.;(Radboud(University(Nijmegen,(RIMLS(

CoZassessor:(Lars(P.(van(der(Heide,(Ph.D.;(University(of(Amsterdam,(SILS(

(

Leigh’s(syndromeZassociated(neurodegeneration:(NDUFS4Zmutations(and(

oxidative(phosphorylation(dysfunction(

(

Abstract(

(

Leigh’s(syndrome((LS)(is(an(infantile(fatal(neurodegenerative(disorder(of(a(clinical(and( genetic(heterogeneous(nature(associated(with(oxidative(phosphorylation((OXPHOS)( dysfunction.(The(NADH(Dehydrogenase((Ubiquinone)(FeZS(Protein(4((NDUFS4)Zsubunit(is(of( particular(importance(to(the(assembly(and(activity(of(Complex(I((CI),(the(largest(and(most( complex(of(the(OXPHOS(system.(Characteristics(of(NDUFS4Zmutations(include(failure(to(thrive( (FTT),(psychomotor(retardation((PMR),(hypotonia,(cardiomyopathy((CM),(accumulation(of( lactate((LA)(and(death(by(respiratory(failure((RF),(often(within(the(first(year((~9(months).( Lesions(to(brain(stem(and(basal(ganglia(are(neuropathological(hallmarks,(central(to(disease( progression(and(death(by(RF.(Clinical(reports(of(LS(feature(premature(termination(codons( (PTCs),(an(absence(of(protein(and(deficient(CI.(CI(has(a(key(regulatory(role(of(cellular( metabolism(by(promoting(the(tricarboxylic(acid((TCA)Zcycle(and( Zoxidation,(a(capacity( lacking(with(NDUFS4Zmutations.(The(consequential(WarburgZlike(metabolic(state(adaptation( results(in(a(reduction(of(metabolic(dynamics,(postulated(to(result(in(nutrient(starvation(and( necrosis.(Several(animal(models(have(helped(delineate(the(cellZ(and(tissueZspecific(effects(of( NDUFS4ZKO,(enabling(a(more(detailed(analysis(of(the(molecular(mechanisms(involved(and( putative(therapeutic(interventions.(A(novel(mechanistic(coupling(is(proposed(that(consists(of( an(intrinsic(failure(of(TCAZcycle(and Zoxidation(activity,(gliosis(and(neuronal(atrophy.( Furthermore,(the(use(of(NDUFS4ZKO(mice(is(assessed(in(models(of(Parkinson’s(disease((PD),( providing(a(window(into(the(role(of(CIZdeficiency(in(ageZassociated(disease.(

(

(

2(

Table(of(contents(

(

Introduction……….

3

2

2

Clinical2genetics2of2NDUFS4:associated2CI:deficiency……….………

3

2

(

Animal(models……….….…….6( ( Table(1:(Clinical,(metabolic(and(neuropathological(features(of(NDUFS4Zpatients………….………..7( Figure(1:(NDUFS4Ztranscript……….….…....8( (

Cellular2metabolic2adaptions2with2NDUFS4:mutations

………11Z152 Glycolysis,(TCAZcycle(and( Zoxidation………..………..……….……..…11( Figure(2:(The(dynamic(interplay(of(glycolysis,( Zoxidation,(TCAZcycle(and(OXPHOS.…….…...12( The(OXPHOS(system……….……..12( Figure(3:(CI(assembly……….……….13( Neuropathology……….………14( (

Molecular2mechanisms2and2therapeutic2interventions2of2LS

………..15Z17( ( Figure(4:(Mechanistic(model(of(neuronal(necrosis(with(NDUFS4Zmutations…….………182 (

NDUFS4:mutations2in2PD:models

……….………..17Z20( (

Concluding2remarks2and2future2perspectives

………20( (

References……….………..

21Z27(

(

3(

Introduction((

( Oxidative(phosphorylation(is(key(to(cellular(viability,(providing(the(cell(with(freely( available(ATP(to(maintain(many(of(its(functions.(Mutations(in(the(OXPHOS(system(genes(can( result(in(mitochondrial(dysfunction,(an(ensuing(reduction(in(ATP(and(apoptosis(induction( (Seppet,(Gellerich(et2al.(2009;(Koopman,(Smeitink(et2al.(2012).(OXPHOS(mutations(cause( monogenic(mitochondrial(disorders(including(Leigh’s(syndrome((Koopman,(Smeitink(et2al.( 2012).(Leigh’s(syndrome((LS)((OMIM#:(256000)(is(a(infantile(neurodegenerative(disorder(of( clinical(and(genetic(heterogenous(nature(that(is(characterized(by(breathing(difficulties,( growth(retardation,(PMR,(FTT,(muscular(hypotonia,(CM,(encephalomyopathy,(lethargy,(and( visual(impairment((Van(den(Heuvel,(Smeitink(et2al.(1998;(Budde,(Smeitink(et2al.(2003;(Iuso,( Papa(et2al.(2006;(Assouline,(Lebre(et2al.(2012).(( ( Neuropathological(features(of(LS(include(spongiform(necrotic(lesions(in(brainstem,( basal(ganglia(and(cerebellum,(although(also(damage(to(the(spinal(cord,(optic(nerve(and( thalamus(is(reported((Lee,(Chen(et2al.(2009;(Koene,(Smeitink(et2al.(2012).(Often( neuropathology(is(concurrent(with(gliosis,(oxidative(stress,(vascular(hypertrophy,(and(changes( in(metabolite(content(in(blood(and(cerebrospinal(fluid((CSF;(e.g.(lactate,(glycolysis( intermediates)((Lake,(Paetau(et2al.(2015).(It(is(assumed(that(the(affected(brain(areas(underlie( subsequent(dysfunction(and(clinical(symptoms,(disease(progression(and(eventual(death.(LS( typically(results(in(death(at(~2.4(years,(with(an(onset(between(the(3rd_(and(12th_{(months(after(} birth,(and(has(been(linked(to(mutations(of(both(mitochondrial(and(nuclear(genomes((Ruhoy( and(Saneto,(2014;(Thornburn,(Stephens(et2al.(2014).(NADH(Dehydrogenase((Ubiquinone)(FeZS( Protein(4((NDUFS4)Zassociated(LS(shows(a(relatively(rapid(disease(progression(with(an( average(life(span(of(~9(months((Assouline,(Lebre(et2al.(2012),(which(make(NDUFS4Zmutations( societally(and(scientifically(of(particular(importance(for(the(prevention(of(loss(of(life(and(the( study(of(OXPHOS(deficiency(in(pathology.(( ( Since(the(first(case(report(by(Denis(A.(Leigh(in(1951((Leigh,(1951),(several(studies(have( elucidated(the(genetic(aetiology(of(LS.(In(this(review,(the(most(recent(findings(concerning(LSZ associated(NDUFS4Zmutations(are(discussed,(followed(by(a(report(of(the(cellular(metabolic( adaptions,(and(the(molecular(mechanisms(and(therapeutic(intervention(strategies(of( pathology.(Lastly,(we(report(on(studies(in(PD,(which(describe(the(contribution(of(NDUSF4ZKO2 models(in(CIZassociated(neurodegeneration.( 2

4(

Clinical(genetics(of(NDUFS4Zassociated(CIZdeficiency

(

2 The(NDUFS4(gene(is(located(on(chromosome(5q11(and(consists(of(676(base(pairs((bp)( spanning(over(5(exons,(forming(a(mature(protein(of(133(amino(acids(with(a(weight(of(18kD( (NCBI,(Nucleotide(GenBank,(2015).(Dispersed(throughout(its(amino(acid(sequence(are(several( conserved(sequences(associated(with(peptide(phosphorylation(and(assembly(characteristics( (Fig.(1)(Budde,(Smeitink(et2al.(2003;(Scacco,(Papa(et2al.(2003).(The(phosphorylation(sites(are( located(in(the(carboxy(terminus(and(the(‘leader(sequence’(of(the(NDUFS4Ztranscript(–the( leader(sequence(encompasses(the(mitochondrial(targeting(sequence(in(exon(1(and(2((Walker,( 1992;(Papa,(Smeitink(et2al.(2001;(Bénit,(Rötig(et2al.(2003).(Mitochondrial(directed(transport(of( NDUFS4(is(mediated(by(the(mitochondrial(targeting(sequence(and(the(ATPZdependent( mitochondrial(outer(membrane((TOM)Zinner(membrane((TIM)(system((Wiedemann,(Pfanner( et2al.(2004;(De(Rasmo,(Papa(et2al.(2008).(The(mitochondrial(membrane(potential((Ψ)( functions(to(activate(the(TOMZTIM(system(and(drives(the(positively(charged(targeting( sequence(to(the(mitochondrial(matrix;(the(PMF(is(determined(by(the(Ψ(and(the(pH(difference( over(the(MIM([Δp(=(ΔΨ(+(ΔpH,(PMF]((Fig.2)(Yadava,(Kim(et2al.(2013).(Although(the(Ψ(targets( NDUFS4(for(mitochondria,(it(does(not(enable(the(peptide(to(cross(the(outer(membrane,( preventing(assembly(into(CI.(It(was(found(that(cyclic(adenosine(monophosphate((cAMP)Z dependent(protein(kinase(A((PKA)Zmediated(phosphorylation(of(the(carboxy(terminal( phosphorylation(site((arginineZvalineZserine,(RVS,(amino(acid(positions(129Z133(of(the(mature( peptide)(stimulated(peptide(translocation,(maturation(and(incorporation(into(CI((De(Rasmo,( Papa(et2al.(2008).(In(contrast,(cytosolic(Mg2+_{Zactivated(serine/threonine(PP2Cγ(type(} phosphatases(antagonise(the(effect(by(PKA((Papa,(2002;(De(Rasmo,(Papa(et2al.(2008).( Consequently,(the(balance(of(phosphorylation/dephosphorylation(regulates(NDUFS4( presence(and(CIZfunctionality((De(Rasmo,(Papa(et2al.(2010).(( ( NDUFS4Zmutations(are(associated(with(PTCs,(an(absence(of(protein(and(an(impaired( CIZactivity.(These(features(are(concurrent(with(brain(stem(and(basal(ganglia(neuropathology( and(several(key(clinical(symptoms:(hypotonia,(PMR,(absence(of(eye(contact,(abnormal(vision,( bradypnea,(feeding(problems,(death(by(RF(and(lactate(increases(in(plasma(and(cerebral(spinal( fluid((CSF)((Van(den(Heuvel,(Smeitink(et2al.(1998;(Budde,(Smeitink(et2al.(2000;(Petruzzella,( Papa(et2al.(2001;(Bénit,(Rötig(et2al.(2003;(Budde,(Smeitink(et2al.(2003;(Anderson,(Rubin(et2al.( 2008;(LeshinskyZSilver,(LermanZSagie(et2al.(2009;(Calvo,(Mootha(et(al(2010;(Assouline,(Lebre( et2al.(2012;(Assereto,(Gazzero(et2al.(2014).(The(first(case(report(of(NDUFS4Zassociated(LS(was( by(Van(den(Heuvel,(Smeitink(et2al.((1998).(The(patient(featured(homozygous(5Zbase(pair( duplication(at(nucleotide(position(466Z470((c.466_470dupAAGTC),(resulting(in(a(frameshift( that(was(expected(to(increase(the(length(by(14(amino(acids(and(removed(the(RVSZsequence( (Van(den(Heuvel,(Smeitink(et2al.(1998).(Although(most(key(features(are(shared(with(the(other( NDUFS4Zmutations,(the(patient(did(not(exhibit(an(increase(in(lactate((Table(1).(A(study(of(an( Ashkenazi(Jewish((AJ)(family(did(describe(a(CZterminal(frameshift(associated(with(increased( lactate(levels((Table(1)(Anderson,(Rubin(et2al.(2008).(In(three(siblings(homozygous(alanine( deletion(at(nucleotide(462((c.462delA)(produced(an(alternative(transcript(for(the(peptide,( replacing(the(last(22(amino(acids(for(34(new(amino(acids(and(removal(of(the(RVSZsequence.( Remarkably,(the(same(c.462delAZmutation(in(a(consanguineous(family(did(not(correspond( with(an(increase(in(lactate(of(both(patients((Table(1)(Assereto,(Gazzerro(et2al.(2014).(This( highlights(the(heterogeneous(nature(of(LS(and(implies(a(complexity(to(pathology(that(extends( beyond(the(genetic(mutation.(( (

