RESEARCH ARTICLE◥

PARKINSON’S DISEASE

Dopamine oxidation mediatesmitochondrial and lysosomaldysfunction in Parkinson’s diseaseLena F. Burbulla,1,2 Pingping Song,1 Joseph R. Mazzulli,1,2 Enrico Zampese,3

Yvette C. Wong,1 Sohee Jeon,1 David P. Santos,1 Judith Blanz,1

Carolin D. Obermaier,4,5,6 Chelsee Strojny,1 Jeffrey N. Savas,1 Evangelos Kiskinis,1

Xiaoxi Zhuang,7 Rejko Krüger,4,6,8 D. James Surmeier,3 Dimitri Krainc1,2*

Mitochondrial and lysosomal dysfunction have been implicated in substantia nigradopaminergic neurodegeneration in Parkinson’s disease (PD), but how these pathways arelinked in human neurons remains unclear. Here we studied dopaminergic neurons derivedfrom patients with idiopathic and familial PD. We identified a time-dependent pathologicalcascade beginning with mitochondrial oxidant stress leading to oxidized dopamineaccumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity,lysosomal dysfunction, and a-synuclein accumulation. This toxic cascade was observed inhuman, but not in mouse, PD neurons at least in part because of species-specificdifferences in dopamine metabolism. Increasing dopamine synthesis or a-synucleinamounts in mouse midbrain neurons recapitulated pathological phenotypes observed inhuman neurons. Thus, dopamine oxidation represents an important link betweenmitochondrial and lysosomal dysfunction in PD pathogenesis.

Parkinson’s disease (PD) is the second mostcommonneurodegenerative disorder (1). Al-though several neuronal populations areaffected in PD, substantia nigra pars com-pacta (SNc) dopaminergic neurons are

among the first neurons to degenerate, leadingto motor symptoms of PD (2). Familial genes inPD point to both mitochondrial and lysosomalmechanisms as contributors to pathology (1).However, how mutations in components of twodistinct essential cellular pathways both result inindistinguishable clinical and pathological pheno-types and finally cause the death of dopaminergicneurons in patients remains unclear. Moreover,becausemost PDmousemodels do notmanifestnigral degeneration (3), it is unclear whether thisinteraction is specific to human neurons.Loss-of-function mutations in DJ-1 cause early-

onset autosomal recessive PD (4), and DJ-1 is

known to orchestrate oxidant defenses becauseits deletion leads to elevated mitochondrial oxi-dant stress (5). To examine whether elevatedmitochondrial oxidant stress triggers downstreampathological phenotypes, fibroblasts from PDpatients with a homozygous loss-of-functionmutation inDJ-1 c.192G>C (6)were reprogrammedinto induced pluripotent stem cells (iPSCs). Thesewere then differentiated into dopaminergic neu-rons, which were grown in long-term cultures(fig. S1, A to D, and fig. S2, A and B). For com-parison purposes, dopaminergic neurons werealso generated from a healthy individual with asingle copy ofmutatedDJ-1, idiopathic PDpatientswithout DJ-1 mutations, and healthy individualswithout PD. These neurons had anatomical andelectrophysiological characteristics of SNc neu-rons (7), including robust, low-frequency auton-omous pacemaking (fig. S2C) and prominentvoltage sag upon brief injection of strong hyper-polarizing currents (fig. S2D). DJ-1 protein amountswere decreased by ~50% in heterozygous and un-detectable in homozygous DJ-1 mutant neurons(fig. S2E).

Neuromelanin and oxidized dopamineaccumulate in dopaminergic neuronsfrom genetic and sporadic PD patients

By day 50 (d50) in culture, mitochondrial oxi-dant stress was elevated in human homozygousDJ-1 mutant dopaminergic neurons (Fig. 1A). Thisincrease was seen by using both a genetically en-coded, mitochondrial matrix redox sensor (mito-roGFP) (5) and by fluorescence measurements

using 2′,7′-dichlorodihydrofluorescein diacetate(H2DCFDA) at d50 and d70 (Fig. 1B). Increasedmitochondrial oxidant stress was accompaniedby a drop in basal respiration in mutant neuronsat d50 (Fig. 1C), suggesting that the observedstress damaged mitochondria.Homozygous DJ-1 mutant neurons also accu-

