Proteasome Mediates Dopaminergic Neuronal Degeneration, and ItsInhibition Causes �-Synuclein Inclusions*□S

Received for publication, August 1, 2003, and in revised form, December 10, 2003Published, JBC Papers in Press, December 12, 2003, DOI 10.1074/jbc.M308434200

Hideyuki Sawada‡, Ryuichi Kohno‡, Takeshi Kihara‡, Yasuhiko Izumi§, Noriko Sakka§,Masakazu Ibi§, Miki Nakanishi§, Tomoki Nakamizo‡, Kentarou Yamakawa‡, Hiroshi Shibasaki‡,Noriyuki Yamamoto§, Akinori Akaike§, Masatoshi Inden¶, Yoshihisa Kitamura¶,Takashi Taniguchi¶, and Shun Shimohama‡�

From the ‡Department of Neurology, Graduate School of Medicine, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507,Japan, §Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto,Shimoadachicho, Sakyoku, Kyoto 606-8501, Japan, and ¶Department of Neurobiology, Kyoto Pharmaceutical University,Kyoto, Misasagi, Yamashinaku, Kyoto 607-8412, Japan

Parkinson’s disease is characterized by dopaminergicneuronal death and the presence of Lewy bodies.�-Synuclein is a major component of Lewy bodies, butthe process of its accumulation and its relationship todopaminergic neuronal death has not been resolved.Although the pathogenesis has not been clarified, mito-chondrial complex I is suppressed, and caspase-3 is ac-tivated in the affected midbrain. Here we report that acombination of 1-methyl-4-phenylpyridinium ion (MPP�)or rotenone and proteasome inhibition causes the appear-ance of �-synuclein-positive inclusion bodies. Unexpect-edly, however, proteasome inhibition blocked MPP�- orrotenone-induced dopaminergic neuronal death. MPP�

elevated proteasome activity, dephosphorylated mito-gen-activating protein kinase (MAPK), and activatedcaspase-3. Proteasome inhibition reversed the MAPKdephosphorylation and blocked caspase-3 activation;the neuroprotection was blocked by a p42 and p44MAPK kinase inhibitor. Thus, the proteasome plays animportant role in both inclusion body formation anddopaminergic neuronal death but these processes formopposite sides on the proteasome regulation in thismodel.

Parkinson’s disease (PD)1 is characterized by selective celldeath of the mesencephalic dopaminergic neurons and by thepresence of Lewy bodies. However, the relationship betweendopaminergic neuronal death and inclusion body formation hasnot been elucidated. Autosomal recessive juvenile parkinson-ism (ARJP) is caused by mutations in the gene encoding Parkin(1), a ubiquitin ligase E3 of the ubiquitin-proteasome system(2), and the mutant gene products lose E3 ligase activity (3).

Furthermore, in patients with sporadic PD, proteasome activ-ity is well preserved in the striatum (4) but reduced in themidbrain (5). Therefore, it has been assumed that insufficientfunction of the ubiquitin-proteasome system plays an impor-tant role in the pathogenesis of PD including ARJP.�-Synuclein, mutation of which causes familial PD (6), is one ofthe major components of Lewy bodies (7, 8). It is degraded bythe proteasome (9), and proteasome inhibition leads to�-synuclein-positive inclusion formation in vitro (10, 11) and�-synuclein accumulation in dopaminergic neurons in vivo (12).Therefore, impairment of the ubiquitin-proteasome system isthought to be associated with inclusion body formation. Thesubstrates of ubiquitin ligase E3 Parkin are thought to beaccumulated in dopaminergic neurons in ARJP; however, Lewybodies have not been detected in patients with ARJP (13, 14)except for one case (15). Despite the absence of Lewy bodiesdopaminergic neuronal degeneration starts earlier in patientswith ARJP than in those with sporadic PD. In this context,inclusion body formation is not always required for dopamin-ergic neuronal death, and the role of Lewy body formation inthe pathogenesis of PD has not been fully elucidated.

Although the pathogenesis of sporadic cases has not beenclarified, mitochondrial complex I is suppressed in sporadic PDpatients (16–18). Exogenous or endogenous neurotoxins suchas 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (19,20) or MPTP analogues (21), which suppress mitochondrialcomplex I activity, may be involved. MPTP is unique in causingchronic progressive dopaminergic neuronal degeneration in thehuman brain. It is converted to MPP� by monoamine oxidaseand is taken up into dopaminergic neurons through the dopam-ine transporter. It inhibits the activity of mitochondrial com-plex I and causes selective dopaminergic neuronal death (22–24). Recently, it was shown that chronic exposure to rotenone,a selective inhibitor of mitochondrial complex I, causes selec-tive neuronal death of mesencephalic dopaminergic neuronswith Lewy-like �-synuclein inclusion body formation (25, 26).Here we report that dopaminergic neuronal death induced bymitochondria complex I inhibition using MPP� or rotenone wasaccompanied by an elevation in proteasome activity and thatproteasome inhibition caused �-synuclein inclusion body for-mation but blocked dopaminergic neuronal death.