5( The(leader(sequence(is(important(for(mitochondrial(targeting;(however,(it(also( contains(a(second(phosphorylation(site((arginineZthreonineZserine,(RTS)(and(a(cleavage(site( for(peptide(maturation((Walker,(1992;(Bénit,(Rötig(et2al.(2003).(Petruzzella,(Papa(et2al.((2001)( showed(a(transition(mutation(in(the(leader(sequence((c.44G>A)(that(changed(a(tryptophan( codon(into(a(stop(codon((TGA),(thereby(introducing(a(premature(termination(codon((PTC)(and( resulting(in(a(transcript(lacking(both(phosphorylation(sites,(absence(of(protein,(and(an( impaired(assembly(and(activity(of(CI((Table(1)((Petruzzella,(Papa(et2al.(2001).(Similarly,(an( homozygous(mutation(in(intron(1((c.99Z1G>A)(introduced(a(PTC(after(39(amino(acids(of(the( predicted(protein,(resulting(in(a(transcript(with(a(modified(mitochondrial(targeting(sequence( that(skipped(exon(2(and(lacked(the(cleavage(site(for(protein(maturation((Bénit,(Rötig(et2al.( 2003;(Assouline,(Lebre(et2al.(2012).(The(case(report(of(the(c.44G>A(has(been(one(the(most( detrimental,(expressing(most(key(clinical(features(with(additional(vomiting,(seizures(and( cardiac(hypertrophy;(however,(it(does(not(correlate(to(the(shortest(lifespan((7(months),(as( others(report(ages(of(death(at(4,(4.5(and(even(3(months((Table(1)(Budde,(Smeitink(et2al.(2000;( Petruzzella,(Papa(et(al(2001;(Bénit,(Rötig(et2al.(2003;(Calvo,(Mootha(et2al.(2010).(Hence,( clinical(features(are(not(always(a(good(predictor(of(underlying(pathology,(further(suggesting( neuropathology(as(the(main(link(to(disease(progression.( ( Mutations(of(NDUFS4(are(expected(to(cause(the(generation(of(dysfunctional( transcripts((Assereto,(Gazzerro(et2al.(2014).(However,(in(most(clinical(studies(a(complete( absence(of(NDUFS4(peptide(was(reported,(suggestive(of(nonsenseZmediated(decay((NMD)(Z NMD(is(a(cellular(mRNA(quality(control(pathway,(which(prevents(the(translation(of(PTCZ containing(transcripts((Kervestin(and(Jacobson,(2012).(NMD(is(initiated(by(the(presence(of(a( PTC.(Specifically,(association(of(the(3’(polyZAA(tail(of(mRNA(with(poly(A)(binding(protein( (PABP)(inhibits(serine/threonineZprotein(kinase(SMGZ1Zmediated(phosphorylation(of(Upf1( (Brooks,(2010).(A(PTC(prevents(a(proper(association(between(PABP(and(Upf1,(resulting(in(the( phosphorylation(of(Upf1(and(the(recruitment(of(the(proteasome(capable(of(degrading(the( truncated(transcript.(However,(NMD(initiation(is(only(possible(when(an(intron(is(present(and( the(‘PTC(is(located(more(than(50Z55(nucleotides(upstream(of(the(last(exonZexon(junction( within(the(mRNA’((Zhang,(Maquat(et2al.(1998;(Carrier,(Eschenhagen(et2al.(2010).(However,( neither(requirement(was(fulfilled(with(c.44G>A(and(c.99Z1G>A(Zmutations.(As(expected,(both( mutations(were(associated(with(an(accumulation(of(the(canonical(and(splice(variants( (Petruzzella,(Papa(et2al.(2005;(Panelli,(Papa(et2al.(2008).(The(absence(of(protein(suggests(that( macroautophagyZassociated(membranes(might(capture(defective(NDUFS4(peptides(for( lysosomal(enzymatic(protein(breakdown,(preventing(detrimental(protein(aggregates( (Rubinsztein,(2003).!( (

6( The(middleZsegment(of(the(NDUFS4:transcript(is(not(associated(with(either( phosphorylation(site(or(the(leader(sequence((Fig.(1);(however,(the(introduction(of(a(PTC(and( absence(of(NDUFS4(peptide(were(causal(to(LS(pathology((Budde,(Smeitink(et2al.(2000,( Assouline,(Lebre(et2al.(2012).(Three(different(homozygous(mutations(are(reported(in( literature:((1)(guanine(deletion(at(nucleotide(position(290(truncating(the(expected(peptide(at( amino(acid(96((c.290delG);((2)(deletion(of(nucleotide(178Z528((c.178_528del)(associated(with( the(loss(of(exons(3(to(5;(and((3)(a(mutation(at(position(316,(changing(arginine((CGA)(to(a(stop( codon(and(truncating(it(at(the(expected(amino(acid(position(106((c.316C>T).(All(mutations( showed(increased(lactate(levels(and(basal(ganglia(lesions,(whereas(age(of(death(ranged(from( 3(to(8(months(of(age((Table(1)(Budde,(Smeitink(et2al.(2000,(Budde,(Smeitink(et2al.(2003;( Assouline,(Lebre(et2al.(2012).(A(second(patient(with(a(c.316C>T(mutation(showed(similar( clinical(features(and(neuropathology(to(the(first(case(report((Table(1)(Budde,(Smeitink(et2al.( 2000;(Budde,(Smeitink(et2al.(2003).(Although(in(both(cases(CM(contributes(to(clinical( outcome,(the(overall(pathology(of(NDUFS4Zassociated(LS(is(aggravated(with(the(additional( presence(of(FTT.(This(is(reflected(by(the(ageZofZdeath((AOD),(3(and(8(months,(respectively,( and(might(reflect(the(extend(of(neuronal(atrophy.(( ( Most(cases(of(LS(are(autosomal(homozygous(recessive(mutations;(however,( heterozygous(mutations(have(also(been(described((LeshinskyZSilver,(LermanZSagie(et2al.(2009;( Calvo,(Mootha(et2al.(2010;(Assouline,(Lebre(et2al.(2012).(So(far(4(combinations(of(double( heterozygous(mutations(have(been(reported:(c.99Z1G>A/c.351Z2A>G,(c.99Z1G>A/c.462delA,( c.355G>C/c.462delA(and(c.291delG/c472_476dupAAGTC.(Both(c.351Z2A>G(and(c.99Z1G>A( mutations(alter(NDUFS4(splicing(and(are(detrimental(to(transcript(stability((Calvo,(Mootha(et2 al.(2010).(Furthermore,(in(comparison(to(the(homozygous(c.462(mutation,(age(of(death(was( delayed(with(c.355G>C/c.462(and(c.291delG/c472_476dupAAGTCZmutations((27.5(and(24( months),(whereas(c.99Z1G>A/c.462(resulted(in(a(disease(duration(similar(to(homozygous( mutations((Table(1).(Increased(survival(might(reflect(chromosomal(recombination.(This( suggests(that(c.99Z1G>A(mutations,(and(the(associated(failure(in(NMD,(are(particularly( detrimental(to(cellular(health((Table(1).(Although(single(heterozygous(mutations(might( predispose(the(carrier(to(some(defects;(however,(presence(of(functional(NDUFS4(does(not( properly(reflect(the(clinical(features(associated(with(LS.( ( Taken(together,(literature(has(described(several(different(mutations(that(all(result(in( a(loss(of(NDUFS4,(CIZdeficiency(and(LS;(suggesting(that(NDUFS4(is(particularly(susceptible(to( deleterious(mutations(and(that(it(can(be(regarded(as(a(‘mutation(hotspot’((Fig.(1)((Petruzzella,( Papa,(2002;(Tucker,(Thorburn(et2al.(2011).(( (