mulated membrane-bound, dense pigmentedaggregates consistent with the appearance ofneuromelanin (Fig. 1D and fig. S2F). Biochemicalproperties of these pigments, such as their in-solubility to strong detergents, further indicatedtheir similarity to neuromelanin extracted fromhuman brain (8). Neuromelanin is a complexaggregate, including oxidized dopamine, pro-teins, and lipids (9). To quantify the amount ofoxidized dopamine in these neurons, a nearinfrared fluorescence (nIRF) assay was used (10).Oxidized dopamine progressively increased fromd70 to d150 in homozygous DJ-1 mutant neu-rons, but not in controls (Fig. 1E). To determinewhether the absence of DJ-1 protein was suffi-cient to induce this pathological phenotype, clus-tered regularly interspaced short palindromicrepeats (CRISPR)–Cas9 was used to generatehomozygous DJ-1 knock out (KO) iPSC-derivedneurons (DJ-1 KO) from controls (fig. S3). Theseneurons recapitulated the phenotype of DJ-1 pa-tient neurons, resulting in the progressive accu-mulation of oxidized dopamine from d70 to d90(Fig. 1F), as well as decreased basal mitochon-drial respiration (Fig. 1G).Dopaminergic neurons from idiopathic PD

patients [iPD1, (11); iPD2, fig. S1, E and F] alsomanifested a similar phenotype, including de-creased basal respiration and oxidized dopamineaccumulation, but at later time points (d150,d180) (Fig. 1H and fig. S4A). We observed partialinactivation of DJ-1 in iPD neurons (fig. S4B),suggesting that decreased DJ-1 function maycontribute to these delayed pathological alter-ations in iPD neurons (12).

Dopamine-mediated modification ofglucocerebrosidase (GCase) contributesto downstream lysosomal dysfunction

Because neuromelanin granules have been foundin lysosomes (13), theaccumulationofneuromelanin-like aggregates in mutant dopaminergic neuronssuggested that lysosomal function might be im-paired. Although there was no detectable impair-ment in lysosomal proteolysis in homozygousDJ-1mutant neurons at d70 (Fig. 2A), the activityof the lysosomal enzyme glucocerebrosidase,an important PD risk factor implicated in lyso-somal dysfunction in PD (14), was decreased inlysosomal fractions (Fig. 2B). By contrast, therewas no drop in the activity of another lyso-somal enzyme, a-iduronate-2-sulfatase (a-i-2-sulf). However, lysosomal proteolysis was sig-nificantly diminished at a later time point (d180)(Fig. 2A and fig. S5), suggesting that accumulatedoxidized dopamine contributed to lysosomaldysfunction in homozygous DJ-1 mutant neu-rons. Idiopathic PD neurons exhibited a delayeddecrease in GCase activity (d180) compared toDJ-1mutant neurons (d70) (Fig. 2C). To determine

RESEARCH

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1Department of Neurology, Northwestern University FeinbergSchool of Medicine, Chicago, IL 60611, USA. 2Department ofNeurology, Massachusetts General Hospital, Harvard MedicalSchool, MassGeneral Institute for Neurodegeneration,Charlestown, MA 02129, USA. 3Department of Physiology,Northwestern University Feinberg School of Medicine,Chicago, IL 60611, USA. 4Department for NeurodegenerativeDiseases and Hertie-Institute for Clinical Brain Research,University of Tübingen, DZNE, German Centre forNeurodegenerative Diseases, Tübingen, Germany. 5GraduateSchool for Cellular and Molecular Neuroscience, University ofTübingen, Germany. 6Clinical and ExperimentalNeuroscience, Luxembourg Center for Systems Biomedicine,University of Luxembourg, Luxembourg. 7Department ofNeurobiology, University of Chicago, Chicago, IL 60637, USA.8Centre Hospitalier Luxembourg, Luxembourg.*Corresponding author. Email: dkrainc@nm.org

