Tipping Points and Endogenous Determinants of Nigrostriatal Degeneration by MPTP

Review

Tipping Points and

Endogenous Determinants of Nigrostriatal Degeneration by MPTP

Stefan Schildknecht,

* Donato A. Di Monte,

Regina Pape,

Kim Tieu,

and Marcel Leist

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a Parkinson’sdisease (PD)-like syndrome by inducing degeneration of nigrostriatal dopaminergic neurons. Studies of the MPTP model have revealed thepathomechanisms underlying dopaminergicneurodegeneration andfacilitatedthedevelopmentof drugtreatmentsforPD.Inthisreview, we provide an update on MPTPbioactivation and biodistribution, reconcile the distinctviewsonenergeticfailureversusreactive oxygenspecies(ROS) for- mation as main drivers of MPTP-induced neurodegeneration, and describe recently identified intrinsic features of the nigrostriatal system that make it particularlyvulnerable toMPTP. We discuss these new perspectives on the endogenoustippingpointsoftissuehomeostasisandthedriversresponsible for viciouscycles in relationtotheir relevance forthedevelopment of novel interventionstrategiesforPD.

MPTP:AnExperimental ParkinsonianToxicant

Almostfourdecadesago,theneurotoxicantMPTPwasidentifiedasanillicitdrugcontaminant thatcancausesymptomsandsignsinhumanssimilartothoseobservedinidiopathicPD[1].

Studiesduringthe1980selucidatedthebasicmechanismsoftoxicityofMPTP,whichhas sincebecomethemost-studiedexperimentalneurotoxicant.Itisnowtextbookknowledgethat theprotoxicant MPTP ismetabolized byastrocytic monoamineoxidase-B (MAO-B;see Glossary) togenerate theactive metabolite 1-methyl-4-phenylpyridinium(MPP⁺)(Figure 1).

MPP⁺istakenupintoneuronsbydopamine(DA)transporters(DATs)[2–5].CytosolicMPP⁺ isaccumulatedincatecholaminergicstoragevesiclesbyvesicularmonoaminetransporter- 2 (VMAT-2)andinmitochondriathrough membranepotential-dependent uptake[6].MPP⁺ accumulatesinthemitochondrialmatrixandinhibitscomplexIoftherespiratorychain,leading to an impairment in mitochondrial ATP generation and an increase in superoxide (O₂) formation[6–8].Furthermore,MPP⁺ uptakeintoDA neuronstriggersvesicular DA release, whichresultsinDAautoxidationandfreeradicalformation(Figure2).Inthepresenceofironand H2O2,extracellular DA can beoxidized to form 6-hydroxy-DA. This toxicant can undergo cyclizationtoyieldaminochrome,whichiscapableofdirectlyinhibitingcomplexI[9].Thus, oxidativestressandenergyfailure areinvolved intricatelyinaviciouscycle.ATP depletion triggeredbyMPP⁺emergedasinsufficienttotriggercelldeath[10].Agrowingbodyofevidence indicatesamoresignificantroleofROSgenerationasconsequenceofcomplexIinhibitionthan initially anticipated [11,12]. For instance, mice overexpressing copper/zinc superoxide

Trends

PD-associatedmotordeficitsarechar- acterized bynigrostriatal dopaminer- gic-neuronloss.

TheparkinsonianprotoxicantMPTPis activatedinthecentralnervoussystem (CNS)byastrocyticmonoamine oxi- dase-B to form the active toxicant 1-methyl-4-phenylpyridinium(MPP⁺).

Membrane-impermeable MPP⁺ is takenupbyneurotransmittertranspor- tersintocatecholaminergicneurons.

Insidecells,MPP⁺inhibitsmitochon- drialcomplexI,reducingmitochondrial ATP output and favoring ROS formation.

In MPTP-exposed brains, different catecholaminergic populations show large sensitivity differences, with a distinct nigrostriatal dopaminergic- neuron degeneration, reflecting the degenerationpatterninPD.

Intrinsicfactors,suchasMPP⁺uptake and/orvesicular-sequestrationkinetics, neuronalmorphology,andintracellular Ca²⁺ handling, distinguish sensitive fromresistantneuronalsubpopulations intheMPTPmodelandinPD.

1InvitroToxicologyandBiomedicine, DepartmentofBiology,Universityof Konstanz,D-78457Konstanz, Germany

2GermanCenterfor

NeurodegenerativeDiseases(DZNE), Sigmund-Freud-Strasse27,D-53127 Bonn,Germany

541 Erschienen in: Trends in Pharmacological Sciences ; 38 (2017), 6. - S. 541-555

https://dx.doi.org/10.1016/j.tips.2017.03.010

dismutase(Cu/Zn-SOD)displayedhigherresistanceagainstMPTP[13],whileknockdownof endogenousCu/Zn-SODelevatedthesensitivityofnigrostriatalDAneuronstowardsMPTP[14]

(Figure3).