Animal2models2

The(mitochondrial(leader(sequence(and(the(carboxy(RVSZsequence(are(two(key( NDUFS4(conserved(phosphorylation(sites(throughout(mammalian(species((Papa,(Signorile(et2 al.(2002).(Although(the(leader(sequence(site(shows(some(variation,(over(the(whole(peptide( there(is(an(expected(sequence(similarity(around(90%(in(mammals((e.g.(Bos2Taurus,2Mus2 musculus).(Comparisons(of(the(sequence(outside(the(mammalian(phylogenic(tree(results(in(a( quick(reduction(of(homology,(dropping(to(45%(in(Caenorhabditis2elegans2and(20%(in( Drosophila2melanogaster.(Because(of(the(genetic(homology(and(the(extensive(transgenic( opportunities(several(mouse(models(have(been(described(in(the(study(of(NDUFS4ZKO.(The( most(influential(models(are(discussed(in(relation(to(their(translatability(to(LS(pathology.2 (

Mutation

Reference

Gender AOO AOD

FTT

PMR

HT

RF

LA

BS

BG

c.44G>A c.99-1G>A c178_529del c.466_470dupAAGTC c.462delA c.290delG c.291delG c.291delG/c.472_476dupAAGTC c.99-1G>A/c.351_2A>G c.99-1G>A/c.462delA Petruzella et al.2001 Bénit et al. 2003 Assouline et al. 2012 Budde et al. 2000 Assouline et al. 2014 Budde et al. 2000 Anderson et al. 2008 Assereto et al. 2014 Van den Heuvel et al. 1998

Calvo et al. 2010 Assouline et al. 2014

F

F

F

F

F

F

F

F

M

M

M

M

M

M

M

N.A.

N.A.

Budde et al. 2003

M

c.316C>T

F

2 wks

7

4

N.A.

N.A.

4.5

3.5

5 dys

3

10 wks

4

1

3

7 wks

6

3

6

3

4

8

3.5

10

5

6

6

10

4

9

5

N.A.

8

16

3

N.A.

4

4.5

8

27.5

Y

N.A.

N.A.

N.A.

Y

N.A.

Y

N.A.

Y

N

Y

N.A.

Y

Y

N.A.

Y

N.A.

N.A.

N.A.

Y

Y

N.A.

Y

Y

Y

Y

Y

Y

Y

N.A.

N.A.

Y

Y

Y

Y

Y

Y

Y

Y

Y

N.A.

Y

Y

Y

Y

N.A.

Y

Y

Y

N.A.

N.A.

Y

N.A.

Y

N.A.

N.A.

Y

+

+

+

+

+

+

+

+

+

+

+

N.A.

+

c

+

c

+

+

N.A.

N.A.

Y

Y

Y

Y

Y

N

N.A.

Y

Y

Y

Y

N.A.

Y

Y

Y

N.A.

N.A.

Y

Y

N

Y

Y

Y

Y

Y

N.A.

Y

Y

Y

Y

N.A.

Y

Y

Y

N.A.

N.A.

Y

Y

N.A.

Y

N.A.

Y

N.A.

N

Y

N

N

N.A.

N.A.

N.A.

N.A.

N.A.

Y

N.A.

Y

Y

C.355G>C/c.462delA Lehinsky-Silver et al. 2009

M

22

24

N.A.

Y

Y

Y

N.A.

Y

N

Table 1; Clinical, metabolic and neuropathological features of NDUFS4-patients. All 12 mutations currently know to the field are depicted

in the table, supplemented with the reference of their first description. AOO and AOD are depicted in months, unless otherwise specificed.

AOO, age-of-onset; AOD, age-of-death; BG, basal ganglia; BS, brain stem; c, constant/unchanged; CM, cardiomyopathy; dys, days; F, female;

FTT, failure to thrive; HT, hypotonia; LA, lactate; N.A., not available; N, no; PMR, psychomotor retardation; RF, respiratory failure; wks, weeks;

Y, yes; +, increased levels.

CM

Y

Y

Y

Y

Y

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N

N

N

N.A.

N.A.

N

N.A.

N.A.

7

1-126

127-205

206-378

379-452

453-676

c.99-1G>A

c.44G>A

c.178_529del

_{c.290/291delG}

c.316C>T

c.351_2A>G

c.355G>C

c.466-470dupAA

GT

C

c.472_476dupAA

GT

C

Figure 1; NDUFS4-transcript. The transcript is composed of 5 exons, each marked by their specific lengths and the approximate locations of several key sites: NDUFS4-mutations, conserved phosphorylation sites (RTS, RVS), and mitochondrial leader sequence (ellipse).

RVS

RT

S

9( The2NDUFS4ZKO(model(described(by(Kruse,(Palmiter(et2al.((2008)(is(the(most(used.(It( features(a(whole(body(KO((WBKO)(and(loss(of(NDUFS4(protein(by(flanking(exon(2(with(loxP( sites((NDUFS4lox/lox_{).(In(this(manner,(NDUFS4ZKO(introduces(a(PTC(after(17(amino(acids(of(the(} predicted(peptide(and(shares(similarities(to(the(effects(of(c.44G>A(and(c.99Z1G>A(Zmutations( (Petruzzella,(Papa(et(al(2001;(Bénit,(Rötig(et2al.(2003;(Kruse,(Palmiter(et2al.(2008).(In(addition,( the(location(of(the(deletion(suggests(that(dysfunctional(transcripts(are(to(be(expected,( although(no(studies(have(reported(on(the(presence(these(transcripts.(Clinical(symptoms(of( the(mice(include(FTT,(abnormal(vision,(PMR(and(alopecia(Zan(autoimmune(disorder( characterized(by(hair(loss.(Alopecia(usually(recovered(in(the(next(hair(cycle(after(week(3,(and( can(be(considered(characteristic(of(NDUFS4ZKO(mice(due(to(the(lack(of(hair(in(human(infants,( although(hair(abnormalities(have(been(reported(in(mitochondrial(disorders((Bodemer,(de( Prost(et2al.(1999).(Similarly,(a(drop(in(body(temperature((~2°C)(after(postnatal(day((P)30(was( not(observed(with(human(patients.(Starting(from(week(5(mice(displayed(a(progressive( worsening(of(ataxia,(ceased(grooming(and(died(after(week(~7.(Furthermore,(isolated(CI(from( hepatocyte(NDUFS4ZKO(submitochondrial(particles((SMP)(lacked(CIZactivity,(whereas(CII( increased(when(compared(to(control(while(CIII(and(CIV(were(unchanged.(Despite(the( significant(reduction(in(CIZactivity,(measures(of(mycoyte(mitochondrial(metabolism(were( without(major(modifications;(resting(ATP(demand,(maximal(ATP(production(and(O2( consumption(were(all(in(the(normal(range((Kruse,(Palmiter(et2al.(2008).(Although(>P40(mice( showed(a(minor(increase(in(lactate(levels,(suggestive(of(changes(in(the(metabolic(profile.(In( contrast,(a(reduction(in(fibroblast(and(myocyte(CIZactivity(in(c.316G>C(and(c.290delGZ mutations(is(concomitant(with(a(reduction(of(activity(in(CIII((Budde,(Smeitink(et2al.(2000;( Budde,(Smeitink(et2al.(2003).(Interestingly,(it(was(suggested(that(only(middleZsegment( NDUFS4Zmutations(were(associated(with(a(loss(of(CIIIZactivity,(thought(to(reflect(a(regulatory( role(for(exon(3(in(OXPHOS(activation((Ugalde,(Nijtmans(et2al.(2004).(( ( LS(disease(progression(is(thought(to(reflect(neuropathological(changes(with(brain( stem(and(basal(ganglia(lesions(believed(to(be(causal(to(RF.(However,(the(first(description(of( NDUFS4lox_{–mice(was(without(major(neuropathological(changes(up(to(week(5.(Recently,(} selective(lox/lox((exon(2)(knockout((KO)(of(neuronal(and(glial(NDUFS4Zmice((NesKO)(were( generated(to(assess(the(role(of(the(CNS(in(phenotypical(disease(progression.(NesKOZmice( were(grouped(in(early((<P26),(middle((P26Z38)(and(lateZstage((>P38)(animals(according(to( detailed(behavioural(phenotyping;(characterized(by(hypothermia,(PMR(and(a(FTT,(without(the( presence(of(alopecia((Quitana,(Palmiter(et2al.(2010).(Indeed,(they(found(that(selective( neuronal(and(glial2NDUFS4ZKO(produces(a(phenotype(that(includes(progressive( neuropathological(decline(and(RF,(correlating(neuropathology(to(LS(progression((Quintana,( Palmiter(et2al.(2010;(Quintana,(Palmiter(et2al.(2012;(Lake,(Paetau(et2al.(2015).(Additionally,( progression(was(associated(with(an(increasing(phagocytic(nature(of(microglia,(concurrent( with(astroglial(and(microglial(reactivity(within(the(affected(areas:(vestibular(nuclei((VN),( inferior(olive((IO),(fastigial(nucleus((FN),(cerebellar(vermis((CV)(and(olfactory(bulb((OB).( Quantitative(analyses(of(neuronal(presence(in(these(areas(support(extensive( neurodegeneration.(Moreover,(astroglial(and(microglial(activity(in(the(VN(was(correlated(to( breathing(difficulties(in(lateZstage(NesKOZmice,(properly(reflecting(human(brain(stem( pathology(associated(with(LS.((Increased(levels(of(oxidative(stress(and(cleaved(caspaseZ8( (CASP8)(were(reported(and(considered(to(underlie(neuropathology(in(NesKOZmice,(suggestive( of(a(role(for(reactive(oxygen(species((ROS)(and(extrinsic(apoptotic(pathway(activation,( respectively.(However,(Kruse,(Palmiter(et2al.((2008)(cultured(fibroblasts(and(neurons(from( their(NDUFS4lox_{–mice(and(found(no(changes(in(ROS,(or(apoptosis((Kruse,(Palmiter(et2al.(2008).(} This(suggests(that(oxidative(stress(is(a(secondary(effect(not(intrinsic(to(these(cell(cultures,(and( supports(neuronal(extrinsic(apoptotic(pathway(activation.((

