Pruning and loss of excitatory synapses by theparkin ubiquitin ligaseThomas D. Heltona,1, Takeshi Otsukaa,2, Ming-Chia Leea, Yuanyue Mua,3, and Michael D. Ehlersa,b,4

aDepartment of Neurobiology and bHoward Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710

Edited by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 25, 2008 (received for reviewMarch 7, 2008)

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD).The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase thatmediates the transfer of ubiquitin onto diverse substrate proteins.Despite progress in defining the molecular properties and substratesof parkin, little is known about its physiological function. Here, weshow that parkin regulates the function and stability of excitatoryglutamatergic synapses. Postsynaptic expression of parkin dampensexcitatory synaptic transmission and causes a marked loss of excita-tory synapses onto hippocampal neurons. Conversely, knockdown ofendogenous parkin or expression of PD-linked parkin mutants pro-foundly enhances synaptic efficacy and triggers a proliferation ofglutamatergic synapses. This proliferation is associated with in-creased vulnerability to synaptic excitotoxicity. Thus, parkin nega-tively regulates the number and strength of excitatory synapses.Increased excitatory drive produced by disruption of parkin maycontribute to the pathophysiology of PD.

excitotoxicity � proteasome � synapse � glutamate

Parkinson disease (PD) is the most common neurodegenerativemovement disorder (1, 2). Although classic clinical symptoms

arise as a result of the loss of dopaminergic neurons of thesubstantia nigra, widespread neurological abnormalities are presentin animal models of PD and in human disease (3–6). Heightenedresponsiveness to the excitatory neurotransmitter glutamate andassociated excitotoxicity has been implicated in the pathogenesis ofPD (7–9). However, the molecular mechanisms linking PD riskfactors to altered excitability and excitotoxic vulnerability remainunclear.

Mutations responsible for rare hereditary forms of PD have beenidentified in several human genes (10, 11). Among these, PARK2encodes a RING domain–containing E3 ubiquitin ligase that iswidely expressed throughout the nervous system but whose cellularfunction is poorly understood (1, 12–17). Among the reportedsubstrates of parkin are several proteins implicated in synaptictransmission, including CDCrel-1 (15), glycosylated �-synuclein,synphilin, synaptotagmin XI (18), Eps15 (19), and protein inter-acting with C kinase 1 (20). Parkin associates with PDZ scaffoldproteins in the postsynaptic density (PSD) (21) and protects post-mitotic neurons from glutamate receptor–mediated excitotoxicity(16, 22), suggesting a link between parkin and glutamatergicsynapse function. Consistent with this notion, mice lacking parkindisplay both motor and cognitive behavioral deficits and alteredexcitability in the hippocampus and striatum (23, 24). We hypoth-esized that parkin may regulate the strength of excitatory synapses.

ResultsPostsynaptic Parkin Dampens Excitatory Synaptic Transmission. Todetermine the effects of postsynaptic parkin on synaptic transmis-sion, GFP-tagged parkin (parkin-WT) was expressed in cultured rathippocampal neurons, and miniature excitatory postsynaptic cur-rents (mEPSCs) and miniature inhibitory postsynaptic currents(mIPSCs) were recorded using a whole-cell voltage clamp. Trans-fection efficiencies were typically �1% and autapses are rare inhigh-density cultures, ensuring that any observed effect of express-ing parkin originates in the postsynaptic neuron. In neurons ex-

pressing parkin-WT, average mEPSC amplitudes and frequencieswere significantly reduced compared with control GFP-transfectedneurons (GFP, 22.3 � 0.8 pA, 2.0 � 0.2 Hz, n � 23; parkin-WT,16.4 � 0.5 pA, 1.1 � 0.4 Hz, n � 18, *, P � 0.001. Fig. 1 A and B).Thus, elevating levels of postsynaptic parkin reduces the strength ofexcitatory synapses.