RecentgroundbreakingstudiesonMPTPmodelshaveprovideddatathatcannotbesatisfac- torilyexplainedbystandardtextbookknowledgeregardingMPTPtoxicokineticsandmodeof action.Take,forexample,modulationofneuroinflammation-induceddamage:blockadeofIL- 1b,TNF-a,orIFN-gsignalingpreventedMPTPneurotoxicity,despiteongoingMPP⁺inhibition ofmitochondrialcomplexI[15–17].Anotherexampleisrelatedtomitochondrialfissionand/or fusion.Geneticsilencingorpharmacologicalsuppressionofdynamin-likeprotein-1(DLP-1),a mitochondrial fission-associated molecule,preventedmitochondrialdysfunctionandneuro- degeneration,despiteMPP⁺-dependentinhibitionofmitochondrialcomplexI[18–21].More- over,adenosineA2AreceptorantagonistswerereportedtoprotectagainstMPTP-triggered neurodegeneration,buttheunderlyingmechanismsremaininadequatelycharacterized[22].

AnothersetofquestionsarisesfromthecelltypespecificityofMPTPand/orMPP⁺-induced damage: motor deficitsinPD andinMPTPmodelscorrelatecloselywith nigrostriatalDA-

3DepartmentofEnvironmental&

OccupationalHealth,Florida InternationalUniversity,Miami,FL 33199,USA

*Correspondence:

Stefan.Schildknecht@uni-konstanz.de (S.Schildknecht).

1,2-MPDP H ₊

H N

N N

N CH ₃

CH ₃

CH ₃ CH ₃

+

+

Autoxidaon

MA O

MPTP MPDP

MPP

Figure1.Structures of1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine(MPTP) andItsMetabolites.The intermediate1-methyl-4-phenyl-2,3-dihydropyridinium(MPDP⁺)isgeneratedthroughatwo-electronoxidation,catalyzed bymonoamineoxidase(MAO).Underphysiologicalconditions,MPDP⁺partiallyexistsinitsconjugatebaseform,1,2- MPDP.Giventhatitislipophilicanduncharged, 1,2-MPDPcan readilycrossmembraneswithouttheneedfora transporter.Inasecond,nonenzymaticstep,theintermediateundergoesautoxidationtoformthestableactivetoxicant 1-methyl-4-phenylpyridinium(MPP⁺).

Glossary

CytochromeP450oxidase (CYP2D6):involvedinthe metabolismandeliminationof xenobiotics.

Dopaminetransporter(DAT):

expressedbydopamineneurons;

enablesre-uptakeofsynaptic dopaminetoterminateitssignaling function.DATalsoallowsuptakeof MPP⁺,thuscontributingtoits preferentialtoxicityintheseneurons.

Monoamineoxidase(MAO):

catalyzestheoxidativedeamination andinactivationofmonoaminergic neurotransmitters.MAOalso catalyzestheactivationofMPTPto formthetoxicantMPP⁺.

Organiccationtransporter(OCT):

allowspassivetransportoforganic cations(incl.MPP⁺)across membranes.

Vesicularmonoaminetransporter 2(VMAT-2):transportof

neurotransmittersandMPP⁺from thecytosolintosynapticvesicles.

neurondegenerationandtheconcomitantdeclineinstriatalDAlevels[23].Thishasobscured the finding that MPTP also markedly affects DA neurons of the mesocortical system or hypothalamictuberomammillary nucleus [24]. Furthermore,DAT-mediated toxicant uptake doesnotexplainwhyMPTPspares,forexample,DAneuronsoftheventraltegmentalarea