10( The(two(NDUFS4ZKO(models(described(here(arise(from(selective(removal(of(exon(2;( however,(LS(is(not(solely(characterized(by(exon(2(mutations,(which(led(to(the(generation(of(a( mouse(model(with(a(homozygous(NDFUS4(B2(SINE(insertion((NDUFS4fky/fky_{)(in(exon(3.(SINEs(} are(transposable(elements(from(the(endogenous(retrovirusZlike(sequences(family,(which( were(used(to(introduce(a(PTC(and(WBKO(of(NDUFS4,(as(assessed(in(brain,(heart,(muscle(and( liver(tissue((Leong,(Scott(et2al.(2012).(Unexpectedly,(some(transcript(variants(were(reported( and(could(reflect(a(nonZspecific(SINE(insertion(in(exon(1(and(2,(preventing(the(stimulation(of( NMD.(Phenotypically(they(were(similar(to(NDUFS4ZKO(characteristics(of(NDUFS4fky_(and( NDUFS4lox_{Zmice(including(FTT,(alopecia(and(PMR((Leong,(Scott(et2al.(2012).(Importantly,(the(} absence(of(NDUFS4(in(these(tissues(was(correlated(with(an(impaired(assembly(and(activity(of( CI,(and(corresponds(to(NDUFS4Zassociated(LS((Budde,(Smeitink(et2al.(2000;(Budde,(Smeitink( et2al.(2003;(Assouline,(Lebre(et2al.(2012).(However,(unlike(LS(patients(and(NesKOZmice,( NDUFS4fky_{(Zmice(were(not(featured(by(major(neuropathological(changes(in(the(brain((Leong,(} Scott(et2al.(2012).(It(was(suggested(that(the(lack(of(neuronal(atrophy(corresponds(to(the( experiment(duration(and(ethical(guidelines(concerning(the(health(of(the(animals.(Quintana,( Palmiter(et2al.((2010)(describe(gliosis(and(pathology(in(lateZstage(NesKOZmice,(a(stage(not( addressed(in(the(study(with(NDUFS4fky_.(( ( Although(LS(is(mostly(recognized(for(its(neuropathological(and(respiratory( characteristics,(CM(is(a(contributing(factor(to(pathology((Budde,(Smetink(et2al.(2000;( Petruzzella,(Papa(et2al.(2001;(Anderson,(Rubin(et2al.(2008;(Assouline,(Lebre(et2al.(2012).(In( order(to(assess(the(associated(CM,(Kamanlidis,(Tian(et2al.((2013)(produced(a(mouse(model( with(a(selective(cardiomyocyte(NDFUS4ZKO((cKO)(that(was(associated(with(a(reduction(in( monomeric(CI(and(supercomplexZactivity((Karamanlidis,(Tian(et2al.(2013).(At(baseline(activity( cKOZmice(had(a(similar(lifespan(and(metabolic(profile(to(control.(However,(cKO(mice( displayed(several(markers(of(congestive(heart(failure(in(conditions(of(stress,(including( fractional(shortening,(left(ventricular(dilation(and(increased(heart(weight((Karamanlidis,(Tian( et2al.(2013).(Oxidative(status(was(unchanged(with(cKO(and(could(not(account(for(mycocyte( atrophy(and(similar(to(the(absence(of(ROS(in(fibroblasts(and(neurons(in(NDUFS4lox_Zmice( (Kruse,(Palmiter(et2al.(2008;(Karamanlidis,(Tian(et2al.(2013).(Albeit(mitochondrial(permeability( transition(pore((mPTP)(sensitization(in(cKO(mice(allowed(for(rapid(mitochondrial(swelling(and( calcium(uptake,(hallmarks(of(mitochondrial(dysfunction(and(predisposing(cells(to(cell(death.(( ( Several(transgenic(mice(models(have(been(created(for(NDUFS4(mutations.(The( combination(of(the(model(by(Kruse,(Palmiter(et2al.((2008)(and(Quintana,(Palmiter(et2al.((2010)( shows(most(promise((i.e.(translatability)(to(NDUFS4Zassociated(LS;(neuropathological(and( phenotypical(features(include(basal(ganglia,(brain(stem,(death(from(RF(and(PMR((Kruse,( Palmiter(et2al.(2008;(Quintana,(Palmiter(et2al.(2010).(Although(NesKOZmice(are(able(to(model( neuropathology(closely(resembling(disease(progression(in(infants,(the(model(fails(to(produce( a(complete(spectrum(inherent(to(the(whole(body(effect(characteristic(of(LS.(An(example(in( NesKOZmice(is(the(absence(of(alopecia.(Taken(together,(the(most(common(mice(models( nicely(simulate(clinical(pathology;(however,(care(must(be(taken(with(generalizing(cell(type( specific(findings,(factoring(in(multiZsystemic(interactions.(

(

11(

Cellular(metabolic(adaptations(with(NDUFS4Zmutations

( ( Brain(metabolism(is(tightly(regulated(by(neurons,(astrocytes(and(cerebral(blood( vessels,(where(a(dynamic(cooperation(between(glycolysis,(TCAZcycle(and(the(OXPHOS(system( determine(cellular(metabolic(profile((Yadava,(Kim(et2al.(2013).(It(allows(for(the(utilization(of( several(different(substrates(in(ATP(formation,(and(does(so(in(response(to(cellular(catabolic( and(anabolic(needs.(In(this(section(we’ll(discuss(the(contribution(of(each(metabolic(system,( and(how(they(relate(to(one(another;(furthermore,(the(role(of(NDUFS4(in(assembly(and( activity(of(CI(is(addressed.((

2

Glycolysis,2TCA:cycle2and2β:oxidation2

Glycolysis(metabolizes(glucose(to(generate(pyruvate/lactate(and(ATP(in(several( enzymatic(reactions((Fig.(2).(Once(glucose(is(transported(into(the(cell(and(converted(to( glucoseZ6Zphosphate((GZ6ZP),(GZ6ZP(can(either(be(stored(as(glycogen(in(astrocytes,(processed( in(the(pentoseZphosphate((PPP)Zpathway(or(converted(into(pyruvate(or(lactate((Schönfeld,( Reiser(et2al.(2013;(Yadava,(Kim(et2al.(2013).(In(aerobic(conditions(pyruvate(enters(the(TCAZ cycle(in(the(mitochondrial(matrix((Yadava,(Kim(et2al.(2013).(In(the(glycolysis(system,(formation( at(glycerylaldehydeZ3Zphosphate((GZ3ZP)(is(NAD+(dependent(–like(FADH2,(NADH(is(a(redox( carrier(enabling(energetic(transport(between(the(metabolic(systems.(This(can(become(a( limiting(factor(in(glycolysis(if(left(untreated;(regeneration(of(NAD+(is(governed(by(LDHZ mediated(oxidation(of(NADH,(converting(pyruvate(to(lactate,(and(corresponds(to(LA(in( aerobic(conditions((Yadava,(Kim(et2al.(2013).(Lactate(is(a(hallmark(of(ageing(and(an(indicator( of(cellular(hypoxia.(Normally(the(hypoxic(response(is(governed(by(hypoxiaZinducible(factor( (HIF);(however,(Lee,(Yeom(et2al.((2015)(show(that(lactate(can(have(a(similar(effect.(Lactate( does(this(by(stabilizing(NZMyc(DownstreamZRegulated(Gene(3(Protein((NDRG3),(allowing(for( cZRaf(binding(and(RafZErk(pathway(activation((Lee,(Yeom(et2al.(2015).(The(activation(of(this( pathway(corresponds(to(the(vascular(hypertrophy(in(LSZpatients((Lake,(Paetau(et2al.(2015).(( ( The(canonical(pathway(of(pyruvate(is(into(the(TCAZcycle(where(it(is(used(as(a( substrate(for(acetylZCoA(and(functions(with(several(central(metabolites((e.g.(αZketoglutarate,( succinylZCoA,(fumarate(and(oxaloacetate)(to(form(NADH(and(FADH2.(βZoxidation(frees(fatty( acids(for(esterification(of(membrane(lipids(or(the(conversion(to(the(central(metabolites(of(the( TCA((Schönfeld,(Reiser(et2al.(2013).(The(energetic(potential(of(longZchain(fatty(acids((39(kJ/g)( is(twice(as(large(as(glucose((17(kJ/g),(although(brain(tissue(hardly(uses(fatty(acids(as(a( substrate.(During(development(fatty(acids(from(the(blood(do(serve(as(a(metabolic(substrate( for(astrocytes,(contributing(up(to(20%(of(total(brain(ATP,(whereas(neurons(and( oligodendrocytes(need(glucose/pyruvate(and(ketone(bodies(to(function((Edmond,(1992,( Ebert,(Walton(et2al.(2003).(Despite(its(importance(in(development,(brain(cells(are(remarkably( ill(equipped(for(βZoxidation(in(comparison(to(other(highZenergy(tissues(like(hearth(and( muscle.(The(activity(of(several(βZoxidation(enzymes(is(low,(particularly(the(fourth(and( terminal(step(mediated(by(3ZketoacylZcoenzyme(A(thiolase;(0.7%(of(its(counterpart(in( cardiomycytes((Schönfeld,(Reiser(et2al.(2013).(Moreover,(βZoxidation(requires(more(oxygen,( increases(the(likelihood(of(oxidative(stress(and(is(too(slow(for(rapid(sustained(neuronal( activity.(The(energetic(potential(of(fatty(acids(is(further(diminished(by(a(high(neuronal(activity( of(cytoplasmic(longZchain(acylZCoA(thioesterease,(which(governs(a(neuroprotective(effect(by( limiting(the(amount(of(fatty(acids(in(neurons(that(are(incapable(of(metabolizing(them( (Schönfeld,(Reiser(et2al.(2013).(In(turn,(glycolysis,(TCAZcycle(and(βZoxidation(all(feed(NADH( and(FADH2(into(CI(and(CIII,(respectively,(where(the(redox(potential(carried(by(these( substrates(is(‘shuttled’(to(their(respective(complexes(by(the(malateZaspartate((MAS)(and( glycerolZ3Zphosphate(redox(shuttles((GPS)((Fig.(2)((Yadava,(Kim(et2al.(2013).(( (