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if other forms of genetic PDmight show similaralterations, we generated iPSC-derived neuronsfrom PD patients with homozygous mutationsin the parkin, PINK1, and LRRK2 (15, 16) genesand neurons carrying triplication of wild-type(WT) a-synuclein (17). We observed a similar in-crease in oxidized dopamine across all genotypes(fig. S4, C and E to G) as well as decreased GCaseactivity in parkinmutant neurons (fig. S4D), fur-ther suggesting the presence of common pheno-types across both sporadic and familial formsof PD.Because elevated oxidant stress and oxidized

dopamine were detected concomitantly withdecreased GCase activity (Figs. 1 and 2, A to C),we next sought to determine if reduced GCase

function could have resulted from oxidation ofthe protein. Indeed, the activity of recombinantGCase, but not a-i-2-sulf, was reduced by incuba-tion with dopamine (Fig. 2D). Using in-gel nIRF,dopamine incubation was found to modify GCasecompared to a-i-2-sulf, and these changes wereblocked upon antioxidant treatment with N-acetylcysteine (NAC) (Fig. 2, E and F). Usingtandem mass spectrometry (MS/MS), we iden-tified cysteine residues in the catalytic site ofGCase that weremodified by dopamine quinones,reactive oxidized forms of dopamine (Fig. 2G).These results suggested that oxidant stress di-rectly modifies and disrupts GCase enzymaticactivity and contributes to lysosomal dysfunctionin PD neurons.

Mitochondrial antioxidants and calciummodulators attenuate the toxic cascadein DJ-1 mutant dopaminergic neuronsWe found that long-term treatment with eitherthe mitochondrial-targeted antioxidant mito-TEMPO or NAC blunted the accumulation ofoxidized dopamine and improved lysosomal GCaseactivity and proteolysis (Fig. 3, A to D). Fur-thermore, because increased Ca2+ entry throughCav1 voltage-gated calcium channels enhancesmitochondrial oxidant stress and dopamine syn-thesis (5, 18), we tested whether the Cav1 channelantagonist isradipine (19) or the calcium-dependentserine-threonine phosphatase calcineurin inhib-itor FK506 (20) could also regulate dopamineoxidation. Both isradipine and FK506 significantly

Burbulla et al., Science 357, 1255–1261 (2017) 22 September 2017 2 of 7

Fig. 1. Neuromelaninand oxidized dopa-mine accumulate inPD patient dopa-minergic neurons.(A) Neuronal expres-sion of mito-roGFPand quantification ofrelative oxidation incontrol (ctrl) andhomozygous DJ-1mutant (hom) neu-rons at d50 (n = 3).Scale bar, 10 mm.(B) H2DCFDA fluores-cence in neurons atd50 and d70 (n = 3).(C) Oxygen con-sumption rate (OCR)in neurons underbasal conditions atd50 (n = 3). (D) Elec-tron microscopy (EM)image of neuromela-nin deposition (arrow)in homozygousDJ-1 mutant neuronat d90. Scale bar,200 nm. (E and F) Oxi-dized dopamine(DA) by nIRF at d90 in(E) homozygousDJ-1 mutant [hom(1)and hom(2)]neurons and controls,quantification atd70, d90, and d150(n = 3), het forheterozygous, or(F) DJ-1 KO and iso-genic control neuronsand quantificationat d70 and d90 (n = 3or 4). (G) OCR inDJ-1 KO neurons andisogenic controls underbasal conditions at d50 (n = 12). (H) Oxidized dopamine in control and twoidiopathic PD (iPD1, iPD2) neurons at d70,d150, andd180 (n=3).Quantificationfor each iPD line is normalized to control for each time point. Equal protein

concentrations were used for nIRFassays. Error bars,means ± SEM. *P <0.05;**P < 0.01; ***P < 0.001; Student’s t test (A), (B), and (F) to (H) or one-wayanalysis of variance (ANOVA) with Tukey post hoc test (C) and (E).

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diminished the accumulation of oxidized dopa-mine (Fig. 3E), highlighting the importance ofCa2+ in regulating dopamine oxidation in DJ-1 mutant neurons.Oxidant stress has also been found to drive