At most excitatory synapses, postsynaptic currents consist of twocomponents: a rapid component mediated by AMPA-type gluta-mate receptors and a slower component mediated by NMDA-typeglutamate receptors. To determine whether parkin independentlyregulates AMPA receptor- or NMDA receptor–mediated excita-tory synaptic currents, we recorded mEPSCs in Mg2�-free extra-cellular solution to prevent NMDA receptor block (25, 26). Wemeasured peak currents (reflecting the contribution of AMPAreceptors) and currents 25 msec after the peak (reflecting thecontribution of NMDA receptors). A change in the NMDA-to-AMPA current ratio would indicate a selective effect of parkin oneither the AMPA or NMDA component. However, we observed nosignificant difference in NMDA-to-AMPA mEPSC ratios betweenneurons expressing parkin-WT and GFP [supporting information(SI) Fig. S1A], despite a significant reduction in peak current inparkin-expressing cells (Fig. 1 B Left and Fig. S1A). Furthermore,scaling mEPSCs obtained from neurons expressing GFP or par-kin-WT to the peak current amplitude revealed no difference inmEPSC rise times or decay kinetics (Fig. S1A Left). Specifically,average mEPSC decay kinetics, fit with two exponentials, werequantitatively indistinguishable between control and parkin-expressing cells (fast [�1] and slow [�2] decay time constants: control,�1 � 6.7 � 0.5 msec, �2 � 78.5 � 7.5 msec, n � 15; parkin, �1 � 6.2 �0.3 msec, �2 � 82.9 � 8.1 msec, n � 15, P � 0.05, respectively).Double immunolabeling of hippocampal neurons for surfaceGluR1 AMPA receptors and the obligatory NMDA receptorsubunit NR1 revealed no difference in AMPA/NMDA receptorfluorescence ratios at synapses on neurons expressing either GFP(0.99 � 0.07 arbitrary fluorescence units, n � 12) or parkin-WT(0.92 � 0.09 arbitrary fluorescence units, n � 11) (Fig. S1 B and C).The number of glutamatergic synapses lacking detectable GluR1AMPA receptors (NMDA receptor–only synapses) was slightlydecreased in neurons expressing parkin-WT relative to GFP con-trol neurons (32.4% � 3.6%, n � 11; and 39.9% � 3.2%, n � 11,respectively; Fig. S1D). These results demonstrate that postsynaptic

Author contributions: T.D.H. and M.D.E. designed research; T.D.H., T.O., M.-C.L., and Y.M.performed research; T.D.H. contributed new reagents/analytic tools; T.D.H., T.O., M.-C.L.,and Y.M. analyzed data; and T.D.H. and M.D.E. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1Present address: Department of Neuroscience, Brown University, Providence, RI 02912.

2Present address: Department of Cerebral Research, National Institute for PhysiologicalSciences, Okazaki 444-8787, Japan.

3Present address: Department of Medicine, Pulmonary Division, Duke University MedicalCenter, Durham, NC 27710.

4To whom correspondence should be addressed. E-mail: ehlers@neuro.duke.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802280105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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parkin coordinately down-regulates both AMPA receptor- andNMDA receptor–mediated excitatory synaptic currents, suggestinga generalized remodeling of the postsynaptic membrane at gluta-matergic synapses.

Disease-Linked Parkin Mutants Increase mEPSC Amplitude and Fre-quency. Mutations in the PARK2 gene are linked to hereditary PD(7, 27, 28). Given the reduction of excitatory synaptic transmissioncaused by elevating postsynaptic parkin (Fig. 1 A and B), we soughtto test the effect of disease-linked parkin mutants. Postsynapticexpression of parkin variants bearing any of several point mutationsknown to cause hereditary PD (Fig. 1D Top) significantly andconsistently increased both mEPSC amplitudes and frequencies(Fig. 1 C and D). For example, expression of parkin bearing anR42P mutation within the ubiquitin-like domain, which disrupts theassociation between parkin and the proteasome (1, 29), led to asignificant increase in mEPSC amplitude and frequency (GFP:21.8 � 0.8 pA, 1.9 � 0.3 Hz, n � 15; parkin-R42P: 27.3 � 1.0 pA,3.5 � 0.4 Hz, n � 15; *, P � 0.001 for both amplitude and frequency,

Fig. 1 C and D). Analysis of mEPSC amplitudes and frequencies incells expressing parkin harboring mutations within or near theRING1 domain (A240R: 25.2 � 1.1 pA and 2.9 � 0.2 Hz, n � 17;R275W: 27.1 � 1.5 pA and 2.9 � 0.5 Hz, n � 18) or the RING2domain (T415N: 26.1 � 1.8 pA, 3.6 � 0.6 Hz, n � 15), which areimplicated in E2 binding and substrate recognition, respectively (1,15, 30), also revealed significantly enhanced excitatory synaptictransmission compared with control neurons (amplitude *, P �0.01, frequency *, P � 0.001 in all cases; Fig. 1E). The magnitudeof the observed increase in mEPSC amplitudes (�30%) andfrequencies (�80%) is similar to that reported for other wellstudied postsynaptic regulatory mechanisms (31, 32).

Parkin is a multi-domain protein containing an N-terminalubiquitin-like domain and a C-terminal RING-IBR-RING triadresponsible for E2 and substrate binding (10) (Fig. 1D Top).Disease-linked mutations occur in each of these domains (33), anddifferent mutations produce different biochemical effects on parkinlocalization, solubility, ubiquitination properties, and inheritancephenotype (28, 34). Supporting an inhibitory effect of disease-linked parkin mutants in some cellular processes, we found thatparkin mutants inhibited WT parkin-induced degradation of theputative parkin substrate CDCrel-1 (septin 5; Fig. S2).