12( ( Figure!2;!The!dynamic!interplay!of!glycolysis,!β:oxidation!,TCA:cycle!and!OXPHOS.!Cytosolic(glycolysis(converts( glucose(into(pyruvate((aerobic)(or(in(lactate((anaerobic).((Lactate(dehydrogenase((LDH)(mediates(the(bidirectional( conversion(of(pyruvate(to(lactate((1).(Redox(potential(is(‘shuttled’((MAS,(GPS)(into(the(OXPHOS(system,(and(is( complemented(by(aerobic(glycolysis(of(pyruvate(in(the(TCZcycle.(βZoxidation(in(the(mitochondrial(matrix(feeds(into( the(pool(of(redox(carriers(and(drives(TCAZcycle(through(AcetylZCoA(formation.(The(OXPHOSZsystem(uses(the(redox( carriers((NADH,(FADH2)(to(produce(a(proton(motive(force((PMF,(Δp),(electron(transport(and(ATP(production.(CI( assembly(and(activity(is(deficient(with(NDUFS4Zmutations,(causal(to(impaired(OXPHOS(function(and(an(expected( pooling(of(NADH.((Adapted(from(Yadava,(Kim(et2al.(2013)!

The2OXPHOS2system2

The(OXPHOS(system(consists(of(5(multiZsubunit(complexes,(encoded(by(the( mitochondrial((mtDNA)(and(nuclear(DNA((nDNA):(complex(I((CI,(NADH:ubiquinone( oxidoreductase;(EC(1.6.5.3),(complex(II((CII,(succinate/ubiquinone(oxidoreductase;(EC( 1.3.5.1),(complex(III((CIII,(ubiqionol:cytochrome(c(oxidoreducatease;(EC(1.10.2.2),(complex(IV( (CIV,(cytochromeZc(oxidase;(EC(1.9.3.1)(and(complex(V((CV,(F0ZF1ZATPZsynthase;(EC(1.9.3.1)( (Koopman,(Willems(et2al.(2013).(The(OXPHOS(system(is(located(within(the(mitochondrial( inner(membrane((MIM)((Fig.(2).(CIZCIV(together(constitutes(the(electron(transport(chain( (ETC),(which(creates(an(inwardZdirected(transZMIM(proton(gradient,(whereas(CV(uses(the( resultant(protonZmotive(force((PMF)(to(produce(ATP.(Although(the(glycolysis(pathway(and( the(TCAZcycle(each(produce(2(ATP(per(glucose(molecule,(the(OXPHOS(system(is(much(more( efficient(and(maximally(generates(30(molecules(per(glucose((Koopman,(Willems(et2al.(2013;( Yadava,(Kim(et2al.(2013).(In(this(sense,(the(OXPHOS(system(represents(the(most(important( ATP(production(pathway(in(a(large(number(of(cell(types(including(neurons(and(myocytes( (Papa,(Sardanelli(et2al.(2012).(( (

13( CI(is(the(largest(complex(of(the(OXPHOS(system,(consisting(of(44(different(subunits( (37(nDNAZ(and(7(mtDNAZencoded).(This(complex(abstracts(electrons(from(NADH(and(donates( them(to(Coenzyme(Q10((CoQ)(to(drive(transZMIM(protonZextrusion((Fig.(2).(Among(the( OXPHOS(complexes,(CI(dysfunction(is(most(often(linked(to(childhoodZonset(mitochondrial( disease((Papa,(TechnikovaZDrobova(et2al.(2002;(Balsa,(Enríquez(et2al.(2012;(PagniezZ Mammeri,(Slama(et2al.(2012)(and(accounts(for(approximately(oneZthird(of(all(mitochondrial( disorders((Loeffen,(Van(den(Heuvel(et2al.(2000).(CI(is(composed(of(14(coreZsubunits,(required( for(catalytic(function,(and(30(supernumerary(accessory(subunits.(CI(consists(of(three( functional(modules:(the(NZmodule((involved(in(NADH(oxidation),(the(QZmodule((involved(in( electron(donation(to(CoQ),(and(the(PZmodule((involved(in(proton(translocation(across(the( membrane)((Zickerman,(Brandt(et2al.(2015).(( ( NDUFS4(is(located(at(the(junction(between(the(peripheral(mass(protruding(in(the( matrix(and(the(membrane(moiety(of(CI((Walker,(1992).(This(subunit(is(encoded(by(the(nDNA,( part(of(the(NZmodule,(and(plays(a(role(in(CI(assembly((Mimaki,(Ryan(et2al.(2012).(Assembly(of( CI(is(a(complex(coordinated(process(of(modular(incorporation;(the(peripheral(and(membrane( intermediates(of(CI(are(formed(independently,(supplemented(with(membrane(arm(subunits,( and(completed(with(the(addition(of(the(NZmodule((Fig.(3)((Mimaki,(Ryan(et2al.(2012).(Subunit( incorporation(of(NDUFS4(is(a(fast(process(featured(by(direct(assembly(into(holoZCI((i.e.( premature(complex(assembly(of(CI)(without(having(to(form(subcomplexes(and/or(monomeric( pools(in(the(mitochondrial(matrix((Dieteren,(Willems(et2al.(2012).((( ( ( Figure!3;!CI!assembly.(CI(assembly(consists(of(the(sequential(addition(of(the(subunits.(NDUFS4Zmutations(prevents( the(addition(of(the(NADHZdehydrogenase(module((NZmodule),(as(depicted(in(the(mitochondrial(matrix(above(the(( ~830(kDa(intermediate(complex.(LSZpatients(lack(the(mature(CIZcomplex((~(980(kDA)(that(carries(the(NADHZ dehydrogenase(capacity,(causal(to(deficient(oxidative(phosphorylation((OXPHOS).((Adapted(from(Mimaki,(Ryan(et2 al.(2012)! 2

14( NDUFS4Zmutations(prevent(the(full(assembly(of(CI;(forming(a((~830(kDa(holoZCI(in( comparison(to(the(mature((~980(kDa(complex((Petruzzella,(Papa(et2al.(2001;(Scacco,(Papa(et2 al.(2003;(Ugalde,(Nijtmans(et2al.(2004),(impairing(CIZdependent(NADHZoxidation(and(ETC( function.(In(support(of(the(function(of(NDUFS4(in(CI,(treatment(of(c.462delAZpatients(with( CoQ(was(unable(to(slow(disease(progression((Anderson,(Rubin(et2al.(2008),(suggesting(that( the(deleterious(effects(of(NDUFS4Zmediated(CIZdeficiency(precede(the(function(of(CoQ.( Normally,(the(direct(assembly(of(NDUFS4(would(allow(it(to(rapidly(exchange(de2novo2 peptides,(ensuring(the(presence(of(mature(and(functional(CI(in(the(presence(of(oxidative( stress;(however,(with(homozygous(NDUFS4Zmutations(subunits(never(incorporate(and( alternative(metabolic(adaptations(must(ensue.(2 ( Interestingly,(several(NDUFS4Zmutation(studies(have(reported(on(a(concomitant( change(of(CI(and(CIII.(These(changes(correspond(to(an(interaction(of(complexes(and(reflect( mammalian(supercomplex(formation((Budde,(Smeitink(et2al.(2000;(Schägger(and(Pfeiffer,( 2000;(Ugalde,(Nijtmans(et2al.(2004).(It(was(shown(that(supercomplex(formation(of(CI(and(CIII( stabilizes(deficient(CI,(giving(CI(a(partial(activity(not(otherwise(brought(about((Calvaruso,( Nijtmans(et2al.(2012).(In(this(manner(cells(could(prevent(pathology,(as(long(as(ATP(production( is(sufficient.(Although(cell(type(dependent(differences(in(residual(activity(have(been(reported( in(NDUFS4ZKO(mice((Calvaruso,(Nijtmans(et2al.(2012);(cardiac,(muscle,(brain,(liver(and(lung( tissue(had(a(residual(activity(of(44,(29,(26,(19(and(9%,(respectively.(This(suggests(that( susceptibility(to(pathology(varies(greatly(between(cell(types,(and(needs(to(be(accounted(for( with(CIZdeficiency.(( (