HO HO

HO HO

HO OH

Fe²⁺ + H₂O₂

H₂O₂

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O₂ O₂ O₂

.O₂^–

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Fe³⁺ + H₂O

Fe³⁺ Fe²⁺

Fe²⁺

NH₂

NH₂ Dopamine

Dopamine Dopamine

Melanin 6-OHDA NH₂

O₂

O₂

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.O₂^–

.O₂

.O₂^– .O

O₂ O

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Aminochrome DA-o-semi-

quinone

DA-o-semi- quinone

Release

.OH + OH^–

Fission/

fragmentaon e^–

Inhibit ed

NADPH NADP

ONOO^– .NO + .O₂^–

DA neuron

.NO/nitrosaon .NO

DOPAC Inflammed glia

i

ii

iii v iv viii

vii vi

ix

MAO

NOS

oxidase

C-III

C-I _{CI CI}

Vesicular DA

Figure2.FreeRadicalSources.1-Methyl-4-phenylpyridinium(MPP⁺)primarilyactsasaninhibitorofmitochondrial complexI(C-I).Atthissite,MPP⁺exposurestimulatesO2 generation(i).Elevatedfreeradicallevelscanleadtooxidative modificationsofC-IandofmitochondrialcomplexIII(C-III)(ii).ThisincreasesandperpetuatesO2 formationbythe respiratorychain.Consequently,mitochondrialfragmentationisaccelerated,leadingtoincreasedO2 formationby fragmentedmitochondria(iii).MPP⁺alsotriggersvesiculardopamine(DA)releaseintoboththecytosolandthesynaptic cleft.CytosolicDAundergoesautoxidation,whichincreasesO2 formation(iv).TheO2 radicalcanbedismutatedto formH2O2,which,inthepresenceofiron,caninitiatetheFentonreactionanddrivetheHaber–Weisscycletoformhighly reactivehydroxylradicals(OH)(v).ExtracellularDAcanalsoundergoautoxidationandtherebyfavorO2 formation.Inthe presenceofironandH2O2,extracellularDAisoxidizedtoformthetoxicant6-hydroxy-DA(6-OHDA),whosesubsequent cyclizationgeneratesaminochromeandO2 (vi).AminochromecandirectlyinhibitC-I and,thus,leadstoaself- perpetuating cycle. Extracellular DA, accumulated by astrocytes, can be detoxified byglial monoamine oxidase (MAO),areactionthatinvolvesH2O2generation(vii).Glialcells,activatedbyinflammatorystimuliordegeneratingneurons, canserveasapotentsourceofO2,formedby,forexample,NADPHoxidases(viii).Glialcellsandneuronsarealso sourcesofnitricoxide(NO),generatedbyeitherconstitutivelyexpressedNOsynthase(NOS)-1orinducibleNOS-2(ix).A fittingexampleofreactiveoxygenspecies(ROS)functionisthefindingthatmicewereprotectedagainst1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine(MPTP)toxicityafteroverexpressionoftheantioxidativeenzymeCu/Zn-superoxide dismutase(Cu/Zn-SOD);conversely,endogenousCu/Zn-SODknockdownelevatedDAneuronMPTPsensitivity.How- ever,therelativecontributionofdifferentROSsourceswarrantsfurtherconsiderationwheninterpretingtheresults obtainedusingdiverseMPTP/MPP⁺modelsandextrapolatingtheresultstoPD.MitochondrialROSgenerationisamore complexcellbiologicalprocessthanpreviouslyassumed:MPTPandMPP⁺leadtomitochondrialfissionthroughoxidant effectsonvariousproteins,suchasdynamin-likeprotein-1(DLP-1),andmitochondrialfragmentationincreasesROS output.Notably,thetippingpointsofallprocesseslinkedtooxidativestressanddisturbedcellularproteostasisare dependentoncounter-regulations(antioxidants,chaperones,etc.).Thesedifferstronglybetweenexperimentalsystems (evenfrommousestraintomousestrain),whichhelpstoexplainwhytheliteratureisoftenapparentlycontradictory,with certainfindingsbeingchallengingtoreproduce,andwhypredictionsfrommodelstothehumandiseaseareassociated withlargeuncertainty.

(VTA).MPP⁺uptakebyothercatecholaminetransportersexplainsthepronounceddegenera- tionofnoradrenergicneuronsofthelocuscoeruleusintheMPTPmodel[25],butisinconsistent withtheresistanceofserotonergic,MAO-B-containingneurons[26].

These examplesofoutstanding questions andinconsistencies illustratehow thesimplified textbookviewisinsufficientforexplainingtheobservationsmade usingMPTPmodels(see OutstandingQuestions).Conversely,newinsightsintoMPTPmetabolismandbiodisposition, togetherwithexpandingknowledgeregardingtheintrinsicfeaturesofneuronalsubpopulations primarilyaffectedintheMPTPmodelandPD,haveledtoamorepreciseunderstandingofthe underlyingmechanisms.Here,wereviewthesenovelfindingstofacilitatetheinterpretationof data obtainedusingMPTPand/orMPP⁺models,andaddressthecriticalquestionofhow observations made inthesetest systems canbroadenourunderstandingof pathogenetic processesinPD.

WhyIsMPP⁺NotAccumulatingwithinAstrocytes?

Inthecentralnervoussystem(CNS),MPTPisconvertedtoMPP⁺almostentirelybyMAO-B,a mitochondrialouter-membraneenzymeexpressedpredominantlyinastrocytesand,toaminor extent, in serotonergic neurons [27,28]. Being charged, MPP⁺ cannot diffuse across cell

MPP ⁺

Inflamma acv tory

of gilaaon

Elavated protein aggregaon

rates oxidaProtonein Elevated requirement

for ATP

Decline in cellular A

TP Disturbances in

cellular Ca

2+

Overstrained

proteostasis Excessiv e acv

aon of repair

pathways Acute imbalance

between A gener TP

aon and consumpon

Cell death Cell

death

Energy Oxidave

Stress Depleon

High Low Low

vi

i ii

iv

iii

v

vii ix

viii

Figure3.1-Methyl-4-Phenylpyridinium(MPP⁺)AsaTriggerofViciousCycles.BindingofMPP⁺tocomplexI inhibitsmitochondrialATPsynthesisandtriggersO2 formation(i).Theseeventsinitiateaseriesofself-amplifyingandself- perpetuatingprocessesthatleadtothedemiseofthecellthroughaviciouscycle.TocompensateforelevatedO2

formation,thecellmustmaintainantioxidantsystems,whichfurtherburdenstheenergybudget(ii).AsaresultofCav1.3- mediatedpacemaking,nigrostriataldopamine(DA)neuronsfaceaconstantinfluxofextracellularCa²⁺.Inappropriate controlofcellularCa²⁺pools,asaconsequenceofanalreadystressedATPbudget(iii),canleadtoCa²⁺accumulationin mitochondriaandtherebytoanincreaseinmitochondrialO2 generation.SuchelevatedratesofROSformationleadto increasedratesofoxidativemodificationsofproteins,lipids,andDNA(iv),whichfurtherreducekeycellularfunctions,such asmitochondrialATPsynthesis.Moreover,cellularfunctionssuchasproteostasisarereducedasaconsequenceof inappropriateATPgeneration(v).Thisleadstoanaccumulationofoxidativelymodifiedandmisfoldedproteins(vi),which furthercompromisesmitochondrialfunction.ActivationofcellularrepairpathwaysleadstoanadditionaldemandforATP (vii),furtherenhancingthealreadyexistingimbalancebetweenATPconsumptionandgeneration(viii).Alloftheafore- mentionedeventsultimatelyresultintheinitiationofcelldamageordeath,andthisleadstoaninflammatoryactivationof surroundingglialcells.Glial-derivedreactiveoxygenandnitrogenspeciesfurtherincreaseoxidativestressintheremaining neurons(ix).