EXPERIMENTAL PROCEDURES

Primary Neuronal Culture of the Ventral Mesencephalon—Culturesof the rat mesencephalon were established according to methods de-scribed previously (27). The ventral two-thirds of the mesencephalonwere dissected from rat embryos on the 16th day of gestation. Thedissected regions included dopaminergic neurons from the substantia

* This work was supported by Grant-in-aid for Scientific Research(to H. S.) from Japan Society for the Promotion of Science 14570591 andfrom the Ministry of Education, Culture, Sports, Science, and Technol-ogy of Japan. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Supplemental Figs. 1–4.

� To whom correspondence should be addressed. Tel.: 81-75-751-3771;Fax: 81-75-751-3265; E-mail: i53367@sakura.kudpc.kyoto-u.ac.jp.

1 The abbreviations used are: PD, Parkinson’s disease; ARJP, auto-somal recessive juvenile parkinsonism; PSI, proteasome inhibitor I;ANOVA, analysis of variance; TH, tyrosine hydroxylase; MPP�,1-methyl-4-phenylpyridinium ion; Ac-DMQD-CHO, acetyl-Asp-Met-Gln-Asp-aldehyde; MAPK, mitogen-activated protein kinase; LDH, lac-tate dehydrogenase; MEK, MAPK kinase-ERK kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 11, Issue of March 12, pp. 10710–10719, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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nigra and the ventral tegmental area but not noradrenergic neuronsfrom the locus ceruleus. Neurons were dissociated mechanically andplated out onto 0.1% polyethyleneimine-coated plastic coverslips at adensity of 1.1 � 105 cells/cm2. The culture medium consisted of Eagle’sminimum essential medium containing 10% fetal calf serum for the first1–4 days in culture and horse serum from the 5th day onwards. Theanimals were treated in accordance with guidelines published in theNIH Guide for the Care and Use of Laboratory Animals.

Treatment of Cultures—To investigate MPP�-induced neurotoxicity,cultured neurons were exposed to 1–100 �M MPP� for 24 h or 10 �M

MPP� for 24–72 h on the 7th day of culture and then fixed. To deter-mine the effects of proteasome inhibitors (lactacystin, proteasome in-hibitor I (PSI), MG-132) on MPP�-induced neurotoxicity, the cultureswere incubated simultaneously with these inhibitors and 10 �M MPP�

for 24 or 48 h on the 7th day of culture. Control experiments were shamoperations, similar to the treatment but using minimum essential me-dium with Earle’s salts containing no drugs. MPP� was dissolved inwater. Rotenone was dissolved in Me2SO. Because incubation with lessthan 0.1% ethanol for 24 h was found to have no effect on neuronalsurvival rates, lactacystin, PSI, and MG-132 were dissolved in ethanol.In pilot studies concentrations of lactacystin greater than 10 �M, PSIgreater than 100 �M, and MG-132 greater than 1 �M were toxic tocultured neurons. Therefore, the drug concentrations used were 10 nM

to 1 �M for lactacystin, 10–100 nM for MG-132, and 0.01–10 �M for PSI.Immunocytochemical Investigation—The numbers of surviving neu-

rons were determined using immunostaining as described in our pre-vious study (28). Briefly, after fixation, cultured cells were incubatedwith anti-tyrosine hydroxylase (TH) antibody (diluted at 1:1000, PA-152, Chemicon) for 24 h, with the secondary biotinylated antibody for1 h, and with avidin-biotin complex solution (Vectastain) for 1 h. Fi-nally, the cultures were reacted with diaminobenzidine solution for 6min. The number of cells stained with anti-TH antibody in the 15randomly selected fields (�200) was counted as the number of survivingdopaminergic neurons by investigators who were blind to the experi-mental treatments. Neurotoxicity was evaluated by the reduction in theneuronal survival rate in each experiment. Statistical analysis wasperformed by ANOVA and post-hoc multiple comparison using New-man-Keul’s method. Statistical significance was defined as p � 0.05.

To investigate the accumulation of ubiquitin and �-synuclein, cul-tured cells were incubated with anti-ubiquitin (diluted 1:300, ZymedLaboratories Inc., San Francisco, CA) and anti-�-synuclein (diluted1:4000, Chemicon International, Temecula, CA) antibodies for 24 h.Anti-cleaved caspase-3 antibody (Cell Signaling Technology, Beverly,MA) was used to detect caspase-3 activation. Cultured cells were ex-posed to 30 �M MPP� for 18 h and then immunocytochemically stainedwith anti-cleaved caspase-3 antibody (1:50) for 24 h. A specific inhibitor