Neuropathology22

Metabolically(astrocytes(are(the(‘big(brother’(of(neurons.(Not(only(is(the(glycolytic( capacity(greater(in(astrocytes,(CIZactivity(is(higher,(adding(to(their(metabolic(prowess( (Schönfeld,(Reiser(et2al.(2013).(Interestingly,(elevated(levels(of(NADH(inhibit(the(activity(of(the( TCAZcycle(and(βZoxidation,(and(needs(CI(to(oxidize(NADH(to(regain(activity.(The(increased( astrocytic(fatty(acid(dependence(during(development(makes(it(particularly(vulnerable(to(an( impaired(βZoxidation.(However,(despite(changes(in(CIZactivity(in(patient(material(and(several( animal(models,(NDUFS4Zmutations(and(NDUFS4ZKO(are(not(associated(with(a(cellular(ATP(loss( (Kruse,(Palmiter(et2al.(2008;(Quintana,(Palmiter(et2al.(2010;(Leong,(Scott(et2al.(2012;(Mimaki,( Ryan(et2al.(2012).(It(suggests(that(cell(death(cannot(be(ascribed(to(cellular(energetic( depression((CED),(a(process(in(which(reductions(in(ATP(induce(apoptosis(or(necrosis( depending(on(the(severity(of(the(chance((Seppet,(Gellerich(et2al.(2009).(Although(bloodZ derived(lactate(is(believed(to(originate(in(the(liver,(as(hepatocyteZspecific(NDUFS4ZKO( correlates(to(changes(in(blood(metabolites((lactate,(lipids)((Jin,(Wan(et2al.(2014).(In(the( central(nervous(system((CNS)(astrocytes(are(the(main(source(of(lactate.(Neuronal(energy( metabolism(is(mostly(aerobic,(astrocyte(metabolic(profile(features(predominantly(anaerobic( glycolysis((Schönfeld,(Reiser(et2al.(2013).(It(is(thought(that(in(physiological(conditions( monocarboxylate(transporters((MCT)(transports(lactate(from(the(extracellular(space(into( neurons,(where(it(is(converted(back(to(pyruvate(for(ATP(production((Bélanger,(Magistretti(et2 al.(2011;(Boumezbeur,(Rothman(et2al.(2010;(Schönfeld,(Reiser(et2al.(2013).(This(coupling(aids( neuronal(glycolytic(capacity,(which(in(itself(is(lower(than(in(astrocytes(due(to(the(reduced( activity(of(glycolysisZpromoting(enzyme(6ZphosphofructoZ2kinase/fructose(2,6Z biphosphatase,(isoform(3,((PFKFB)((Schönfeld,(Reiser(et2al.(2013).(In(addition,(lactate(as(a( substrate(improves(the(ATP(production(by(the(PPPZpathway,(an(anabolic(pathway(mostly( involved(in(anabolic(conditions(that(does(not(aggravate(the(oxidative(status(of(the(cell.( Lactate(might(serve(a(protective(role(by(enabling(astrocyteZneuronal(shuttling(to( accommodate(neuronal(metabolism.(Similarly,(NADH(could(feed(into(the(ETC(with(the(help(of( GPS,(circumventing(CI(and(stimulate(continued(ATP(production((Iuso,(Papa(et2al.(2006).((

15( ( In(conditions(of(CIZdeficiency(cells(become(dependent(on(glycolysis,(a(metabolic( profile(that(in(tumour(cells(has(been(known(as(the(‘Warburg(effect’;(named(after(the( observation(made(in(1924(by(Otto(Warburg((Van(der(Heiden,(Thompson(et2al.(2009).(The( proliferative(conditions(in(early(childhood((0Z5)(stimulate(glycolysis,(although(there(is(an( increasing(CIZdependence(with(maturation((Kuzawa,(Lange(et2al.(2014;(Yadava,(Kim(et2al.( 2013).(The(inflexibility(of(metabolism(with(CIZdeficiency(might(predispose(cells(to(pathology.( Therefore,(lesions(to(brainstem(and(basal(ganglia(in(LS(patients(could(correspond(to(the(early( maturation(of(these(areas(in(brain(development,(and(corresponds(to(the(cell(typeZ(and(tissueZ specific(differences(in(mitochondrial(OXPHOS(function(in(NDUFS4ZKO(mice((Calvaruso,( Nijtmans(et2al.(2012;(Morgan,(Sedensky(et2al.(2013).(To(further(elucidate(if(OXPHOS( differences(play(a(role(in(LSZpathology,(future(studies(could(include(imaging(methods(to( measure(tissue(CIZdependence(through(lactate(and(NADH(accumulations.(In(addition,( concurrent(mathematical(modelling(of(cell(specific(metabolic(profiles(help(correlate( metabolite(concentrations(to(pathology((Alam,(Koopman(et2al.(2015).(

(

Molecular(mechanisms(and(putative(therapeutic(interventions(of(LS(

( In(the(previous(sections(we’ve(addressed(the(clinical(heterogeneity(and(the(cellular( metabolic(adaptions(associated(with(NDUFS4Zmutations.(Here(we(discuss(the(molecular( mechanisms,(and(their(therapeutic(potential,(with(a(particular(focus(the(role(of(metabolism,( oxidative(stress(and(gliosis(in(disease(progression.( ( Mature(CI(stimulates(βZoxidation(and(TCA(in(macrophages(while(suppressing( inflammatory(activity((Yadava,(Kim(et2al.(2013),(a(dynamic(in(metabolism(missing(with( NDUFS4Zmutations.(In(accordance,(NDUFS4ZKO(mice((Kruse,(Palmiter(et2al.(2008)(were(found( to(feature(accumulations(of(fatty(acids(in(lipid(droplets((LDs)((Jin,(Wan(et2al.(2014).(These(LDs( were(localized(to(the(brain(stem,(cerebellum,(midbrain(and(OB(in(early(stage(mice(without( any(apparent(lesions((Liu,(Bellen(et2al.(2015).(Lesions(only(became(apparent(in(middle(and( late(stage(mice(where(neuropathy(was(correlated(with(progressive(gliosis.(Gliosis(is(an( inflammatory(process(characterized(by(the(proliferation(of(glial(cells,(microglial(phagocytosis( and(astrocytic(scar(formation((Gao,(Cai(et2al.(2013).(Phagocytosis(is(believed(to(have(resulted( in(the(removal(LDs(and(the(activation(of(glia((ROS(formation(and(inflammation).(Indeed,( NDUFS4ZKO(macrophages((Kruse,(Palmiter(et2al.(2008)(express(higher(levels(of(ROS(that(were( aggravated(by(the(presence(of(LDs((Kruse,(Palmiter(et2al.(2008;(Karamanlidis,(Tian(et2al.(2013;( Jin,(Wan(et2al.(2014).(These(studies(provide(a(novel(mechanistic(coupling(of(impaired(βZ oxidation,(glial(ROS(formation(and(neuronal(loss.( (

ROS(production(by(CI(a(is(product(of(O2Z(and(reduced(flavin([FMN(⇌(FMNHZ(→(O2Z],(an( electron(carrier(in(the(NZmodule(that(gives(rise(to(more(O2Z(with(in(conditions(where( [NADH]>>[NAD+]((Hirst,(2013).(Oxidation(of(NADH(skews(the(NADH/NAD+(ratio(towards(a( more(favourable(state(and(reduces(ROSZformation,(thereby(linking(CIZactivity(to(cellular( oxidative(health.(AntiZoxidative(and(NADH/NAD+(targeted(therapies(might(therefore( ameliorate(pathology(of(NDUFS4Zassociated(LS.(Surely,(antiZoxidative(treatment(with(NZacetyl( cysteine(amide((AD4)(improved(clinical(symptoms(and(delayed(neurodegeneration;(whereas( treatment(with(nicotinamide(mononucleotide((NMN),(a(NADZprecursor,(reduced(the( NADH/NAD+(ratio(and(improved(mitochondrial(dysfunction((Karamanlidis,(Tian(et2al.(2013;( Liu,(Bellen(et2al.(2015).(Moreover,(increased(NADH(levels((i.e.(a(lowering(of(the(NADH/NAD+( ratio)(inhibits(NADZdependent(Sirtuin3((Sirt3).(Inhibition(of(Sirt3(results(in(the(acetylation(of( several(mitochondrial(peptides(and(sensitizes(the(mitochondrial(permeability(transition(pore( (mPTP)((Karamanlidis,(Tian(et2al.(2013).(Opening(mPTP(sensitization(is(causal(to(a(loss(of(PMF,(