a-synuclein aggregation, the primary structuralcomponent of Lewy bodies and a pathologicalhallmark of PD (21). Furthermore, dopamineadducts can modify a-synuclein and promote itsaggregation, and a recent study reports that lossof DJ-1 function is associated with Lewy bodypathology (22). Soluble a-synuclein was elevatedat d70 in both homozygous DJ-1 mutant andCRISPR-Cas9–generated DJ-1 KO neurons (Fig. 3,F and G). We observed a time-dependent in-crease in oxidized, insoluble a-synuclein fromd70 to d100 in mutant neurons (fig. S6, A and B),but not in heterozygous DJ-1 neurons or controls.NAC treatment also decreased a-synuclein accu-mulation in DJ-1 mutant neurons (Fig. 3H andfig. S6, C and D). To determine whether dopa-mine contributed to this phenotype, neurons weretreated with a-methyl-p-tyrosine (AMPT), whichcompetitively inhibits tyrosine hydroxylase andprevents dopamine synthesis. AMPT treatmentdiminished mitochondrial oxidant stress, as wellas oxidized dopamine (Fig. 3, I and J), and re-duced soluble a-synuclein amounts in DJ-1mutantneurons (Fig. 3K), suggesting that mitochondrial

oxidant stress and oxidized dopamine contrib-ute to a-synuclein accumulation in PD neurons.

Increasing dopamine or a-synuclein inmouse neurons recapitulates pathologyin human neurons

We next examined whether this toxic cascadeobserved in human PD neurons also occurredin SNc dopaminergic neurons from PD mousemodels. In contrast to human neurons, SNcfrom DJ-1 KO mice showed negligible amountsof oxidized dopamine at 3 and 12 months of age(Fig. 4, A and B). In addition, soluble and in-soluble a-synuclein amounts were normal in SNcfrom DJ-1 KO mice (Fig. 4C and fig. S7, A and B),and lysosomal GCase activity was not altered(Fig. 4G) compared toWTmice. Moreover, tyrosinehydroxylase (TH) immunoreactivity in the SNc(Fig. 4I) was not decreased in DJ-1 KO mice,indicating a lack of nigral pathology.Because mutations in a-synuclein that result

in its accumulation lead to familial PD, we thenasked whether increasing amounts of mutanta-synuclein in vivomight drive this toxic cascadein DJ-1 KO mice. We generated mice with bothdopamineneuron-specific overexpressionofhumana-synuclein A53T (23) and constitutive DJ-1 defi-ciency (DASYN53 × DJ-1 KO) (fig. S8A). These miceshowed elevated amounts of oxidized dopamine in

nigral neurons (Fig. 4D) and decreased lysosomalGCase activity (Fig. 4E) compared toDJ-1 KOmice.Heterozygous expression of DJ-1 in DASYN53 micewas sufficient to ameliorate pathological pheno-types observed inDASYN53×DJ-1KOmice (fig. S8, Bto D) suggesting that a combination of a-synucleinaccumulation and loss of DJ-1 contributes to nigraldegeneration in mice.We next investigated whether increasing do-

pamine amounts in vivomight also trigger pathol-ogy in mice. Chronic feeding of WT or DJ-1 KOmicewith levodopa (L-dopa)–supplemented chowled to oxidized dopamine accumulation in thenigra (Fig. 4F and fig. S7C). Treatment with L-dopa in DJ-1 KOmice, but not in WT mice, wassufficient to disrupt SNc GCase enzymatic ac-tivity (Fig. 4G), increase a-synuclein accumu-lation (Fig. 4H and fig. S7D), and trigger the lossof dopaminergic neurons (Fig. 4I and fig. S7E).Treatment with L-dopa had no effect on homo-cysteine amounts (fig. S7F).

Altered calcium and dopaminemetabolism contribute to differencesbetween human and mouse neurons

To directly compare human neurons withmouseneurons, we extended these experiments tomouseiPSC-derived dopaminergic neurons generatedfrom DJ-1 KO and WT fibroblasts (fig. S9A).

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Fig. 2. Dopamine-mediated modification of GCase and lysosomal dys-function in PD patient neurons. (A) Lysosomal proteolysis in control(ctrl) and homozygous DJ-1 mutant (hom) neurons at d70 and d180 (n =3). (B) GCase and a-i-2-sulf activity in lysosomal fractions from controland homozygous DJ-1 mutant neurons at d70 (n = 3). (C) GCase activityin lysosomal fractions from control and idiopathic PD (iPD) neurons atd70 and d180 (n = 3). (D) Recombinant GCase and a-i-2-sulf activityafter incubation with DA or phosphate-buffered saline (PBS) (n = 5).