FIG. 1. Ubiquitin and �-synuclein deposition during MPP�-induced neuronal death. A, B, and C, cultured neurons were exposed to 10�M MPP� for 24, 48, and 72 h and fixed for immunocytochemical analysis of TH, ubiquitin, and �-synuclein (A). The number of survivingTH-positive neurons, representing dopaminergic neurons, was reduced by MPP� in a manner dependent on exposure time (left column). After 24 hof exposure, small deposits of ubiquitin were seen in the cytosol (higher magnification seen in B), which diminished after exposure to MPP� for48 or 72 h (middle column). In contrast, �-synuclein-positive deposits were not seen after 24 or 48 h of MPP� exposure but were seen after 72 hexposure (right column, higher magnification in C). Scale bars represent 100 �m (tyrosine hydroxylase (A)), 50 �m (ubiquitin (A)), 50 �m(�-synuclein (A)) and 10 �m (B and C). D, quantitative analysis revealed that the number of dopaminergic neurons (TH-positives) was reduced ina manner dependent on MPP� exposure time. Ubiquitin-positive cells were detected only after 24 h of exposure to MPP�, and the number of�-synuclein-positive cells was increased after 72 h of exposure. n � 4 coverslips/experiment. E and F, double-labeled immunostaining usinganti-ubiquitin (dark blue) and anti-TH (brown) antibodies after 24 h of exposure to 10 �M MPP�. Ubiquitin was detected in the TH-positive neurons(E), and quantitative analysis revealed that about half of the ubiquitin-positive cells were TH-positive, dopaminergic neurons (F). G and H, doublestaining with anti-�-synuclein (dark blue) and anti-TH (brown) antibodies after 72 h of exposure to 10 �M MPP�. �-Synuclein-positive granuleswere seen in the TH-positive cells (G). Quantitative analysis revealed that about 80% of the �-synuclein-positive cells were TH-positive,dopaminergic neurons (H). Scale bars represent 10 �m (E and G).

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of caspase-3 (Ac-DMQD-CHO) was obtained from Peptide Institute Inc.(Osaka, Japan).

Immunocytochemical Analysis by Double Labeling—A double-immu-nostaining method was used to investigate the co-localization of

�-synuclein and TH, a marker of dopaminergic neurons. On the 8th dayin culture cells were fixed and incubated with mouse monoclonal an-ti-TH antibody (1:600, MB318, Chemicon International) for 2 h at roomtemperature. Cells were then incubated with biotinylated anti-mouse Ig

FIG. 2. Effects of proteasome inhibitors on MPP�-induced neurotoxicity. A and B, proteasome activity after MPP� exposure. Proteasomeactivity was significantly elevated by exposure to 10 or 100 �M MPP� for 2 h in a dose-dependent manner. The proteasome activity was significantlyelevated by 2–48 h of exposure to 10 �M MPP�. *, p � 0.05; **, p � 0.01 compared with control. n � 4 dishes/experiment); C, effect of lactacystinon proteasome activity. Proteasome activity that was elevated by exposure to 30 �M MPP� for 6 h was suppressed by the co-administration of0.01–1.0 �M lactacystin in a dose-dependent manner. (*, p � 0.001 compared with control; #, p � 0.001 compared with 30 �M MPP� by ANOVAand Newman-Keul’s post-hoc comparison. n � 4 coverslips/experiment); D, quantitative analysis of the effect of lactacystin on MPP�-inducedneurotoxicity. Dopaminergic neuronal survival was significantly reduced by exposure to 10 �M MPP� for 48 h. The dopaminergic neuronal deathinduced by MPP� was significantly blocked by the simultaneous administration of lactacystin in a dose-dependent manner. Lactacystin (1.0 �M)alone did not have any effect on dopaminergic neuronal survival. *, p � 0.001 compared with control; #, p � 0.001 compared with 10 �M MPP� byANOVA and Newman-Keul’s post-hoc comparison. n � 4 coverslips/experiment; E, dose-response curves for proteasome inhibition and neuropro-tection by lactacystin. Lactacystin provided suppression of the proteasome activity and neuroprotection against MPP�-induced dopaminergicneuronal death. ED50 was 79.0 � 44.9 nM (mean � S.E.). Emax was 53.3 � 7.65% (mean � S.E.) for neuroprotection. ED50 and Emax for proteasomeinhibition was 99.37 � 35.71 nM (mean � S.E.), and 81.64 � 7.8% (mean � S.E.), respectively. F, effect of MG-132 on proteasome activity.Proteasome activity, which was elevated by exposure to 30 �M MPP� for 6 h, was suppressed by the co-administration of 100 nM MG-132. *, p �0.001 compared with control; #, p � 0.001 compared with 30 �M MPP� by ANOVA. n � 4 dishes/experiment; G, effect of MG-132 on MPP�-inducedneurotoxicity. Exposure to 10 �M MPP� for 48 h caused significant neuronal death. Simultaneous co-administration of MG-132 significantlyblocked MPP�-induced dopaminergic neuronal death. MG-132 (100 nM) alone did not have any effect on dopaminergic neuronal survival. *, p �0.001 compared with control; #, p � 0.001 compared with 10 �M MPP� by ANOVA. n � 4 coverslips/experiment; H, immunocytochemical stainingusing anti-TH antibody. Dopaminergic neurons with long neurites were detected in the control experiment. Exposure to 10 �M MPP� for 48 hreduced the number of surviving dopaminergic neurons and induced shortening of the neurites. Co-administration of 1.0 �M lactacystin or 100 nM