Parkin Acts Postsynaptically and Selectively Regulates ExcitatorySynapses. We next tested the specificity of the postsynaptic effect ofparkin on excitatory synaptic transmission. Using the uptake andrelease of the recycling styryl dye FM4–64 to monitor presynapticfunction, we found that postsynaptic expression of parkin-WT orparkin mutants had no effect on depolarization-induced FM4–64loading or on stimulus-dependent release of FM4–64 from boutonscontacting parkin-expressing postsynaptic cells (Figs. S3A and B).The postsynaptic effect of parkin on synaptic transmission is thusnot a result of a retrograde effect on transmitter release. We alsotested whether the parkin-induced reduction in synaptic efficacywas selective for excitatory synapses by examining the effect ofparkin-WT on mIPSCs. Whole-cell recordings revealed that, unlikefor mEPSCs, the amplitudes and frequencies of mIPSCs wereunaffected by parkin-WT (parkin-WT, 38.7 � 2.2 pA, 3.8 � 03. Hz,n � 19; GFP control, 37.8 � 2.1 pA, 3.7 � 0.5 Hz, n � 20; Fig. 1E and F). Thus, parkin selectively decreases excitatory but notinhibitory synaptic transmission, thereby regulating the balance ofexcitation and inhibition.

Parkin Manipulation Alters Glutamatergic Synapse Number. Thedecrease in mEPSC frequency caused by elevating postsynapticparkin (Fig. 1 A–B), combined with a lack of effect on presynapticfunction (Fig. S3 A and B) and preserved AMPA/NMDA ratios(Fig. S1 A and C), suggested a loss of glutamatergic synapses. To testthis possibility, we examined whether postsynaptic expression ofparkin influences the number of synaptic contacts. For this purpose,we used hippocampal neurons at 14 days in vitro (DIV), a periodof active synapse formation and stabilization in culture. The num-ber of excitatory glutamatergic presynaptic terminals contactingdendrites was analyzed by immunocytochemical staining for thevesicular glutamate transporter VGLUT1, which marks discretesites representing presynaptic glutamatergic boutons, and for thePSD protein PSD-95, which labels postsynaptic sites. VGLUT1labeling of control neurons decorated punctate sites along thedendrite and soma representing individual presynaptic terminals.Postsynaptic expression of parkin-WT resulted in a significant lossof contacting VGLUT-positive glutamatergic terminals (control,2.5 � 0.8 puncta per 10 �m, n � 18; parkin-WT, 1.7 � 0.5 punctaper 10 �m, n � 18; *, P � 0.001; Fig. 2 A Left and Middle and B).Parkin-WT also reduced the number of labeled PSD-95 puncta(puncta per 10 �m dendrite: control, 2.7 � 0.4, n � 13; parkin-WT,1.9 � 0.4, n � 11; *, P � 0.01; Fig. S4). This reduction in VGLUT1puncta coincided with a loss of functional presynaptic terminals asvisualized by FM4–64 loading (Fig. S3C). Conversely, expression of

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Fig. 1. Parkin attenuates excitatory synaptic transmission. (A) RepresentativemEPSCs recorded from a control GFP-expressing hippocampal neuron (DIV21;Upper) and a neuron expressing GFP–parkin-WT (Lower). (B) Quantitative anal-ysis of mEPSCs. Data represent mean � SEM, n � 18, *, P � 0.001 by t test. (C)Examples of mEPSCs recorded from neurons expressing GFP (Upper) or thehereditary PD mutant parkin-R42P (Lower). (D) Quantitative analysis of mEPSCs.Data represent mean � SEM of mEPSC amplitudes (Left) and frequencies (Right),n � 15, *, P � 0.001 by t test. Top, model of the domain structure of parkin(ubiquitin-like domain; in-between RING domain [IBR]), including disease-linkedmutations examined in the current study. (E) Parkin has no effect on inhibitorysynaptic transmission. mIPSCs recorded from neurons expressing GFP–controlneuron (Top) and GFP–parkin-WT (Lower). (F) Quantitative analysis revealed noeffect of parkin on the amplitude and frequency of mIPSCs relative to GFP-expressing control neurons.

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PD-linked point mutants of parkin resulted in a robust increase inthe number of contacting VGLUT-positive presynaptic terminals(R42P, 3.8 � 0.2 puncta per 10 �m; A240R, 3.4 � 0.5 puncta per10 �m; R275W, 4.0 � 0.4 puncta per 10 �m; T415N, 3.9 � 0.9puncta per 10 �m, n � 18; *, P � 0.01; Fig. 2 A and B), and likewiseincreased the number of PSD-95 puncta (puncta per 10 �mdendrite: R42P, 4.2 � 0.5; A240R, 3.7 � 0.4; R275W, 3.9 � 0.6;T415N, 4.4 � 0.7, n � 15; *, P � 0.01; Fig. S4 A and B) consistentwith the increase in mEPSC frequency in neurons expressing theseparkin mutants (Fig. 1 E and F).