(E and F) Oxidized DA by in-gel nIRF of (E) recombinant GCase or (F)a-i-2-sulf after incubation with DA, DA+NAC, or PBS. Coomassie brilliantblue (CBB) was used to visualize total protein. (G) MS/MS spectrum ofGCase treated with DA. Modified cysteine is indicated with mass adduct inparentheses. Prominent b (blue) and y (red) ions and fragments containingthe additional mass (bold) are indicated. Error bars, means ± SEM. *P <0.05; ***P < 0.001; one-way ANOVA with Tukey post hoc test (A) orStudent’s t test (B) to (D).

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Fig. 3. Mitochondrial antioxidants and calcium modulators attenuatethe toxic cascade in DJ-1 mutant dopaminergic neurons. (A andB) Oxidized DA in homozygous DJ-1 mutant (hom) neurons treated with(A) mito-TEMPO or (B) NAC compared to vehicle (veh) at d70 (n = 3).(C) GCase and a-i-2-sulf activity in lysosomal fractions from homozygousDJ-1 mutant neurons treated with mito-TEMPO or vehicle at d70 (n = 3).(D) Lysosomal proteolysis in homozygous DJ-1 mutant neurons treatedwith NAC or vehicle at d180 (n = 3). a.u., arbitrary units. (E) Oxidized DA inhomozygous DJ-1 mutant, heterozygous DJ-1 carrier (het), and controlneurons treated with isradipine, FK506, or vehicle (DMSO, dimethyl sulfoxide)at d90 (n = 3 to 6). (F and G) Immunoblot analysis of a-synuclein (syn211antibody) at d70 in Triton X–100 (T)–soluble neuronal lysates from (F) control,

heterozygous DJ-1 carrier, and two homozygous DJ-1mutant lines (n = 4) or (G)a gene-edited DJ-1 KO line (n = 3 or 4). b-III-tubulin, synapsin, and GAPDH(glyceraldehyde-3-phosphate dehydrogenase) were used as loading controls.(H) T-insoluble a-synuclein (C20 antibody) at d70 in homozygous DJ-1 mutantneurons treated with NAC or vehicle. CBB was used as a loading control (n = 4).(I) Homozygous DJ-1 mutant neurons expressing mito-roGFP treated with AMPTat d50 (n = 3). (J and K) Homozygous DJ-1 mutant neurons treated with AMPTand analyzed at d70 for (J) oxidized DA by nIRF (n = 3) or (K) T-soluble a-synuclein(syn211 antibody). GAPDH and b-III-tubulin were used as loading controls (n = 3).Treatment was applied for 30 days (A) to (C), (E), and (H) to (K) or 140 days(D). Error bars, means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test(A) to (D) and (G) to (K) or one-way ANOVA with Tukey post hoc test (E) and (F).

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Consistentwith the in vivo results,DJ-1KOmouseneurons did not manifest increased amounts ofoxidized dopamine at d40, d70, or d90 (Fig. 5A).However, treatment of these neurons with L-doparesulted in increased oxidized dopamine (Fig. 5B),disrupted lysosomal GCase activity (Fig. 5C), anddecreased neuronal survival (fig. S9B). There wereno pathological changes observed in L-dopa–treatedmouse WT neurons (Fig. 5, B and C), furthersuggesting that both mitochondrial oxidant stressand increased oxidized dopamine contribute toneurotoxicity of dopaminergic neurons.Using high-performance liquid chromatog-

raphy (HPLC) for detection of dopamine and

its metabolites, we found that human controlneurons showed elevated amounts of dopaminecompared to mouse WT neurons (Fig. 5F), sug-gesting a difference in dopaminemetabolism be-tween species. Consistent with this notion is theobservation that L-dopa treatment was sufficientto increase oxidized dopamine in human control(fig. S10), but not mouse WT, neurons (Fig. 5B).Dopamine can either be converted into non-toxic downstream metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), or formtoxic dopamine quinones and aminochrome,which are normally sequestered in neuromelanin(9). We hypothesized that increased oxidative

stress in DJ-1 KO mouse neurons may preferen-tially result in oxidized dopamine and neuro-melanin upon treatment with L-dopa. Althoughthe baseline amounts of total dopamine werecomparable between mouse WT and DJ-1 mu-tant neurons (Fig. 5G), mutant neurons showeda dramatically decreased DOPAC/dopamineratio when treated with L-dopa compared toWT neurons (Fig. 5H). Thus, decreased dopa-mine degradation into nontoxic metabolites maybe responsible for the accumulation of oxidizeddopamine.Because the calcineurin inhibitor FK506 di-