MG-132 protected the dopaminergic neurons from MPP�-induced dopaminergic neuronal death. Bar � 100 �m; I, effect of PSI on proteasomeactivity. Proteasome activity, which was elevated by exposure to 30 �M MPP� for 6 h (*, p � 0.05 compared with control by ANOVA) was suppressedby the co-administration of 0.01–0.1 �M PSI. #, p � 0.01 compared with 30 �M MPP� by ANOVA and Newman-Keul’s post-hoc comparison, n �4 dishes/experiment; J and K, effect of PSI on MPP�-induced neurotoxicity against dopaminergic neurons. Exposure to 10 �M MPP� for 48 causedsignificant dopaminergic neuronal death. Co-administration of 0.01–0.1 �M PSI provided a neuroprotective effect against MPP�-induced neuro-toxicity. *, p � 0.001 compared with control by ANOVA; #, p � 0.001 compared with MPP� by ANOVA and post-hoc multiple comparison usingNewman-Keul’s test. n � 4 coverslips/experiment.

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G antibody for 1 h at room temperature, with peroxidase-conjugatedavidin-biotin-complex solution (Vectastain) for 1 h and finally withdiaminobenzidine solution containing hydrogen peroxide. Cells werethen incubated with rabbit polyclonal anti-�-synuclein antibody (over-night incubation at 4 °C, 1:4000) and with biotinylated anti-rabbit Ig Gantibody and reacted with avidin-biotinylated alkaline phosphatasecomplex (Vectastain). Finally, they were visualized using an anti-alka-line phosphatase kit solution (Vectastain) for 15 min.

Immunoblotting—Cells were fixed on the ninth day in culture todetermine the protein levels of �-synuclein, total and phosphorylatedmitogen-activating protein kinase (MAPK) (New England Biolabs Inc.),and Bcl-X (Transduction Laboratories) by Western blotting. Cells werewashed twice with cold Tris-buffered saline, harvested using a cellscraper, and lysed in buffer containing Tris (40 �M), �-glycerophosphate(50 mM), EGTA (0.8 mM), Triton X-100 (2%), phenylmethylsulfonylfluoride (1 mM), aprotinin (1%), dithiothreitol (2 mM), and vanadate (1mM) on ice. Lysates were centrifuged at 150,000 rpm at 4 °C for 30 min.The protein was denatured by boiling at 100 °C for 4 min. An aliquot (10�g of protein) of the supernatant was loaded onto a sodium dodecylsulfate polyacrylamide gel, separated electrophoretically, and trans-ferred to a polyvinylidene difluoride membrane (Bio-Rad). The polyvi-nylidene difluoride membrane was incubated with 10 mM Tris-bufferedsaline containing 1.0% Tween 20 and 5% dehydrated skim milk (Difco)to block nonspecific protein binding. Then the membrane was incubatedwith primary antibodies (anti-�-synuclein antibody (1:2000), anti-totalMAPK antibody (1:1000), anti-phosphorylated MAPK antibody (1:1000), or anti-Bcl-X antibody (1:2000)) and with secondary antibody.

Subsequently, membrane-bound horseradish peroxidase-labeled antibod-ies were detected by an enhanced chemiluminescence detection system(ECL-plus, Amersham Biosciences) and exposed to Fuji x-ray film.

Lactate Dehydrogenase (LDH) Release Assay—The release of LDHwas measured from culture medium using an LDH assay kit (KyokutoMTX-LDH assay kit, Tokyo, Japan). The culture medium samples werecollected 48 h after the onset of drug exposures and incubated with asubstrate solution containing nitro blue tetrazolium, diaphorase, andNAD at 37 °C for 45 min. Then the reaction was terminated with a stopsolution (0.5 M HCl). The absorbance measurements at 560 nm weretaken as LDH release. LDH contained in the 10% horse serum mini-mum essential medium with Earle’s salts was subtracted from eachexperiment.

Proteasome Activity Assay—Proteasome activity was quantified bymeasurement of the release of 7-amino-4-methylcoumarin from thefluorogenic peptide Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarinusing an assay kit, 20 S proteasome assay kit (Affinity Research Prod-uct, Exeter, UK). Cultured cells were washed twice with Tris-bufferedsaline, harvested on ice, and resuspended into a buffer containing 25mM Hepes and 0.5 mM EDTA. Cells were centrifuged and lysed by briefsonication, added to the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, and incubated at 37 °C for 30 min. Protea-some activity was detected by changes in fluorescence intensity at 355nm of excitation and 460 nm of emission using an automatic multi-wellplate reader. The relative activity was standardized by protein concen-tration, which was determined using the Bio-Rad protein assay kit(Bio-Rad).

FIG. 3. LDH release assay demon-strated that exposure to 30 �M MPP�

for 48 h caused significant elevationof LDH release, which was blockedby lactacystin (A), MG-132 (B), or PSI(C). *, p � 0.05 compared with control byone-way ANOVA, n � 4 dishes/experi-ment; #, p � 0.05 compared with MPP�

alone by one-way ANOVA, n � 4dishes/experiment.