Unlike its effect on excitatory synapses, neither WT parkin norPD-linked mutant parkin affected the density of inhibitory synapsesvisualized by staining for the vesicular GABA transporter (VGAT;puncta per 10 �m dendrite: GFP, 3.9 � 0.2; parkin-WT, 4.1 � 0.3;R42P, 4.0 � 0.3; A240R, 3.8 � 0.2; R275W, 4.3 � 0.5; T415N, 4.3 �0.5, n � 15; Fig. 2 C and D;) and the inhibitory postsynapticscaffolding protein gephyrin (puncta per 10 �m dendrite: GFP,4.4 � 0.4; parkin-WT, 4.2 � 0.3; R42P, 4.5 � 0.6; A240R, 3.9 � 0.4;R275W, 4.2 � 0.4; T415N, 4.3 � 0.3 puncta per 10 �m, n � 13neurons each; Fig. S5 A and B). These data are in agreement withthe lack of effect of parkin on mIPSCs (Fig. 1 E and F), and showthat elevating postsynaptic parkin causes a selective loss of excita-tory synapses whereas disease-linked parkin mutants cause a pro-liferation of excitatory synapses.

Reduction of Endogenous Parkin Augments mEPSCs and IncreasesExcitatory Synapses. To examine the effect of endogenous parkin onexcitatory synaptic transmission, we used an RNAi strategy. Two

short 29-bp hairpins were generated against the rat PARK2 genesequence (GenBank accession no. NM�020093) and cloned intoGFP-expressing vectors allowing bicistronic expression of parkinshRNA and GFP. This approach produced a robust and reliableknockdown of parkin expression (Fig. S6). Whole-cell voltage-clamp recordings were then performed on hippocampal neurons(DIV14) expressing GFP (control), parkin-WT, or parkin shRNA.After 3 to 5 days, knockdown of endogenous parkin resulted in asignificant increase in mEPSC amplitude and frequency comparedwith GFP control neurons (control, 21.0 � 1.1 pA, 2.0 � 0.1 Hz, n �17; parkin shRNA, 28.2 � 1.9 pA, 2.5 � 0.04 Hz, n � 22; *, P �0.01; Fig. 3 A and B). Importantly, the effect of parkin knockdownwas completely reversed by coexpression of a parkin cDNA con-taining silent third codon mutations (parkin*) that rendered theparkin mRNA resistant to the specific shRNA sequence (Fig. 3 Aand B). Specifically, when parkin* was expressed alone or inconjunction with parkin shRNA, mEPSC amplitudes and frequen-cies were reduced compared with control (parkin-WT, 13.5 � 0.9pA, 1.1 � 0.1 Hz, n � 19; *, P � 0.001; parkin*, 11.8 � 1.4 pA,0.94 � 0.1 Hz, n � 15; *, P � 0.01; shRNA � parkin*, 12.9 � 1.6pA, 1.1 � 0.1 Hz, n � 14; *, P � 0.01; Fig. 3 A and B). Expressionof a scrambled shRNA sequence failed to alter mEPSC amplitudeor frequency relative to GFP-expressing control neurons (scram-bled shRNA, 20.9 � 0.9 pA, 2.1 � 0.1 Hz, n � 17; Fig. 3 A and B).These findings demonstrate that loss of endogenous parkin in-creases excitatory synaptic transmission, similar to the effect ob-served upon expression of disease-linked parkin mutants (Fig. 1 Cand D).