16( further(inhibiting(OXPHOS(capacity((Zong(and(Thompson,(2006).(The(link(between(NADH(and( mPTP(sensitization(might(proof(an(interesting(therapeutic(target(for(CIZdeficiencyZassociated( LS;(blocking(mPTP(with(Cyclosporin(A(abrogated(dysfunction((Karamanlidis,(Tian(et2al.(2013).( Alternatively,(it(is(possible(to(promote(mitochondrial(fusion.(Fusion(increases(the(size(of( mitochondria(and(reduces(mtDNAZmutations(while(improving(ATP(production(and(stress( resilience((Youle(and(Bliek(et2al.(2012;(Varanita,(Scorrano(et2al.(2015).(Future(studies(should( include(measures(of(mitochondrial(morphology(in(CIZdeficient(patients,(as(it(might(proof(to( be(an(endogenous(manner(to(cope(with(the(loss(of(the(subunit.( ( The(therapeutic(potential(of(AD4(an(NMN(is(likely(governed(through(modulation(of( gliosis,(as(NDUFS4ZKO(in(neuron,(myocyte(and(fibroblast(cultures(all(lacked(changes(in(ROS( (Kruse,(Palmiter(et2al.(2008;(Karamanlidis,(Tian(et2al.(2013;(Jin,(Wan(et2al.(2014).(However,(IPZ treatment(with(NZ(6ZoxoZ5,6ZdihydrophenanthridinZ2Zyl)Z(N,NZdimethylamino)acetamide( hydrochloride((PJ34,(20mg/kg),(an(inhibitor(of(poly(adenine(ribose(polymerase((PARP),( reduced(astrogliosis(and(scar(formation(without(an(effect(on(neuronal(viability(or(life( expectancy(in(NDUFS4ZKO(mice((Kruse,(Palmiter(et2al.(2008)((Felici,(Chiarugi(et2al.(2014).(This( finding(is(unexpected,(as(PARP(promotes(necrosis(by(limiting(NAD(availability(and(ATP((Zong( and(Thompson,(2006).(PJ34Ztreatment(was(able(to(reduce(neurological(impairment(and( increased(motor(skills.(These(results(suggest(that(limiting(astrogliosis(is(insufficient(to(stop( disease(progression.(Astrocytic(scar(formation(is(protective(at(first(by(localizing(pathology;( however,(if(it(is(maintained(longZterm(it(prevents(neuronal(synaptic(plasticity(and(recovery(of( function((Sofroniew,(2009).(By(abrogating(scar(formation(PJ34(has(a(limited(therapeutic( potential,(albeit(an(important(one(if(this(allows(for(an(improved(quality(of(life.(Interestingly,( apoptotic(initiation(inhibits(PARPZinduced(necrosis,(which(might(explain(the(limited(effect(by( PJ34((Zong(and(Thompson,(2006).( ( Recently(mammalian(target(of(rapamycin((mTOR)(has(received(considerable(attention( in(the(treatment(of(LS.(It(was(shown(that(oral(and(intraperitoneal((IP)(treatment(with( rapamycin,(an(inhibitor(or(mTOR,(significantly(increased(survival(and(overall(health(in( NDUSF4ZKO(mice((Kruse,(Palmiter(et2al.(2008)((Johnson,(Kaeberlein(et2al.(2013,(Johnson,( Kaeberlein(et2al.(2015).(Although(the(reduction(of(gliosis(by(rapamycin(was(thought(to( correspond(to(its(immune(modulatory(function,(disease(progression(did(not(alleviate(with( tacrolimus((FKZ506),(an(antiZinflammatory(equivalent(of(rapamycin.(It(was(found(that(the( neuroprotective(effect(was(governed(through(its(modulation(of(nutrient(signalling.( Specifically,(rapamycin(stimulates(a(metabolic(shift(from(glycolysis(to(protein(catabolism,(and( stimulates(autophagy((Johnson,(Kaeberlein(et2al.(2013).(In(this(way(rapamycin(restricts(the( toxic(buildZup(of(glycolytic(intermediates(and(lactate,(while(increasing(free(amino(acids(and( fatty(acids(for(anabolism((Johnson,(Kaeberlein(et2al.(2013).(A(side(effect(of(the(intervention( was(a(significant(reduction(in(body(weight,(reflecting(a(systemic(effect(of(the(drug.(Albeit(it( was(not(detrimental(to(health,(the(selective(release(of(rapamycin(in(inflammatory(lesions( could(provide(targeted(aid(to(where(it(is(needed(without(impinging(on(tissues(lacking(an( immune(activation((Bibee,(Wickline(et2al.(2014).(In2vivo(modelling(should(resolve(the( developmental(impact(of(such(an(approach.(In(addition,(it(would(be(interesting(to(see(how( rapamycin(in(combination(with(dietary(adaptions(would(serve(health(in(LSZpatients.((( ( Inhibition(of(the(TCAZcycle(and(βZoxidation(impairs(synthesis(of(key(proteins(and( membrane(lipids.(The(freeing(of(these(nutrients(enables(a(continued(cellular(synthesis(of( several(key(developmental(molecules((Zong(and(Thompson,(2006).(In(contrast,(the(absence(of( membrane(lipids(causes(a(loss(of(membrane(integrity,(a(known(stimulatory(factor(for(necrosis( and(immune(activation.(Mitochondria(still(carry(many(damageZassociated(molecular(patterns( (DAMPs)(capable(of(initiating(an(immune(response((e.g.(extracellular(mitochondrial(DNA(

17( (mtDNA),(NZformyl(peptides(for(mitochondrial(protein(synthesis,(ATP)((West,(Ghosh(et2al.( 2010).(The(uncontrolled(release(into(the(extracellular(space((ESS)(serves(as(a(promoter(of( inflammatory(activation,(oxidative(stress(and(neuronal(loss.(In(turn,(oxidative(stress(promotes( immune(activation,(setting(in(motion(a(selfZpropagating(cascade(of(damage,(whereas(the( selective(removal(of(dysfunctional(mitochondria((i.e.(mitophagy)(ameliorates(ROS(production( and(inflammation.( ( The(intervention(studies(support(a(cumulative(nature(of(disease,(affecting(the(brain(in( a(cellZ(and(functionZdependent(manner(contributing(to(the(overall(pleiotropy(of(the(gene.( More(importantly,(mechanistic(insights(from(these(studies(correspond(to(the(metabolic( profile(and(neuropathological(characteristics(of(NDUFS4Zassociated(LS,(providing(novel( therapeutic(targets(to(ameliorate(disease(progression(and(extend(life.(Although(antiZ oxidative,(antiZgliosis(and(metabolic(approaches(reduce(pathology,(it(is(the(new(availability(of( nutrients(with(rapamycin(that(prevents(necrosis.(AD4,(NMN,(PJ34(and(rapamycin(have(all( been(used(as(a(monotherapy.(However,(the(potential(of(polydrug(therapy(involving(these( could(be(synergistic,(affecting(multiple(mechanisms(concurrently(to(have(the(greatest(overall( effect.(The(molecular(mechanisms(discussed(suggest(that(neuropathology(is(causal(to(an( initial(defect(in(the(TCAZcycle(and(βZoxidation,(which(translates(in(gliosis(and(neuropathy((Fig.( 4).( (

NDUFS4Zmutations(in(PDZmodels(((

( Mitochondrial(dysfunction(in(LS(is(correlated(with(an(infantile(pathogenesis;(however,( several(ageing(associated(neurodegenerative(disorders(have(been(linked(to(CIZdeficiency((e.g.( Alzheimer’s,(Huntington’s,(PD)((Koopman,(Willems(et2al.(2013;(Federico,(Radi(et2al.(2012).( Clinical(and(neuropathological(hallmarks(of(LS(share(commonalities(with(PD,(making(it( particularly(relevant.(PD(is(featured(by(a(selective(loss(of(dopaminergic(substantia(nigra(pars( compacta((SNpc)(neurons,(Lewy(body(accumulations,(motor(dysfunction((e.g.(rigidity,( tremors),(memory(loss(and(problems(with(executive(functioning((Brookes(and(Picini,(2006;( Song(and(Cortopassi,(2015).(The(recognition(of(similarities(has(led(to(the(use(of(NDUSF4ZKO( models(in(combination(with(CIZinhibitors(capable(of(inducing(PDZlike(symptoms((e.g.( rotenone(and(MPTP((1ZmethylZ4ZphenylZ1,2,3Ztetrahydropyridine)).((Choi,(Xia(et2al.(2011;( Zhang,(Ding(et2al.(2014;(Sterky,(Larsson(et(al(2012).(( ( Studies(with(NDUFS4ZKO(have(shown(that(SNpc(lacks(obvert(neuropathological( changes,(despite(lesions(in(the(basal(ganglia((Kruse,(Palmiter(et2al.(2008;(Quintana,(Palmiter( et2al.(2010).(Even(dopaminergic(specific(NDUFS4:KO(did(not(result(in(neurodegeneration( and/or(motor(deficits(in(mice(aged(up(to(24(months,(and(correspond(to(the(absence(of(SNpc( neuropathy(in(NDUFS4Zassociated(LSZpatients((Lee,(Chen(et2al.(2009;(Choi,(Xia(et2al.(2011;( Koene,(Smeitink(et2al.(2012;(Sterky,(Larsson(et2al.(2012;(Kim,(Xia(et2al.(2015;).(These(results( argue(that(NDUSF4ZKO(alone(is(insufficient(to(induce(neurodegeneration(in(SNpc,(supporting( the(tissueZ(and(cellZspecific(vulnerability(to(NDUFS4ZKO.(However,(despite(an(absence(of( neurodegeneration(in(the(SNpc,(several(studies(have(reported(on(metabolic(alterations(in( dopamine((DA)(production(with(NDUFS4ZKO.(The(main(enzyme(responsible(for(DA(production( is(tyrosine(hydroxylase((TH),(it(catalyses(the(conversion(of(LZtyrosine(to(LZ3,4Z dihydrophenylalanine((LZDOPA).(Due(to(its(central(role(in(DA(synthesis(TH(is(often(used(as(a( marker(for(dopaminergic(neurons((Choi,(Xia(et2al.(2014).(It(was(found(that(the(expression(of( TH(is(reduced(in(the(dopaminergic(system(of(late(stage(mice((Kruse,(Palmiter(et2al.(2008),( including(SNpc,(striatum(and(midbrain((Song(and(Cortopassi(et2al.(2015).((In(addition,( expression(of(catecholZOZmethyltransferase((COMT),(an(enzyme(important(for(DA((

Figure 4; Mechanistic model of neuronal necrosis with NDUFS4-mutations. NDUFS4-patients lack the N-module of CI

(depicted in red), thereby impairing cellular metabolism. Glycolysis-derived redox potential is transferred from NADH to GPS-shuttle, enabling continued OXPHOS function. In parallel, pyruvate forms a substrate for the TCA-cycle, improving nutrient availability and ATP production. However, CI-defiency inhibits the TCA-cycle and β-oxidation through an accumulation of NADH. In turn, increased levels of NADH promote ROS-production and inhibit Sirtuin3 (Sirt3) function, resulting in mPTP opening, additional inhibition of OXPHOS function and mitochondrial dysfunction. In microglia ROS-levels correlate to the release of inflammatory mediators that neuronal health. Furthermore although astrocytes are able to feed neurons with lactate, the intrinsic low β-oxidation capacity of neurons without a functional CI predispose it to nutrient starvation, loss of membrane integrity and necrosis. Glycolysis associated pathways are highlighted with an ellipse. GPS, glycerol-3-phosphate; IMS, intermembranal space; LDH, lactate dehydrogenase; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; mPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; TCA, tricarboxilic acid.