FIG. 4. Temporal profile of the neuroprotective effect of lactacystin against MPP�-induced dopaminergic neuronal death. A andB, immunostaining with an anti-TH antibody revealed that MPP� (10 �M, 48 h) reduced the number of surviving dopaminergic neurons.Co-administration of 0.1 �M lactacystin at the beginning of MPP� exposure (Lacta (0–48H)) provided significant neuroprotection. Administrationof lactacystin at 12 h after the beginning of MPP� exposure (Lacta (12–48H)) also provided a neuroprotective effect. In contrast, administrationmore than 24 h after the beginning of MPP� exposure (Lacta (24–48H) and Lacta (36–48H)) did not have any neuroprotective effect. *, p � 0.001compared with control by ANOVA. #, p � 0.001 compared with MPP� by ANOVA and post-hoc multiple comparison using Newman-Keul’s test.n � 4 coverslips/experiment. NS, not significant.

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Dose Relationship of Neuroprotection and Proteasome Inhibition byLactacystin—Rate of neuroprotection was defined as

Neuroprotection �[Survival]lacta � [Survival]MPP�

[Survival]sham � [Survival]MPP�� 100 (Eq. 1)

where [Survival]lacta is survival after MPP� exposure with lactacystin,[Survival]MPP� is survival after MPP� exposure without lactacystin,and [Survival]sham is survival after sham treatment.

The dose-response curve for neuroprotection was fitted to the curveusing following the Michaelis-Menten equation,

Neuroprotection �Emax � [Lactacystin][Lactacystin] � ED50

� 100 (Eq. 2)

where [Lactacystin] is the lactacystin concentration (nM), ED50 is theED50 of neuroprotection, and Emax is the maximal effect of neuropro-tection. The values of ED50 and Emax were determined by the computersoftware (GraphPad Prism 4.0, GraphPad software Inc.). The rate ofproteasome inhibition was defined as the following formula,

Proteasome inhibition

�[P.activity]MPP� � [P.activity]lacta

[P.activity]MPP�� 100 (Eq. 3)

where [P. activity]lacta is proteasome activity after MPP� exposure withlactacystin and [P. activity]MPP� is proteasome activity after MPP�

exposure without lactacystin. The curve fitting for proteasome inhibi-tion was performed similarly as neuroprotection.

Intranigral Microinjection of MPP� and Proteasome Inhibitors—Male Wistar rats weighing �180 g were used. The rats were fastedovernight with free access to water. For stereotaxic microinjection ratswere anesthetized (sodium pentobarbital, 50 mg/kg, intraperitoneal)and immobilized in a Kopf stereotaxic frame. Subsequently, the ratswere injected with MPP� (3 �g) and lactacystin (0.12 �g) or MG-132(0.19 �g) in a final volume of 4 �l of sterilized physiological saline(containing 1% Me2SO) (n � 4). This was injected into the substantianigra via a motor-driven 10-�l Hamilton syringe. After 7 days treatedrats were perfused through the aorta with 150 ml of 10 mM phosphate-buffered saline followed by 300 ml of a cold fixative consisting of 4%

paraformaldehyde, 0.35% glutaraldehyde, and 0.2% picric acid in 100mM phosphate buffer under deep anesthesia with pentobarbital (100mg/kg, intraperitoneal). After perfusion the brain was quickly removedand post-fixed for 2 days with paraformaldehyde in 100 mM phosphatebuffer and then transferred to a 15% sucrose solution in 100 mM phos-phate buffer containing 0.1% sodium azide at 4 °C. 20-�m-thick nigralsections were cut in a cryostat and collected into 100 mM phosphate-buffered saline containing 0.3% Triton X-100. After several washes, thenigral slices were incubated with rabbit polyclonal TH antibody (1:20000, AB-151, Chemicon International) for 3 days at 4 °C. The anti-body was detected by an ABC Elite kit (Vector Laboratories) usingdiaminobenzidine with nickel enhancement. Subsequently, the numberof TH-positive neurons in the nigral sections was counted blind to theexperiments.

RESULTS

Protein Ubiquitination in MPP�-induced Dopaminergic Neu-ronal Degeneration—Using rat mesencephalic cultured neu-rons, we investigated dopaminergic neuronal death, proteinubiquitination, and �-synuclein accumulation in MPP�-in-duced neurotoxicity by immunostaining using anti-TH (amarker for dopaminergic neurons), anti-ubiquitin, and anti-�-synuclein antibodies, respectively. Exposure to MPP� (1–100�M) for 24 h caused dopaminergic neuronal death in a dose-de-pendent manner but did not cause �-synuclein aggregation(data not shown). Long term exposure to 10 �M MPP� (for 24,48, and 72 h) caused dopaminergic neuronal death, which in-creased with exposure time. A 24-h exposure caused fine cyto-plasmic depositions of a ubiquitin-positive substance but didnot cause �-synuclein aggregation (Fig. 1, A and B). Cytoplas-mic depositions of ubiquitin were not detected after MPP�

exposure for 48 h or longer (Fig. 1A). In contrast to ubiquitindepositions, �-synuclein-positive fine granules were seen in thecytosol after 72 h exposure (Fig. 1, A and C). Quantitativeanalysis revealed transient ubiquitination followed by�-synuclein deposition (Fig. 1D). Double-labeled immunocyto-