To test whether the loss of endogenous parkin affected thenumber of contacting presynaptic terminals, we expressed parkinshRNA and performed immunocytochemical staining for excita-tory (VGLUT1, PSD-95) and inhibitory (VGAT, gephyrin) syn-apses. Knockdown of endogenous parkin resulted in a significantincrease in VGLUT1 and PSD-95 puncta compared with controlcells, whereas expression of parkin-WT, parkin*, and shRNA �parkin* resulted in a marked reduction in VGLUT1-positive ter-minals (puncta per 10 �m dendrite: GFP, 2.8 � 0.2, n � 14;parkin-WT, 2.0 � 0.2, n � 16; *, P � 0.01; parkin shRNA, 3.8 �0.2, n � 19; *, P � 0.01; scrambled shRNA, 2.7 � 0.2, n � 13;parkin*, 1.9 � 0.1, n � 14; *, P � 0.01; shRNA � parkin*, 1.9 �0.2, n � 15; *, P � 0.01; Fig. 3 C and D Left) and PSD-95 puncta(puncta per 10 �m dendrite: GFP, 2.7 � 0.4, n � 12; parkin-WT,1.9 � 0.3, n � 11; *, P � 0.01; R42P, 4.2 � 0.5, n � 12; *, P � 0.01;A240R, 3.7 � 0.4, n � 9; *, P � 0.01; R275W, 3.9 � 0.6, n � 11;*, P � 0.01; T415N, 4.4 � 0.7, n � 9; *, P � 0.07; parkin shRNA,4.2 � 0.6, n � 14; *, P � 0.01; parkin*, 1.7 � 0.3, n � 11; *, P �0.01; shRNA � parkin*, 1.6 � 0.4, n � 9; *, P � 0.01; Fig. S4). Incontrast, the number of contacting GABAergic inhibitory synapseswas unaffected by increasing or decreasing parkin in postsynapticcells as measured by VGAT staining (puncta per 10 �m dendrite:GFP, 4.1 � 0.2, n � 11; parkin-WT, 4.3 � 0.2, n � 13; parkinshRNA, 3.9 � 0.3, n � 16; scrambled shRNA, 3.9 � 0.3, n � 13;parkin*, 4.4 � 0.2, n � 12; shRNA � parkin*, 4.2 � 0.2, n � 12;Fig. 3D Right) and gephyrin staining (puncta per 10 �m dendrite:GFP, 4.4 � 0.4, n � 10; parkin-WT, 4.2 � 0.3, n � 12; R42P, 4.5 �0.6, n � 9; A240R, 4.5 � 0.6, n � 13; R275W, 4.2 � 0.5, n � 11;T415N, 4.3 � 0.2, n � 11; parkin shRNA, 4. � 0.5, n � 13; parkin*,4.3 � 0.4, n � 10; shRNA � parkin*, 3.8 � 0.6, n � 10; Fig. S5).These results indicate that endogenous parkin expressed in postsyn-aptic neurons selectively limits or constrains the number of gluta-matergic synapses.

Parkin Reduces Excitotoxicity by Synaptically Released Glutamate.Glutamatergic transmission in many brain regions is altered in bothanimal models of PD and the human disease (6, 35), which may leadto undue excitation and neuronal degeneration through excitotox-icity (6–9). Given our findings of enhanced glutamatergic synaptictransmission and excitatory synapse proliferation in neurons ex-

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Fig. 2. Parkin promotes excitatory synapse loss. (A) Hippocampal neurons(DIV14) expressing GFP, GFP–parkin-WT, or the PD-linked parkin mutant GFP–parkin-A240R for 3 days were fixed and stained with anti-VGLUT1 antibody.Arrows indicate glutamatergic terminals contacting transfected dendrites. Ar-rowheads indicate glutamatergic terminals contacting neighboring untrans-fected dendrites in the same field. Boxed regions (Upper) are also magnified(Lower). (Scale bar, 10 �m, Upper; 5 �m, Lower.) (B) Quantitative analysis of thenumber of glutamatergic terminals contacting dendrites on neurons expressingGFP, GFP–parkin-WT, or PD-linked parkin mutants R42P, A240R, R275W, andT415N. Data represent mean � SEM of VGLUT1 puncta per 10 �m dendrite, n �18, *, P � 0.001 relative to GFP control by t test. (C) Parkin has no effect on thenumber of inhibitory synapses. GABAergic presynaptic terminals were visualizedby immunocytochemical staining for the vesicular GABA transporter (VGAT).(Scale bar, 5 �m.) (D) Quantification of VGAT puncta number along dendrites ofneuronsexpressedGFP,parkin-WT,orthe indicatedparkinmutants (n�15each).Data represent mean � SEM, P � 0.05.

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pressing familial PD-linked mutants of parkin (Figs. 1 and 2) orupon parkin knockdown (Fig. 3), we hypothesized that defects inparkin could lead to increased excitotoxic vulnerability by augment-ing excitatory synaptic transmission. To test this hypothesis, wemeasured the effect of expressing WT parkin, PD-linked mutantparkin, or parkin-shRNA on excitotoxic cell death induced by

synaptically released glutamate. Glutamate release was triggered bya brief depolarization using a high K� solution (90 mM, 5 min) (36).Cell death was assessed 4 to 6 h later by trypan blue dye staining(Fig. 4A) or annexin V labeling of externalized phosphatidylserine(Fig. 4B). To allow for sufficient cell counts, high-efficiency ex-pression of parkin constructs in hippocampal neuron cultures wasachieved by using lentivirus. In control neurons expressing GFP,brief depolarization by high K� elicited significant cell death within4 to 6 h (39% � 3% trypan blue–positive cells, n � 5; Fig. 4A;42.3% � 2% annexin V–labeled cells, n � 6; Fig. 4B). Neuronsexpressing parkin-WT were resistant to excitotoxicity (23% � 3%trypan blue–positive cells, n � 5; *, P � 0.001; 27.5% � 3% annexin