membranes,andwouldbeexpectedtoaccumulateandcausetoxicitywithinMPP⁺-producing cells.ThiscontrastswiththelackofastroglialdamagetypicallyobserveduponMPTPexposure [29,30], although astrocytesare not MPP⁺resistant. In astrocytes,genetically modifiedto expressDAT,MPP⁺accumulatesintracellularlyandelicitsatoxicresponsecomparabletothat inDAneurons[31,32].ThesefindingssuggestthatMPP⁺toxicityinanycelltypeisdetermined mainly by thetoxicant concentration reached insidemitochondria. Cellshave beenwidely demonstratedtoexhibitdistinctsusceptibilitieslargelyduetovariationsinMPP⁺uptakeacross the plasma membrane, availability of export mechanisms, and intracellular deposition in organelles.AstrocytesmightbesparedfromMPTP-inducedcelldeathbecauseofanefficient exportofMPTPmetabolites[32](Figure4).

OnekeycarrierthatenablesMPP⁺equilibrationacrosscellularmembranesistheorganic cationtransporter-3(OCT-3)[33].OCT-3ispreferentiallyexpressedinglialcellslocatednear theDAneurons,andOCT-3-deficientmicewerehighlyresistanttoMPTPtoxicity[33].This suggestsacriticalroleofOCT-3inMPTP-dependentneurodegeneration,althoughthemech- anisticbasisiscomplexbecauseOCT-3allowsnotonlytheexportofcationiccompounds, suchasMPP⁺ormonovalentparaquat,butalsotheiruptake[33,34].Dependingonthebrain regionandphaseofMPTPmetabolism,OCT-3-expressingcellsmightactasatransient‘sink’ forhighlevelsofextracellularMPP⁺,orOCT-3mightallowMPP⁺effluxfromcellsinwhichMPP⁺ ishighlyaccumulated.ThiscomplexMPP⁺distributionisalsoaffectedbytherelativeaffinitiesof thetransporters,asexemplifiedbytheK_mofDATbeinglowerthanthatofOCT-3(Figure4).

Near DAT-expressing neurons, this lower Km of DAT for MPP⁺ favors MPP⁺ uptake and retentionwithindopaminergicneurons[33].

MPTPmetabolites arealsoexportedthrough atransporter-independentmechanism[32].

UnderstandinghowMPP⁺isremovedfromastrocyteslackingOCTorrelatedtransporters requirescomprehensionofthemetabolicstepsinvolvedinMPTPbioactivation.Thistwo- stepprocessstartswithMAO-B-catalyzedtwo-electrona-carbonoxidationofMPTP,which generates the unstable intermediate 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) [35,36]. In the second step, which is nonenzymatic,MPDP⁺ undergoes autoxidation to formthestableMPP⁺[37](Figures1and4).AlthougharoleofMPDP⁺inextracellularMPP⁺ formationandaccumulationwassuggestedinearlyMPTPstudies[3,37],MPDP⁺wasonly recentlymeasuredinbraintissueandidentifiedasatransporter-independentexportmetab- oliteinitsuncharged,membrane-permeablefree-baseform:1,2-MPDP[32].Specificcon- ditions in braintissues have been shown to stabilize the intermediate intracellularlyand concurrently promote its conversion to MPP⁺ extracellularly. The intracellular milieu is characterizedbyloweroxygentensionduetomitochondrialconsumptionandmoderately moreacidicconditions comparedwith theextracellular environment[38].Whereasthese conditionspreventMPP⁺formationwhenMPDP⁺/1,2-MPDPisinsidecells,conversioninto MPP⁺ispromotedbythemorealkalineandoxygen-richenvironmentandtheiron-containing complexesintheextracellularspace[32](Figure4).IdentificationofMPDP⁺/1,2-MPDPasa membrane-permeableMPTPmetaboliteandelucidationoftheextracellularconditionsthat favoritsautoxidationtoMPP⁺provideaconvincingmechanismforexplainingtheresistance ofMPTP-convertingastrocytes(Figure4).TherapidclearanceofextracellularMPP⁺outof the brain guarantees a constant steep concentration gradientbetween intracellular and extracellularcompartmentsand,thus,asustainedeffluxofMPDP⁺/1,2-MPDPfromastro- cytes.ThismolecularexplanationforpreferentialMPP⁺formationintheextracellularspaceis alsorequiredforunderstandingthemechanismunderlyingthelong-recognizedDAT-depen- dentuptakeofextracellularMPP⁺intonigrostriatalDAneurons[32].Thenotionthatalarge fraction of MPP⁺ formed in the brain prevails in the extracellular space is support by microdialysis and mass spectrometry studies in MPTP-exposed mice and rats [39,40]:

MPP⁺levelsroserapidlythroughoutthebrain,butMPP⁺wasalsoclearedrapidly(1–2h)

(A)

(B)

(C)

1:2000 1:600

1:50 1:1

1:1 1:15

MPP ⁺

MPP ⁺ MPP ⁺

MPP ⁺ MPP ⁺

MPP⁺ MPP⁺

MPP⁺

MPP⁺ MPP⁺

MPP⁺ MPP⁺

MPDP+/

1.2-MPDP MPDP+/

1.2-MPDP

MPP⁺

ATP O ₂ ^–

MPP ⁺

MPP ⁺

MPP+

MPP+

MPP+

MPP+

MPP+

MPP ⁺

_ _ _

__ _ _ _ _ _ _

_ _ _

_ _

_ _ _ _ _ _ _

_ _ _

+ + +

+

+

+ _

_ _

Transporter-mediated uptake

Facilitated diffusion

Extracellular

Mitochondrial matrix Cytosol

Mitochondrium DA vesicles

ΔΨ

ΔΨ

DAT

DA neuron

VMAT-2

DAT OCT3

Passive carriers Diffusion

Other cells

MPTP

Metabolism of MPTP

+ –

DA derivaves Iron

Low pH Low O

tension -

- - -

MAO-B Mitochondrium

++ + ++

as trocyt e

STOP

OCT3

OCT3

Figure 4.

(Figurelegendcontinuedonthebottomofthenextpage.) Metabolism of1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) and Distribution of1- Methyl-4-Phenylpyridinium (MPP⁺).(A) Inthe brain,most MPTP isconverted byglialmonoamine oxidase-B (MAO-B)intotheintermediateMPDP⁺/1,2-MPDP,whichfreelydiffusesacrossbiologicalmembranes.Onceoutside astrocytes,theintermediatemighteitherdiffuseintoothercellsorundergononenzymaticautoxidationtoformthestable toxicantMPP⁺.SeveralfactorsfavorapreferentialformationofMPP⁺intheextracellularspace:insidecells,therelatively

in most areas, except regions rich in neurons that actively accumulate MPP⁺ via their catecholaminetransporters.

WhichEnzymesinNeurons orAstrocytesContributetoMPTPOxidation?

Chibaetal.[35]discoveredtheMAO-dependentactivationofMPTPtoformMPP⁺.Knockout studiesandpharmacologicalinterventionstudiesusingMAO-A-/MAO-B-selectiveinhibitors indicatedthatMAO-BhasadominantroleinMPTPactivationintheCNS[41].Analternative activation pathway, MPTP conversion by cytochrome P-450 2D6 (CYP2D6), was also suggested [42,43].Indeed,geneticassociation studiesrevealedalinkbetweenapolymor- phismatthelocusCYP2D6andPDsusceptibility[44].However,biochemicalactivitystudies indicatedthathighCYP2D6activitieswerecorrelatedwithadiminishedriskofPD[45,46].A potentialmolecularexplanationfortheseobservationsisthedetoxificationofendogenously formedb-carbolinesandisoquinolinesandinactivationofpesticidesbyCYP2D6[42,47].

TheaforementionedobservationsinPDraisethisquestion:doesCYP2D6haveaprotectiveor toxicity-enhancingroleintheMPTPmodel?ThemajormetabolitesproducedbyCYP2D6,1- methyl-4-(4-hydroxyphenyl)-1,2,3,6-tetrahydropyridine (OH-MPTP; p-hydroxylation) and 4- phenyl-1,2,3,6-tetrahydropyridine(PTP;N-demethylation)[47],arenot neurotoxic,andonly approximately10%ofMPTPistransformedintoMPDP⁺[48].Thisagreeswiththefindingthat thecytochromeP450inhibitordiethyldithiocarbamateincreasesMPTPtoxicityinmice[49].

Furthermore,CYP2D6upregulationprotectedagainstMPTPalsoinvitro[50].Analysisofthe respectivecontributionsofMAO-BandCYP2D6toMPTPoxidationrevealedthattheoxidation ratebyMAOisapproximatelythreefoldhigher[48].ThefactthatCYP2D6hasaminorroleonly inMPP⁺formationissupportedbyresultsobtainedusingMAO-B-knockoutmice,whichwere almostcompletelyprotectedagainstMPTP[41].Thus,evenifCYP2D6contributestoMPDP⁺ formation,theextentofthisreactionwouldlikelybeinsufficienttodamageandkillDAneurons.

Inconclusion,CYP2D6expressioninthenigrostriatalsystemcanberegardedasaprotective factorinidiopathicPDandintheMPTPmodel.

HowDoesSequestrationinside NeuronsDetermineMPP⁺Toxicity?