FIG. 5. Neuroprotective effect of lactacystin and MG-132 in vivo. A, coronal sections of the rat midbrain. Immunohistochemical analysisrevealed that unilateral injection of vehicle had no effect on the dopaminergic neurons. Microinjection of MPP� (3 �g/animal) caused loss ofdopaminergic neurons in the ipsilateral substantia nigra pars compacta (SNc) and reticulata (SNr). Co-administration of lactacystin (0.12�g/animal) or MG-132 (0.19 �g/animal) with MPP� caused mild dopaminergic neuronal loss in the substantia nigra pars compacta and reticulata.Bar, 200 �m. B, semiquantitative analysis of TH-positive neurons in the mesencephalon. MPP� caused significant neuronal loss (*) that wasblocked by the co-administration of lactacystin and MG-132 (#). Each value is the mean � S.E. (%), taking the number of TH-positive neurons inthe contralateral side as 100%. *, p � 0.01, compared with vehicle by ANOVA; #, p � 0.05; ##, p � 0.01, compared with MPP� by ANOVA andpost-hoc multiple comparison using Newman-Keul’s test. n � 4 experiments.

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chemical analysis using anti-ubiquitin (dark blue) and anti-TH(brown) antibodies revealed cytoplasmic protein ubiquitinationin dopaminergic neurons (Fig. 1E). About half of the ubiquitin-positive cells were TH-positive neurons (Fig. 1F). After a 72-hexposure to MPP�, double-labeled immunocytochemical anal-ysis using anti-�-synuclein (dark blue) and anti-TH (brown)antibodies revealed that more than 80% of the �-synuclein-positive neurons were dopaminergic neurons (Fig. 1, G and H).

MPP�-induced Dopaminergic Neuronal Death and the Pro-teasome—MPP�-induced dopaminergic neuronal death was ac-companied by cytoplasmic protein ubiquitination, which can bea target of the proteasome. Therefore, we investigated protea-some activity after exposure to MPP�. Although MPP� did notdirectly elevate the proteasome activity in a cell-free assay(Supplemental Fig. 1), exposure of cultured mesencephalic cellsto MPP� for 2 h significantly elevated proteasome activity (Fig.2A). The proteasome activity was significantly elevated at 2–48h after exposure to MPP� (Fig. 2B). The elevated proteasomeactivity was suppressed by 0.1–1.0 �M lactacystin (A. G. Scien-tific Inc. San Diego, California), a cell-permeable and irrevers-ible inhibitor of both the 20 S and 26 S proteasomes (Fig. 2C).Therefore, 0.1–1.0 �M lactacystin was used to investigate theeffect of proteasome inhibition on MPP�-induced neurotoxicity.Simultaneous administration of lactacystin significantlyblocked MPP�-induced dopaminergic neuronal death at dosesof 0.1–1.0 �M (Fig. 2D). A dose-response curve of neuroprotec-tion by lactacystin was similar to that of proteasome inhibition(Fig. 2E). A reversible proteasome inhibitor, benzyloxycar-bonyl-Leu-Leu-leucinal (MG-132, Calbiochem, 100 nM) alsosuppressed the proteasome activity (Fig. 2F) and provided sig-nificant neuroprotection against MPP�-induced toxicity (Fig.2G). 48 h of exposure to 1.0 �M lactacystin or 100 nM MG-132alone had no effect on the number of surviving dopaminergic

neurons (Supplemental Fig. 2). Immunocytochemical stainingrevealed that 10 �M MPP� alone reduced the number of sur-viving dopaminergic neurons and shortened the neurites andthat co-administration with lactacystin or MG-132 blocked theMPP�-induced neurotoxic effect (Fig. 2H). A cell-permeableirreversible proteasome inhibitor, benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI, Carbiochem) significantlyblocked proteasome activity (Fig. 2I) and also blocked dopa-minergic neuronal death (Fig. 2, J and K). LDH release assayrevealed that exposure to 30 �M MPP� for 48 h elevated LDHrelease, and the LDH release elevation was blocked by protea-some inhibitors (Fig. 3). Furthermore, dopaminergic neuronaldeath by 72 h of exposure to MPP� was also blocked by lacta-cystin or MG-132 (Supplemental Fig. 3). The time course of theprotective effect of a proteasome inhibitor against MPP�-in-duced dopaminergic neuronal death was investigated. Lacta-cystin was administered at 0, 12, 24, and 36 h after the begin-ning of a 48-h exposure to 10 �M MPP�. Administration at 0 or12 h after the beginning of MPP� exposure provided significantneuroprotection, but administration at 24 h or later did notprovide any protection (Fig. 4, A and B).

The Proteasome and Dopaminergic Neuronal Death inVivo—We investigated the effect of proteasome inhibitors ondopaminergic neuronal death in vivo. MPP� was injected ste-reotaxically into the unilateral mesencephalon, and the ratswere sacrificed 7 days after microinjection to investigate do-paminergic neuronal loss. MPP� caused dopaminergic neuro-nal loss in the ipsilateral substantia nigra, and this wasblocked by the co-administration of lactacystin or MG-132 withMPP� (Fig. 5).