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Fig. 3. Knockdown of endogenous parkin results in enhanced excitatorytransmission and a proliferation of glutamatergic synapses. (A) RepresentativemEPSC traces from hippocampal neurons expressing GFP, parkin-WT, parkinshRNA, scrambled shRNA, parkin*, and parkin shRNA � parkin*. (B) Quantitativeanalysis of mEPSC amplitudes and frequencies from neurons expressing theindicated constructs. Data represent mean � SEM, n � 15, *, P � 0.01 relative toGFP control by t test. (C) Hippocampal neurons (DIV14) expressing GFP, GFP–parkin-WT, or parkin-shRNA for 5 days were fixed and stained with anti-VGLUT1antibody. Boxed regions (Upper) are also magnified (Lower). (Scale bar, 10 �m[Upper] and 5 �m [Lower].) (D) Quantitative analysis of the number of glutama-tergic (VGLUT1-positive, Left) and GABAergic (VGAT-positive, Right) terminalscontacting dendrites on neurons expressing GFP, parkin-WT, parkin shRNA,scrambled shRNA, parkin*, and parkin shRNA � parkin*. Data represent mean �SEM of puncta per 10 �m dendrite, P � 0.05.

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Fig. 4. Disruption of parkin enhances neuronal vulnerability to excitotoxicityelicited by synaptically released glutamate. (A and B) Neurons expressing par-kin-WT or parkin* exhibit reduced cell death whereas neurons expressing dis-ease-linked parkin mutants (R42P, A240R, R275W, T415N) or parkin-shRNA(RNAi) exhibit enhanced cell death in response to a high K� excitotoxic challenge.Data represent mean � SEM of the number of cells labeled by trypan blue dye (A)orannexinV–Alexa568 (B),bothmarkersofcelldeath. Inclusionof the ionotropicglutamatereceptorantagonists6-cyano-7-nitroquinoxaline-2,3-dione(CNQX,25�M) and D-AP5 (50 �M) blocked excitotoxicity, indicating a requirement forglutamatergic receptoractivation. (C)Proposedmodel for synapticenhancementby disease-linked mutations in parkin. Under normal physiological conditions,parkin restricts the number of excitatory synapses (Glu) and decreases synapticstrength. Mutations in parkin that cause hereditary PD or loss of WT parkin leadto an increase in the strength and number of excitatory synapses without affect-ing inhibitory synapses (GABA).

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V–labeled cells, n � 6 coverslips; *, P � 0.01), consistent with thedown-regulation of excitatory transmission mediated by parkin(Figs. 1 and 2). In contrast, expression of multiple disease-linkedparkin mutants increased neuronal vulnerability to synaptic exci-totoxicity (trypan blue–positive cells: parkin-R42P, 50% � 2%, n �6; *, P � 0.01; parkin-A240R, 50% � 2%, n � 5; *, P � 0.01;parkin-R275W, 54.9% � 2%, n � 5; *, P � 0.01; parkin-T415N,52% � 3%, n � 5; *, P � 0.01; Fig. 4A; annexin V–labeled cells:parkin-R42P, 58% � 2%, n � 5; *, P � 0.01; parkin-A240R, 56%� 2%, n � 5; *, P � 0.01; parkin-R275W, 56.3% � 2%, n � 5; *,P � 0.01; parkin-T415N, 57% � 2%, n � 6; *, P � 0.01; Fig. 4B).Similarly, shRNA knockdown of endogenous parkin increasedneuronal vulnerability to synaptic excitotoxicity (54% � 3% trypanblue–positive cells, n � 5; *, P � 0.01; 54% � 2% annexinV–labeled cells, n � 6; *, P � 0.01; Fig. 4 A and B), whereasexpression of the RNAi-resistant mutant recovered the parkinneuroprotective effect (parkin*, 25% � 2% trypan blue–positivecells, n � 6; *, P � 0.01; 31% � 3% annexin V–labeled cells, n �6; *, P � 0.01; shRNA � parkin*, 27.4% � 3% trypan blue–positivecells, n � 6; *, P � 0.01; 32.1% � 2% annexin V–labeled cells, n �6; *, P � 0.01; Fig. 4 A and B).