minished the accumulation of oxidized dopamine

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Fig. 4. Increasing dopamine synthesisor amounts of a-synuclein in mousemidbrain neurons recapitulatespathological phenotypes observed inhuman neurons. (A and B) OxidizedDA in substantia nigra (SNc) fromDJ-1 KO and WTmice at 3 and 12 monthscompared to human control neurons atd70. (C) T-soluble a-synuclein (C20antibody) in SNc of WT and DJ-1 KOmice. GAPDH and NSE (neural-specificenolase) loading controls (n = 3 pergroup). (D and E) SN from DJ-1 KO andDASYN53 × DJ-1 KO mice analyzed at8 months of age for (D) oxidized DA and(E) GCase activity (n = 5 per group).(F to I) WT and DJ-1 KO mice were fedL-dopa–supplemented or vehicle-treatedchow for 6 months and substantia nigraanalyzed at 14 months for (F) oxidized DA(n = 3 or 4 per group), (G) GCase activity(n = 3 or 4 per group), (H) T-insolublea-synuclein (C20 and syn202 antibodies)with CBB loading control (n = 3 or 4per group), and (I) number of DAB(3,3′-diaminobenzidine)–stainedTH-positive neurons (n = 3 per group).Equal protein concentrations were usedfor nIRF assays. Error bars, means ± SEM.*P < 0.05; **P < 0.01; ***P < 0.001;Student’s t test (C) to (E), and (H), orone-way ANOVA with Tukey post hoc test(B), (F), (G), and (I). n.s., not significant.

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(Fig. 3E), we examined if calcineurin might bealtered between mice and humans. Control hu-man neurons showed both increased proteinamounts and activity of calcineurin (Fig. 5, Dand E) compared to mouse WT neurons, furthersuggesting that alterations in calcium homeosta-sis may also contribute to interspecies differencesin dopamine metabolism.

Discussion

Here we have found that elevated mitochon-drial oxidant stress in human SNc dopaminergicneurons triggers a dopamine-dependent toxic

cascade leading to lysosomal dysfunction anda-synuclein accumulation—causally linking threemajor pathological features of PD. This discoverycomplements previous work demonstrating thatlysosomal dysfunction leads to deficits in mito-phagy and the buildup of dysfunctional mito-chondria (24) and raises the possibility of apathogenic positive feedback between thesetwo organelles. The pathogenic cascade frommitochondria to lysosomes was only evident inhuman—notmouse—dopaminergic neurons. Thisspecies difference was inferred to be a conse-quence of higher amounts of dopamine in hu-

man neurons on the basis of several lines ofevidence, including the selective accumulation ofneuromelanin in human neurons and the abilityof L-dopa, the synthetic precursor of dopamine,to induce the pathological cascade in mouse do-paminergic neurons modeling PD. In addition,our data also suggest that increased cytosolicdopamine contributes to elevated mitochondrialoxidant stress, indicating a vicious cycle of do-pamine and mitochondrial oxidation in humanmidbrain neurons.Although increased a-synuclein inhibits GCase

trafficking and lysosomal function in various celltypes (19), our data suggest that increased oxi-dant stress and dopamine adduct formationmodified the catalytic site of GCase and loweredits activity preferentially in midbrain dopamin-ergic neurons. Because similar alterations inoxidized dopamine and GCase were seen in neu-rons obtained frompatients with idiopathic andfamilial PD, we propose that this mechanism ap-plies across various forms of PD. In support of thisnotion, we showed that elevation of a-synucleinin the substantia nigra of DJ-1 KO mice also re-sulted in increasedoxidizeddopamine, diminishedactivity of GCase, and neurodegeneration.Our long-termmidbrain cultures demonstrated

that early treatment with mitochondrial anti-oxidants reduced downstream accumulation ofoxidized dopamine and a-synuclein and rescuedlysosomal dysfunction, highlighting the impor-tance of early therapeutic intervention in thepathogenic cascade. The inherent differencesbetween human andmouse dopaminergic neu-ronal vulnerability emphasize the value of studiesin human neurons to identify pathways and tar-gets for therapeutic development in PD and re-lated synucleinopathies.