Efficient inhibitionof isolatedcomplex Iofthemitochondrial respiratorychain wasfound to require10–20mMMPP⁺[6],concentrationsthatare500–2000-foldhigherthanthosemea- suredinbrainhomogenatesafterMPTPtreatment[51,52].ThisraisesthequestionofhowMPP⁺ isaccumulatedintracellularlyatconcentrationssufficientforcomplexIinhibition(Figure4).

lowerpHandlowoxygentensioncontributetoastabilizationoftheintermediate,whereasintheextracellularspace,the availabilityofeithercatalyticallyactiveiron-containingcomplexesordopamine(DA)autoxidationproductscanstrongly acceleratetheautoxidationofMPDP⁺intoMPP⁺.ExtracellularavailabilityofMPP⁺isaprerequisiteforitscellspecific, transporter-dependentaccumulationincatecholaminergicneurons.InDAneurons,cytosolicMPP⁺iseitheractively accumulatedinneurotransmittervesiclesbyvesicularmonoaminetransporter-2(VMAT-2),orisenrichedinthemito- chondrialmatrix,drivenbythemitochondrialtransmembranepotential.Inmitochondria,MPP⁺inhibitscomplexIofthe respiratorychain,whichleadstothelimitationofmitochondrialATPproductionandtoanincreaseinO2 formationat complexI.MPP⁺distributioninthebrainisalsoaffectedbypassivetransporters,suchasorganiccationtransporter-3 (OCT-3).Suchbidirectionaltransportersallowamembranepotential-dependentuptakeofMPP⁺.Underconditionsoflow extracellularMPP⁺,thesetransportersalsofacilitatetheexportofintracellularMPP⁺.(B)TransportofextracellularMPP⁺ intothemitochondrialmatrixrequirespassageacrossthe(1)cellmembraneandthe(2)innermitochondrialmembrane.

PassivetransportersallowanintracellularaccumulationofthecationMPP⁺,accordingtotheNernstequation.Atagiven membranepotentialof 70mV,anapproximately15-foldconcentrationofMPP⁺cantheoreticallybeachieved.(C)Active transporters,suchastheDAtransporter(DAT),enablea unidirectionalbuild-upofMPP⁺inthecytosolagainsta concentrationgradientbyafactorofapproximately1:50.Inasecondstep,cytosolicMPP⁺isaccumulatedinthe mitochondrialmatrix,mostlydrivenbythetransmembranepotentialoftheinnermitochondrialmembrane.Thisstep permitsanadditionalconcentrationbyafactorofapproximately1:40.WhenextracellularMPP⁺ispresentinthelow micromolarrange,OCTexpressionisinadequate toenablequantitativecomplexIinhibition,whereasatthesame concentrations,DATexpressionallowsmillimolartoxicantconcentrationstobereachedinmitochondria,whichissufficient forinhibitingtherespiratorychain.

Whenpassivetransportersarepresent,theplasmamembranepotentialof 70mVwouldlead to an approximately 15-fold concentration of the monovalent cation MPP⁺ intracellularly, accordingtotheNernstequation[32].Moreover,isolatedrespiringmitochondriaaccumulate MPP⁺byafactorofapproximately40[6].GiventhattissueMPP⁺concentrationsof>10mM can bereached in standard MPTP models in vivo [51,52], this two-step electrochemical accumulationwouldallowMPP⁺toreachmillimolarconcentrationsinthemitochondrialmatrix (Figure4).However,suchconcentrationswouldbemaintainedonlytransiently:MPTPintoxi- cationischaracterizedbyrapidMPP⁺clearance fromthe brain(half-life inthe1-h range).

Moreover,cytosolicMPP⁺effluxthroughabidirectionalcarrierwouldreduceitsmitochondrial concentration,whichwouldleadtothesparingofcellsfromMPP⁺toxicity.

Theaforementionedscenarioiscontrastedbywhatoccursincatecholamineneurons,which expresstwoactivetransporters:aplasmamembranecatecholaminetransporter,suchasDAT;

and the vesicular transporter VMAT-2. Unlike the OCT-transporter-mediated bidirectional passiveflux, catecholaminergictransporters driveactive,unidirectionalMPP⁺accumulation (Figure 4). Once MPP⁺ is inthe cytosol, its further sequestration, either by mitochondrial accumulationorbyVMAT-2-mediatedvesicularaccumulation,largelydeterminesMPP⁺tox- icity.WhereasDAToverexpressionenhancedneuronalMPTPsensitivity[53],pharmacological inhibition of DAT protected against MPTP toxicity [5]. VMAT-2 mediates vesicular MPP⁺ sequestration, which prevents MPP⁺ toxicity and, accordingly, VMAT-2 overexpression resultedindiminishedtoxicity[54],whereasitsgeneticablationorpharmacologicalinhibition elevatedtheMPTPsensitivityofDAneurons[55].AnalysisofDATandVMAT-2expressionin distinctneuronalpopulationsrevealedthattheDAT:VMAT-2ratiowashigherinMPTP-sensitive striatalterminalsofnigralDAneuronsthanincomparativelylesssensitiveVTAneurons[56].

DisturbancesinvesicularstorageaffectnotonlyDAneurons,butalsonoradrenergicneuronsof thelocuscoeruleus.Inalow-VMAT-2-expressionmousemodel,locuscoeruleusnoradrener- gicneurons exhibited progressive degeneration even before the onset of nigrostriatal DA neurodegeneration[57].Conversely,polymorphismsidentifiedinthehumanVMAT-2promoter region indicatedthatelevatedVMAT-2expressioncorrelatedwith reducedPDrisk[58,59].