To confirm that the observed cell death was a result of synap-tically released glutamate acting on postsynaptic receptors, weincluded glutamate receptor antagonists (25 �M CNQX, 50 �MD-AP5) during the high K� depolarization. In the presence ofglutamate receptor antagonists, cell death induced by high K� wassignificantly reduced (percent trypan blue–positive cells: GFP,12.2% � 1%; parkin-WT, 11.1% � 2%; parkin-R42P, 11.9% �2%; parkin-A240R, 11.4% � 1%; parkin-R275W, 15.4% � 2%;parkin-T415N, 12.2% � 1%; parkin shRNA, 12.8% � 2%; parkin*,12.8% � 2%; shRNA � parkin*, 14.3% � 3%, n � 6; percentannexin V–positive cells: GFP, 14.2% � 2%; parkin-WT, 11.1% �2%; parkin-R42P, 15.9% � 2%; parkin-A240R, 11.9% � 1%;parkin-R275W, 16.5% � 2%; parkin-T415N, 13.5% � 2%; parkinshRNA, 13.6% � 2%; parkin*, 17.0% � 2%; shRNA � parkin*,19.5% � 2%, n � 6; Fig. 4 A and B). These findings demonstratethat loss of endogenous parkin or expression of PD-linked parkinmutants renders neurons vulnerable to excitotoxicity by enhancingexcitatory synaptic transmission.

DiscussionIn the present study, we have demonstrated that the ubiquitinligase parkin regulates the strength and abundance of excitatorysynapses. When parkin is expressed postsynaptically, excitatorysynaptic transmission is dampened and glutamatergic synapticinput is significantly reduced. In this regard, parkin acts to limitexcitatory synaptic input. This decrease in synaptic strength andconnectivity is opposed by disease-linked mutants of parkin orby loss of endogenous parkin. By refining or containing synapticcircuitry, the reduction of glutamatergic synapses by parkinprovides a mechanism for adjusting excitation. The winnowingof excitatory synapses may be a developmental phenomenon orcould reflect ongoing synaptic fine-tuning that continues intoadult life. Indeed, parkin is expressed early in brain developmentand its expression is maintained into adulthood (14).

Although classically considered autosomal recessive, the geneticsof parkin-linked PD is more complex than a simple model ofrecessive inheritance (11, 37). Parkin is a multi-domain protein andPD-linked mutations are found throughout the gene, including inthe ubiquitin-like, RING1, and RING2 domains (28). These mu-tations in different functional domains can produce either loss offunction or dominant inhibitory cellular phenotypes (15, 16, 34, 38,39). Our observed dominant effect of mutant parkin on excitatorytransmission was unexpected, as disease-linked mutations in parkinhave been linked to recessively inherited juvenile PD, and suggestedthat parkin mutants exert gain of function effects on glutamatergicsynapses. Here, we have demonstrated that the disease-linkedparkin mutants R42P, A240R, R275W, and T415N produce a

synaptic phenotype—proliferation of excitatory synapses—thatmimics parkin loss of function and opposes the gain-of-functionphenotype of decreased excitatory synapses upon parkin overex-pression. Further, we have shown that the same parkin mutants canact in a dominant inhibitory fashion to prevent the degradation ofthe putative parkin substrate CDCrel-1 (septin 5) by coexpressedparkin-WT (Fig. S2). It is important to note that recessive inheri-tance patterns in juvenile PD associated with parkin mutations aredefined based on classic signs and symptoms of nigrostriataldopaminergic insufficiency, and such patterns of inheritance neednot imply that the same mutations cause parkin loss of function inall cellular phenotypes (28, 34). Our results suggest a distinct rolefor parkin in glutamatergic synapse function and elimination thatmay occur in parallel with its regulation of dopaminergic neuronsurvival.

Consistent with a synaptic deficit, parkin-null mice show subtledeficiencies in non–motor-related behaviors including spaciallearning and memory, as well as startle and anxiety responses (24,40, 41). Interestingly, parkin-null mice show no abnormalities ingross brain morphology and no loss of dopaminergic neurons (23,24, 40, 41). These results indicate possible compensation of parkinloss by developmental mechanisms that are not well understood. Toavoid complications resulting from developmental compensationmanifested in parkin-knockout mice, we acutely knocked downendogenous parkin using RNAi. Similar to overexpressing mutantparkin, reduction of endogenous parkin caused a proliferation ofexcitatory synapses (Fig. 3) and an increase in excitotoxic vulner-ability (Fig. 4).

An intriguing aspect of the present study is that mutation or lossof parkin leads to the strengthening and proliferation of glutama-tergic synapses, and the resulting unchecked excitatory drive in-creases neuronal vulnerability to synaptic excitotoxicity (Fig. 4).Previous studies identifying a neuroprotective effect of parkinproposed that parkin regulates downstream apoptotic signaling (16,42, 43). Our results suggest that parkin reduces excitotoxic vulner-ability at an additional earlier step by reducing overall excitatorysynaptic input. The excitatory amino acid glutamate, acting as botha neurotoxin and a neurotransmitter, has long been posited to playa central role in the pathophysiology of PD (7–9). Interestingly, inrodent striatum, dopamine depletion produces an imbalance ofglutamatergic synaptic input onto striatopallidal versus striatonigralmedium spiny neurons (44). However, a direct mechanistic linkbetween PD pathogenic factors and alterations in glutamatergicfunction has remained elusive. Our findings suggest that disease-linked mutations in parkin cause enhancement of excitatory syn-apses, which may contribute to neuronal loss and altered neuralcircuitry in PD.