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Fig. 5. Alterations in calcium homeostasis and dopamine metabolism contribute to intrinsicdifferences between human and mouse dopaminergic neurons. (A to C) WT and DJ-1 KO mouseiPSC-derived dopaminergic neurons were analyzed for (A) oxidized DA at d40, d70, and d90 (50 mMand 500 mM standards shown) or treated with L-dopa or vehicle and analyzed at d55 for (B)oxidized DA (n = 3) and (C) lysosomal GCase activity (n = 3). (D to F) Control human and WTmouseiPSC-derived dopaminergic neurons analyzed for (D) T-soluble calcineurin (n = 5 or 6), (E)calcineurin activity (n = 5), and (F) DA amounts by HPLC [n = 5 (mouse); n = 10 (human)]. HPLCchromatogram in a 4 mM standard sample is shown. Dihydroxybenzylamine (DHBA) was used asan internal standard. (G) WTand DJ-1 KO mouse iPSC-derived dopaminergic neurons were analyzedfor total DA content at d55 (n = 5). (H) The ratio of DOPAC/DA for WT and DJ-1 KO mouseiPSC-derived dopaminergic neurons treated with L-dopa (n = 8). Equal protein concentration wasused in nIRF assays. Error bars, means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test(D) to (H) or one-way ANOVA with Tukey post hoc test (B) and (C). n.s., not significant.

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24. M. E. Gegg, A. H. Schapira, Neurobiol. Dis. 90, 43–50(2016).

ACKNOWLEDGMENTS

We are grateful to D. Sulzer, E. V. Mosharov, J. W. Shim, G. Caraveo,and J. Zheng for helpful discussions and suggestions. We thankthe Northwestern Stem Cell Core Facility for the generation ofmouse iPSC lines (supported by P30 NS081774). We aregrateful to K. Hamada as well as M. K. V. Suresh for technicalassistance and C. Leong for help with the assessment of off-targetgene editing in the CRISPR-edited lines. The mito-roGFP construct

was a gift from P. Schumacker (Northwestern University). Thiswork was supported by NIH (grants R01NS076054 to D.K.,NS047085 to D.J.S., R01NS092823 to J.R.M., R00DC013805-02 toJ.N.S., and T32NS041234 and F32NS101778 to Y.C.W.); The JPBFoundation and The Michael J. Fox and IDP Foundations to D.J.S.;German Academic Exchange Service to L.F.B.; Les Turner ALSFoundation, Target ALS, and MDA to E.K.; and the GermanResearch Council grant KR2119/8-1 and the Fond National deRecherche PEARL program grant FNR/P13/6682797 to R.K. Alldata necessary to support this paper’s conclusions are available inthe supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/357/6357/1255/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S10References (25–36)

7 February 2017; resubmitted 10 August 2017Accepted 23 August 2017Published online 7 September 201710.1126/science.aam9080

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diseaseDopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's

Surmeier and Dimitri KraincBlanz, Carolin D. Obermaier, Chelsee Strojny, Jeffrey N. Savas, Evangelos Kiskinis, Xiaoxi Zhuang, Rejko Krüger, D. James Lena F. Burbulla, Pingping Song, Joseph R. Mazzulli, Enrico Zampese, Yvette C. Wong, Sohee Jeon, David P. Santos, Judith

originally published online September 7, 2017DOI: 10.1126/science.aam9080 (6357), 1255-1261.357Science 

, this issue p. 1255Scienceand related synucleinopathies.neurons emphasize the value of studying human neurons to identify relevant targets for treatment of Parkinson's diseasespecies-specific differences in dopamine metabolism. Inherent species-specific differences between human and mouse

-synuclein, but it was not found in Parkinson's mouse models, owing toαaccumulation of oxidized dopamine and and lysosomal dysfunction in human neurons derived from patients with Parkinson's. The dysfunction was mediated by

identified a toxic cascade of mitochondrialet al.protecting our brain from degeneration in Parkinson's disease. Burbulla Pathways involved in energy metabolism and removal of cellular debris by lysosomes play an important role in

Human-derived neurons provide the answers

ARTICLE TOOLS http://science.sciencemag.org/content/357/6357/1255

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REFERENCES

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