Notably,miceexposedduringdevelopmenttodieldrin,anenvironmentaltoxicant,displayedan increasedDAT:VMAT-2ratiointheadultnigrostriatalsystemand,consequently,weremore vulnerabletoMPTPtoxicitythanwereunexposedcontrolmice[60].Inconclusion,notonlythe activityofcellsurfacecatecholaminetransporters,butalsointracellularvesicularsequestration determinesthewidelyrecognizedvariationsinMPTPsensitivityofdistinctcatecholaminergic populationsofthebrain.Thesefactorsmust becarefullyconsidered wheninterpretingthe region-selectivedifferencesinneuronaldegenerationobservedinMPTPmodels.

FeaturesofNigrostriatalDA NeuronsThatAffect theTippingPointof MPP⁺ Toxicity

Asdescribedabove,mechanismsofMPP⁺production,accumulation,andsequestrationare nottheonlydeterminantsoftheMPTPsusceptibilityofneuronalsubpopulations.Thisconcept isexemplifiedbytheevidencethatnotallcatecholaminergicneurons(i.e.,notallneuronsthat cantakeupMPP⁺)areequallytargetedbyMPTPneurotoxicity[23,24,26].Specificneuronal featureshaverecentlybeenidentifiedasmodulatorsoftheselectiveeffectsofMPTP,andthese susceptibilityfactorscouldalsohaveacriticalroleinPDpathogenesis.

IntracellularCa²⁺andItsInfluenceonEnergyExpenditure

AmongtheendogenousfactorsthatcontributetoselectiveneuronaldegenerationintheMPTP model,dysregulation ofintracellularCa²⁺homeostasishas a central role[61] (Figure5),a conclusion supported by evidence from studies illustrating a neuroprotective influence of intracellularCa²⁺regulation[62].Accordingly,theendogenousCa²⁺-bindingproteincalbindin appears topromoteneuroprotection:MPTP-resistantVTAneuronsexpresshigherlevelsof

calbindinthandoneuronsintheMPTP-sensitivesubstantianigraparscompacta(SNpc),which agreeswiththepossibilitythatcalbindinconfersresistanceagainstdegenerationintheMPTP model [63]. Notably, the ranking of DA neuron susceptibility in the retrorubral area (A8), substantia nigra (A9),and VTA (A10) inverselycorrelates with calbindin expression inthe respectiveregionsinboth miceand monkeys[64].Moreover,thesame relativerankingis foundinPDandinthehumanbrain,andcorrelateswiththepatternofcalbindinexpression [63,65].

AmajordevelopmentinresearchontherelationshipbetweenCa²⁺homeostasisandselective neurodegenerationwastheidentificationofanautonomouspacemakingactivityofSNpcDA neuronsanditsrelianceontheL-typeCa²⁺channelCav1.3[66,67].PDisassociatedwithcell lossinnotonlytheSNpc,butalsothelocuscoeruleusandhypothalamictuberomammillary nucleus[68].Intriguingly,theseothertworegionsalsoexhibitautonomouspacemakingactivity, againmediatedbyCav1.3[69,70].Theseobservationsrevealapotentialcorrelationbetween theuseofextracellularCa²⁺forpacemakingandelevatedsensitivitybothinPDandinMPTP models.Forinstance,olfactorybulbdopaminergicneuronsarerelativelysparedfromdegen- erationinPDandintheMPTPmodel;unlikeSNpccells,theseneuronsexpressT-typeCa²⁺

channelsanddonotrelyonoscillatoryCa²⁺wavesforpacemakingactivity[71].Analysisofthe pacemakingactivityofVTAneurons,apopulationthatdisplayshigherresiliencethandoesits nigrostriatalcounterpart,indicatedtheirrelianceonNa⁺insteadofCa²⁺channels[72].

(i) DA autoxidaon

(ii) Glial environment

(iii) Large neurite field

(iv) Ca_v 1.3 pacemaking

(v) Metabolic phenotype

ROS and cytokines

High ener demand gy

No back

up ener gy

Cell death

ASYN

Figure5.EndogenousSusceptibilityFactorsofNigrostriatalDopamine(DA)Neurons.NigrostriatalDAneurons arecontinuouslyexposedtoaseriesofstressors,suchaselevatedlevelsoffreeradicals,derivedfromDAautoxidation,(i) andfromreactiveoxygenandnitrogenspecies,whichcanbegenerateduponinflammatoryactivationofmicrogliain responsetoinitialneuronaldamage(ii).NigrostriatalDAneuronsdifferfromseveralotherneuronalpopulationsbyvirtueof theirlargetotalneuritelength,ahigherdegreeofneuritebranching,and,consequently,alargenumberofsynapsesper neuron(iii).Thisleadstoaparticularlyhighenergydemand,whichisfurtherincreasedbecausenigrostriatalDAneuronsare autonomouspacemakersanduseCav1.3L-typeCa²⁺channelsforpacemaking(iv).MaintenanceofintracellularCa²⁺

homeostasisrepresentsacontinuouschallengetothecellularATPbudget.Theindividualmetabolicphenotype(i.e.,the highrelianceonmitochondrialmetabolisminsteadofglycolysis)furthercompromisesDAneuronsurvival(v).Noneofthese factorsindividuallycompromisestheviabilityofhealthyneurons,buttheircombinationincreasesthesusceptibilitytoany additionalexogenousstressor.Thisisalsothecasewiththehighlyabundantproteina-synuclein(ASYN),whichis expressedwidelythroughoutthenervoussystem.Whereasthenativeproteinmostlikelyperformsantioxidantand protectivefunctions,itengagesinmultiplereciprocalinteractionswithcytosolicDA,elevatedcalcium,orsurroundingglia, andtheuniquecombinationofASYN,Cav1.3,andDAmightrepresentamajorsusceptibilityfactorfornigrostriatal neurons.