Inclusion Body Formation in Dopaminergic Neurons afterCombined Treatment with MPP� and Proteasome Inhibitors—The effect of the simultaneous addition of proteasome inhibi-

FIG. 6. Effect of proteasome inhibitors on �-synuclein accumulation and inclusion body formation in primary culture of the ratmesencephalon. A and B, cultured cells were immunostained using anti-�-synuclein antibody to detect �-synuclein accumulation. �-Synucleinwas not detected in the control experiment or after exposure to 10 �M MPP� for 24 h. Exposure to a combination of 1.0 �M lactacystin (Lacta) andMPP� for 24 h resulted in the detection of round-shaped accumulations of �-synuclein in the cytoplasm (arrow). Combined treatment with MPP�

and either 10 �M PSI or 10 nM MG-132 for 24 h caused round-shaped dark-staining with the anti-�-synuclein antibody (arrows). To reveal thelocalization of �-synuclein accumulation in dopaminergic neurons, cells were double-immunostained using anti-TH (brown) and anti-�-synuclein(dark blue) antibodies (TH/�-Synuclein). In the control experiment the cytoplasm of the dopaminergic neurons was stained brown with the anti-THantibody and was not stained with the anti-�-synuclein antibody (dark blue). After exposure to MPP�, neurites were shortened, and the cell bodywas shrunk but not stained with anti-�-synuclein. Combination exposure to MPP� and either 1.0 �M lactacystin, 10 �M PSI, or 10 nM MG-132caused dark blue �-synuclein accumulation in the cytoplasm of the dopaminergic neurons. Bars represent 10 �m. Exposure to 10 �M MPP� for 24 hcaused fine cytoplasmic deposition of ubiquitin (ubiquitin, arrowheads). Combined treatment with MPP� and proteasome inhibitors causedmassive inclusions that were ubiquitin-positive (ubiquitin, arrows). Exposure to proteasome inhibitors alone did not cause the appearance of�-synuclein positive inclusions (B). C, Western blotting analysis of �-synuclein. Cultured cells were treated with 10 �M MPP� with or withoutlactacystin (0.1 �M). In the control experiment �-synuclein was detected as a trace of a band at 19 kDa. MPP� treatment elevated the protein levelof �-synuclein, and the elevation was not affected by co-administration of 0.1 �M lactacystin. �-Actin was not affected by the experimentaltreatments.

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tors to 24 h of MPP� treatment was investigated to determinethe role of the proteasome system in �-synuclein aggregation.Combination of MPP� and lactacystin caused the formation of�-synuclein-positive inclusions in the cytoplasm (Fig. 6A,�-synuclein). Co-administration of PSI or MG-132 with MPP�

also caused �-synuclein-positive inclusion formation. In con-trast to treatment with MPP� alone, massive round-shapedinclusions (5–8 �m in diameter) were seen in the cytoplasm.We then investigated the localization of the �-synuclein inclu-sions by double-labeled immunostaining using anti-TH (brown)and anti-�-synuclein (dark blue) antibodies (Fig. 6A, TH/�-synuclein). In the control experiment, TH was stained in thecytoplasm and neurites, and 24 h of MPP� treatment causedshortening of the neurites. However, �-synuclein was not de-tected in either experiment. Combined treatment with MPP�

and proteasome inhibitors (lactacystin, PSI, or MG-132) caused�-synuclein inclusion formation (dark blue) in the cytoplasm ofdopaminergic neurons (brown). These �-synuclein-positive in-clusions were seen in about 3–8 neurons/cm2, corresponding toabout 1% of dopaminergic neurons. The inclusion bodies causedby a combination of MPP� and proteasome inhibitors werestained with anti-ubiquitin antibody (Fig. 6A, ubiquitin). He-matoxylin and eosin staining showed that the inclusions werestained with eosin but not with hematoxylin (data not shown).

Treatment with proteasome inhibitors alone did not cause theappearance of �-synuclein inclusions (Fig. 6B). Western blot-ting analysis revealed that MPP� treatment with or withoutlactacystin caused an elevation in the �-synuclein protein level(Fig. 6C).

Rotenone-induced Dopaminergic Neuronal Death and theProteasome—Furthermore, we investigated dopaminergic neu-ronal death induced by rotenone, a pharmacological inhibitor ofmitochondrial complex I in rat mesencephalon primary culture.Rotenone caused dopaminergic neuronal death in a dose- andtime-dependent manner (Fig. 7, A and B). Exposure to rotenonecaused a slight but significant elevation in proteasome activity(Fig. 7, C and D). Although rotenone-induced dopaminergicneuronal death (0.1–10 �M for 24 h or 0.1 �M for 24–72 h) wasnot accompanied by �-synuclein aggregation (data not shown),at 0.1 �M for 48 h it was significantly blocked by the co-administration of the proteasome inhibitors, lactacystin, orMG-132 (Fig. 7, E and F). Combined treatment with rotenoneand either lactacystin or MG-132 caused the appearance ofmassive round inclusions stained with �-synuclein (Fig. 7E).