Although the basic biochemical properties of parkin have beenelucidated (7, 41), its normal cellular function is controversial (23,24, 41). Recently, parkin has been implicated in several cellularpathways potentially involved in the regulation of synaptic strengthand neuronal survival. Parkin interacts with Eps15, an adaptorprotein involved in epidermal growth factor receptor endocytosisand trafficking (19). Parkin ubiquitination of Eps15 disrupts Eps15epidermal growth factor binding, delaying receptor internalizationand promoting phophatidylinositol-3 kinase–Akt signaling (19),thereby enhancing cell survival. Parkin also mono-ubiquitinatesprotein interacting with C-kinase (PICK) 1 (20), a protein knownto regulate excitatory synaptic strength by the trafficking of AMPAreceptors (45–49). In principle, the loss of excitatory synapsesinduced by parkin could be a result of direct ubiquitination ofpostsynaptic proteins (50), or to an indirect effect on downstreamsignaling. Complicating matters is the relatively diffuse distributionof parkin in axons, soma, dendrites, and spines (Fig. S6C and Fig.S7). Similarly, we observed no specific subcellular distribution ofGFP-tagged parkin-WT or parkin mutants, whose signal wascorrespondingly diffuse throughout the neuron (Fig. 2 A and C andFigs. S4A and S5A; and data not shown for GFP-tagged parkin

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mutants R42P, R275W, and T415N). As with most ubiquitin ligases,the full range of substrates of parkin, and the validity of mostreported substrates, is unclear. It will be important for future studiesto determine which combination of the numerous identified andunidentified parkin substrates contribute to the parkin-dependentloss of excitatory synapses.

MethodsCell Preparation and Transfection. Hippocampal and cortical neurons fromembryonic rat were cultured and transfected as previously described (50). ForVGAT staining, neurons were obtained from newborn P0-P1 rats and cultured for12 days. For electrophysiology, recordings were obtained 2 to 7 days aftertransfection. For immunocytochemistry, neurons were incubated for 3 days aftertransfection.

Immunocytochemistry and DNA Constructs. Anti-VGLUT1 (Synaptic Systems;1:2.000),anti-VGAT(SynapticSystems;1:2,000),anti-parkin (Abcam;1:1,000),andanti–PSD-95 antibodies (Transduction Laboratories, 1:1,000) were used for im-munocytochemistry. See SI Methods for details.

GFP-Parkin and GFP-parkin R275W were kindly provided by Edward Fon(Montreal, Canada) and Ted Dawson (Baltimore, MD). Mutations in GFP-parkin(R42P, T240R, T415N, and parkin*) were generated using a Stratagene site-directedmutagenesiskitaccordingtothemanufacturer’s instructions.Parkin253

and 635 shRNA constructs were generated against the rat PARK2 gene sequence(GenBank accession no. NM�020093) and cloned into the pRNAT-H1.3/Hygrovector (GenScript). The scrambled shRNA construct was cloned into the samepRNAT-H1.3/Hygro vector. See SI Methods for sequence details.

Electrophysiology. Whole-cell voltage clamp recordings were performed onDIV17–24 rat hippocampal neurons cultured at high density on polylysine-coatedglass coverslips. Glutamate receptors were blocked with 25 �M 6-cyano-7-nitroquinoxaline-2,3-dione and 50 �M D-AP5. See SI Methods for details.

Excitotoxicity Assays. Neuronswere infectedatDIV14or15withGFP,parkin-WT,mutant parkin (A240R, T415N), or parkin-shRNA lentiviral constructs. One weekafter infection, neurons were exposed to high potassium solution containing (inmM): 90 KCl, 31.5 NaCl, 2 CaCl2, 25 Hepes, and 30 glucose (pH 7.4). After 5 minexposure, cells were further incubated for 4 to 6 h in standard neurobasalmedium (Gibco). Cell viability was examined using trypan blue dye exclusion orannexin-V–Alexa 568 fluorescence (Roche). Approximately 600 to 900 neuronswere scored per coverslip. For additional information, including supplementalfigures, see SI Methods.

ACKNOWLEDGMENTS. This work was supported by grants from the NationalInstitutes of Health (M.D.E.). M.D.E. is an Investigator of the Howard HughesMedical Institute.

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