Cytosol mPTP TCA NADH NADH NAD+ NAD+ β-oxidation GPS SIRT3 ROS Pyruvate Lactate Glucose LDH IMS MOM MIM Mattrix Necrosis OXPHOS

18

19( breakdown,(was(reduced(in(midbrain.(Thus,(NDUFS4Zassociated(CIZdeficiency(is(detrimental( to(cellular(DA(content,(implying(a(stimulatory(role(for(CI(in(DA(synthesis.(( ( MPTP(induction(of(Parkinson’s(features(is(governed(through(the(accumulation(of(its( metabolite((MPP+)(in(dopaminergic(neurons.(The(accumulation(of(MPP+(is(toxic(to(CI(and( inhibits(its(activity.(NDUFS4ZKO(mice((Kruse,(Palmiter(et2al.(2008)(were(not(associated(with(an( increased(susceptibility(to(apoptosis(with(MPTP,(although(it(enhanced(striatal(DA(reduction( (Kim,(Xia(et2al.(2015;(Sterky,(Larsson(et2al.(2012).(Moreover,(the(reduction(in(DA(content(was( associated(with(increases(in(DAZmetabolites(in(relation(to(DA,(indicative(of(a(high(DA( turnover;(3,4Zdihydroxyphenylacetic(acid((DOPAC)(and(homovanillic(acid((HVA)((Sterky,( Larsson(et2al.(2012).(The(study(by(Song(and(Cortopassi((2015)(nicely(compliments(the(work( done(by(Sterky,(Larsson(et2al.((2012).(NDUFS4ZKO((Kruse,(Palmiter(et2al.(2008)(impairs(DA( synthesis(throughout(the(dopaminergic(system;(however,(the(midbrain(has(a(selective( reduction(breakdown,(resulting(in(a(higher(DA(content(in(this(tissue.(Neural(atrophy( associated(with(MPTP(could(not(reflect(its(effect(on(CI,(as(NDUFS4ZKO(did(not(result(in(a( larger(susceptibility.(It(is(possible(that(these(effects(are(modulated(by(the(accumulations(of(αZ synuclein,(or(changes(in(the(ubiquitinZproteasome(system((UPS)((Fornai,(Südhof(et2al.(2005).( Moreover,(MPTP(is(not(known(for(big(effects(on(motor(function,(suggesting(that(MPTP(only( models(changes(in(DA(levels,(more(characteristic(of(early(stage(PD((Meredith,(Chesselet(et2al.( 2008). ( Lewy(bodies((LBs)(–hallmarks(for(PD(pathologyZ(are(composed(of(ubiquitin(and(αZ synuclein((Iwatsubo,(2003;(Stefanis,(2012).(αZsynuclein(is(a(presynaptic(protein(with(a(genetic( and(pathologic(link(to(PD,(whereas(ubiquitin(binds(peptides(to(select(them(for(proteasomal( and(autophagic(removal((Webb,(Rubinsztein(et2al.(2003;(Stefanis,(2012).(However,( proteasomal(function(was(inhibited(in(late(stage(mice,(as(the(activity(of(a(key(component(of( the(complex((20S)(was(impaired.(It(removed(the(selective(pressure(of(the(UPS(and(allowed(for( an(increase(of(ubiquitin(and( Zsynuclein((Webb,(Rubinsztein(et2al.(2003).(Dopaminergic( NDUFS4ZKO(strengthened( Zsynuclein(accumulations(in(the(SNpc(of(24(month(old(mice,(and( featured(a(loss(of(striatal(dopamine(in(mice(9(months(and(older((Kim,(Xia(et2al.(2015).(Since( improving(the(autophagic(removal(of(protein(might(reduce(the(accumulation(of(ubiquitin(and( Zsynuclein,(rapamycin(could(have(a(therapeutic(benefit(in(PD((Malagelada,(Green(et2al.( 2010).(( ( Despite(the(similarities(between(PD(and(LS,(the(diseases(discriminate(from(each(other( by(ageZofZonset.(LS(symptoms(typically(arise(in(the(first(year(of(life,(whereas(risk(of(PD( increases(with(age;(1Z2%(of(the(population(over(65(years(of(age(to(3Z5%(over(85(years((Alves,( Larsen(et2al.(2008;(Ruhoy(and(Saneto,(2014).(During(aging(cellular(NAD+(content(drops,(and( mirrors(a(deteriorating(mitochondrial(function(and(NDUFS4Zassociated(OXPHOS(dysfunction( (Choi,(Xia(et2al.(2011;(Stein,(Imai(et2al.(2012).(Remarkably,(clinical(reports(of(NDUFS4Z associated(LS(haven’t(measured(NADH/NAD+(ratios(and(characterize(CIZactivity(with(different( parameters((e.g.(BN–PAGE,(mitochondrial(complex(I(inZgel(activity(assays)((Ugalde,(Nijtmans( et2al.(2004).(Choi,(Xia(et2al.((2011)(were(able(to(show(that(basal(NADH(levels(are(indeed( increased(with(NDUFS4ZKO.(( ( Rotenone(is(another(drug(commonly(used(for(the(induction(of(Parkinson’s(disease.( Unlike(MPTP,(rotenone(is(associated(with(motor(deficits((Meredith,(Chesselet(et2al.(2008).( Although(rotenone(impairs(CIZactivity,(pathology(in(NDUFS4ZKO(mice(is(by(a(reduction(of( microtubule(stability,(believed(to(underlie(defective(vesicular(DA(transport,(synaptic(DA( release(and(neuronal(loss;(factors(corresponding(to(PD(pathology((Miller,(Caron(et2al.(1999;( Ren,(Feng(et2al.(2005;(Choi,(Xia(et2al.(2008;(Zhang,(Ding(et2al.(2014).(Treatment(with(piericidin(

20( A,(a(CIZinhibitor(that(does(not(affect(microtubule(stability,(did(not(induce(dopaminergic(cell( loss(in(NDUSF4ZKO(mice,(suggesting(that(rotenoneZinduced(microtubule(instability(is(the(main( driving(force(behind(the(pathological(derangements.(Indeed,(taxolZmediated(microtubule( stabilization(of(rotenoneZinduced(injury(reduced(DA(accumulations((Choi,(Xia(et2al.(2011).( Future(studies(could(consider(using(colchicine(to(specifically(destabilize(microtubules(in( NDUFS4ZKO,(as(the(inhibitory(role(of(rotenone(might(confound(the(effects(of(NDUFS4ZKO.( Specifically(cytosolic(DA(exacerbates(oxidative(stress,(whereas(vesicular(DA(does(not((Choi,( Xia(et2al.(2011).(Rotenone(is(thought(to(induce(a(disruption(of(vesicular(monoamine( transporter((VMAT)(–a(vesicular(transport(proteinZ(thereby(preventing(vesicular(DA(loading( and(increase(toxic(cytosolic(DA((Ren,(Fen(et2al.(2005;(Choi,(Xia(et2al.(2008).(Treatment(with( radical(scavenger(NZacetylcysteine((NAC)(or(pyrroloquinoline(quinine((PQQ)(reduced( apoptosis((Choi,(Xia(et2al.(2011;(Zhang,(Ding(et2al.(2014).(Interestingly,(a(rotenoneZinduced( reduction(of(NDUFS4(mRNA(was(ameliorated(by(PQQ,(an(effect(mediated(by(Erk1/2( activation.(However,(striatal(Erk1/2(activation(is(associated(with(dyskinesia(in(LZDOPA( treatment,(and(might(proof(a(difficult(target(for(further(modulation((Pavón,(Moratalla(et2al.( 2006).(Activation(of(the(cAMP/PKA(pathway(could(be(an(alternative(therapeutic(approach.(As( stated,(the(cAMP/PKA(pathway(has(a(key(role(in(regulating(translocation,(maturation(and( assembly(of(NDUFS4((De(Rasmo,(Papa(et2al.(2008).(Activation(of(the(cAMP/PKA(pathway( promotes(the(presence(of(NDUFS4,(which(in(turn(increases(CI(activity(and(βZoxidation,(and( reduces(oxidative(stress((Papa,(Santeramo(et2al.(2008;(GerhartZHines,(Puigserver(et2al.(2011;( Valenti,(Vacca(et2al.(2013).(( ( ( ( The(study(of(NDUSF4ZKO(in(relation(to(PD(has(revealed(several(interesting( mechanisms(that(could(underlie(SNpc(dopaminergic(neuronal(loss,(including(oxidative(stress,( increasing(NADH/NAD+(ratio,(UPS(activity,(and(changes(in(dopamine(metabolism.(All( contributed(to(pathology(with(exposure(to(rotenone(and(MPTP.(However,(none(alone(was( enough(for(neuronal(loss.(Of(note,(MPTP(and(rotenone(model(PD(at(early(and(late(disease( progression(phases,(respectively,(and(are(therefore(limited(in(the(applicability.(These(studies( provide(mechanistic(insight(into(the(role(of(OXPHOS(deficiency(in(PD.(Moreover,(these(studies( allow(us(to(better(understand(the(cell(type(specific((metabolic)(differences,(and(broaden(the( scope(and(relevance(of(NDUFS4ZKO(models.( (

Concluding(remarks(and(future(perspectives(

( We(have(shown(that(cellular(metabolic(changes(are(central(to(the(clinical(and( neuropathological(characteristics(of(NDUFS4Zassociated(LS.(Although(all(NDUFS4Zmutations( introduce(a(PTC,(lack(of(protein(and(CIZdeficiency,(some(might(be(more(pathogenic(than( others(due(to(the(accumulation(of(dysfunctional(transcripts.(The(multisystemic(nature(of(the( disease(promotes(genetic(pleiotropy(and(clinical(phenotypical(variation,(while(disease( progression(reflects(lesions(in(the(brain(stem(and(basal(ganglia.(Defective(assembly(and( activity(of(CI(with(NDUFS4Zmutations(impairs(metabolic(profile(plasticity,(‘locking’(cells(in(a( WarburgZlike(state(that(is(detrimental(to(cellular(health(in(a(tissueZ(and(cellZdependent( manner.(NDUFS4Zmediated(CIZdeficiency(plays(a(key(role(in(cellular(metabolism,(oxidative( stress(and(inflammatory(profile.(Several(therapeutic(interventions(have(addressed(the( potential(of(targeting(these(processes;(Rapamycin(shows(great(potential(for(both(infantile( and(ageZassociated(diseases,(whereas(cAMP/PKA(pathway(activation(might(proof(beneficial( for(Parkinson’s.(Future(studies(need(to(tailor(to(the(needs(of(the(individual(through( microprofiling(and(‘mitochondrial(medicine’.( ( (

21(

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