InvolvementofextracellularCa²⁺forpacemakingisdemandingmetabolically,becauseexport ofthedivalentionCa²⁺requiresahigherATPinvestmentperchargethandoestheuseof monovalentions[61,73].Acorrelationbetweenpacemaking-dependentenergyexpenditure andMPTPvulnerabilitywas elegantlydemonstratedinacellmodelinwhichendogenous Cav1.3 was either pharmacologically inhibited or genetically ablated; both conditions resulted in a ‘rejuvenation’ of neurons, which indicated that their pacemaking activity switchedfromadependenceonCa_v1.3toadependenceonvoltage-gatedNa⁺channels [74]. These rejuvenated neurons were considerably more resistant to MPTP/MPP⁺ or rotenonetoxicitythanweretheirCav1.3-expressingcounterparts[74,75].Collectively,these observations identifiedCav1.3channelsas potentialtargets fortherapeuticinterventionin PD.Notably,humandataonhowchronictreatmentwithCa²⁺-channelblockersmightaffect PDdevelopmentarealreadyavailable,becausethesecompoundsarefrequentlyadminis- teredtotreatpatientswithhypertension.Analysisoftheseinitialclinicalandepidemiological dataindicatedthatPDriskwasreducedinpatientschronicallytreatedwithCa²⁺-channel blockers[76,77].

MorphologyofSNpcDANeuronsAsaDeterminantofMPP⁺Toxicity

Thepacemaker-associatedoscillatoryCa²⁺influxisnot theonlyintrinsicfeature underlying increasedenergydemand:theuniquearchitectureofSNpcDAneurons,whichhadlongbeen largelyignored,iscurrentlysuggestedtorepresentanotheressentialfeaturethatcontributesto selectiveneurodegenerationinPDandintheMPTPmodel [78](Figure5).NigrostriatalDA neuronsarecharacterizedbyunmyelinated,highlybranchedaxonsthatcanhaveatotallength ofupto0.5m(500000mm)perhumanneuron[79,80].Tomaintainsuchlongprojections, neuronsmustexpendahighamountofenergy,whichpredisposestheneuronstoanenergy crisisinthepresenceofmetabolicinhibitors.Moreover,thenumberofsynapsesformedby nigrostriatalDAneuronsisatleasttwoordersofmagnitudehigherthanthatformedbytheless vulnerableDAneuronsintheVTA[78,80,81].TheratSNpccontainsapproximately12000DA neurons[80],eachofwhichformsapproximately100000–250000synapses;inthehuman brain,this is anothertenfold higher (1 million–2.4 million synapses per neuron) [78,82]. A computationalmodelofnigrostriatalDAneuronsindicatedthattheneurite-treesizeincombi- nationwith the high synapse number represents anotable burden for thecellular energy budget: the energy cost increases exponentially with the total length and the degree of branchingofthesynapticfield[83].Consequently,nigrostriatalDAneuronscontinuouslyface anenergydemandthatisbarelymetbytheoxidativecapacityoftheirmitochondria[84].These anatomicalfeatures,inadditiontothehighenergyrequirementcreatedbyCav1.3-mediated influxofextracellularCa²⁺,renderSNpcDAneuronsenergetically‘ontheedge’[78].Insucha scenario,evenmoderatestressorscouldleadtoanimbalanceofcellularenergyprovisionand consumption(Figures 3and5).The conceptofa lowresidualenergycapacitycould also provide a mechanisticbasisfor explainingtheselective lossofnigrostriatalDA neurons in modelsofsystemicallyappliedrotenone,which,unlikeMPP⁺,isnotselectivelyaccumulatedin definedcelltypes.

SynergyofAlpha-SynucleinandOtherSusceptibilityFactorsAsaDrivingForce

Besidestheuniquecalciumhandlingandmorphologyofnigrostriatalneurons,othersuscepti- bilityfactorshavebeenproposed.Oneoftheimportantcontributorsisa-synuclein(ASYN).

ASYNhasbecomerecognizedoverthepasttwodecadesasakeyplayerinPDpathogenetic processes [85–87]. The proteinis expressed at high levels in normal brains, is a primary constituent ofLewybodies(theintraneuronalinclusionsfoundinmostPDbrains),andcan spread in a prion-like pathology across cells [85,88]. Single-point mutations, as well as multiplicationsofthegeneencodingASYN(SNCA),arecausallylinkedtofamilialPD.Besides thegeneticassociationsofPDandASYN,thereisalsoanintriguingrelationshipbetweenASYN andtheoutcomeofPD-relevanttoxicexposures,suchastoMPTP.Vilaandcolleagueswere