Neuroprotective Mechanism of Proteasome Inhibition—Toelucidate the mechanism of neuroprotection by proteasomeinhibitors, we investigated the effects of lactacystin on proteinsmediating survival signals. Among them, MAPK (29, 30) and

FIG. 7. Rotenone-induced dopaminergic neuronal death and proteasome inhibitors. A and B, rotenone-induced dopaminergic neuronaldeath. Rotenone (0.1–10 �M for 48 h) caused dopaminergic neuronal death in a dose-dependent manner (A). Exposure to 0.1 �M rotenone causeddopaminergic neuronal death that increased with exposure time (B). *, p � 0.001 compared with control by ANOVA and post-hoc multiplecomparison using Newman-Keul’s test. n � 4 coverslips/experiment; C and D, elevation of proteasome activity by rotenone. Proteasome activitywas significantly elevated by exposure to rotenone for 2 h (C). Proteasome activity was significantly elevated by 0.03 �M rotenone in atime-dependent manner (D). #, p � 0.05; ##, p � 0.01 compared with control by ANOVA and post-hoc multiple comparison using Newman-Keul’stest. n � 4 dishes/experiment; E, effect of proteasome inhibitors on rotenone-induced dopaminergic neuronal death and �-synuclein inclusion bodyformation. Exposure to rotenone (0.1 �M for 24 h) caused significant neuronal death that was blocked by either 0.1–1.0 �M lactacystin (lacta) or10–100 nM MG-132. *, p � 0.01 compared with control by ANOVA. #, p � 0.01 compared with rotenone by ANOVA and post-hoc multiplecomparison using Newman-Keul’s test. n � 4 coverslips/experiment. F, immunocytochemical staining using anti-tyrosine hydroxylase (left) andanti-�-synuclein antibodies (right). Exposure to 0.1 �M rotenone for 24 h reduced the number of surviving dopaminergic neurons and inducedshortening of the neuritis compared with control experiment. Co-administration of 1.0 �M lactacystin or 100 nM MG-132 protected the dopamin-ergic neurons from rotenone-induced dopaminergic neuronal death. Combined treatment for 24 h with rotenone and either 1.0 �M lactacystin or100 nM MG-132 caused the appearance of inclusions immunostained with �-synuclein (arrows). Bar, 100 �m (TH), 10 �m (�-synuclein).

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Bcl-2 family proteins (31) are reported to be involved in Lewybodies, and therefore, we investigated these proteins. MPP�

treatment slightly reduced the protein level of total and phos-phorylated p42 and p44 MAPK and significantly reduced therelative ratio of phosphorylated MAPK to total MAPK. Theprotein level of phosphorylated MAPK and the relative level ofphosphorylated MAPK to total MAPK were significantly ele-vated by the addition of 1.0 �M lactacystin (Fig. 8A). Theaddition of MG-132 or PSI to MPP� also elevated the proteinlevel of phosphorylated MAPK, and this was blocked byPD098059, an inhibitor of MEK1/2, the kinase of p42 and p44MAPK (Fig. 8B). Furthermore, the neuroprotective effect ofproteasome inhibitors was significantly blocked by PD098059(Fig. 8C). Western blotting analysis revealed that the proteinlevel of Bcl-X, but not that of Bcl-2, was elevated by lactacystin,but the elevation of Bcl-X was not affected by PD098059 (Sup-plemental Fig. 4). These data suggest that neuroprotection byproteasome inhibitors was not linked directly with up-regula-tion of Bcl-X because the neuroprotection was completely

blocked by PD098059. Then we investigated the relationshipbetween proteasome activation and caspase-3 cleavage. MPP�-induced neuronal death was significantly blocked by a specificcaspase-3 inhibitor (Ac-DMQD-CHO) (Fig. 9, A and B). Immu-nocytochemical analysis using an anti-cleaved caspase-3 anti-body revealed that MPP� caused caspase-3 cleavage, whichwas blocked by 0.1 �M lactacystin (Fig. 9, C and D).

DISCUSSION

Exposure to MPP� for 24 h caused dopaminergic neuronaldeath accompanied by the cytosolic deposition of an ubiquiti-nated substance that was not detected after MPP� exposure for48 h or longer. These data suggest that MPP� exposure causedthe ubiquitination of proteins within 24 h that was eliminatedwithin the following 24 h. Furthermore, we found that MPP�

exposure elevated the proteasome activity. Protein ubiquitina-tion is a marker targeting proteolysis by the proteasome sys-tem, and ubiquitinated proteins might be proteolyzed by acti-vated proteasome and eliminated within the subsequent 24 h.

FIG. 8. Effect of lactacystin on MAP kinase. A, a typical demonstration of immunoblotting and semi-quantitative analysis of the ratio ofphosphorylated MAP kinase to total MAP kinase. The protein level of MAP kinases (p44 and p42) was slightly reduced by 24 h of treatment with10 �M MPP�. The protein level of phosphorylated MAP kinases was also slightly reduced by MPP� treatment but was elevated by combinedtreatment with MPP� and 1.0 �M lactacystin. The protein levels of phosphorylated and total MAP kinases were determined using a densitometer.The ratio of phosphorylated MAP kinases to total MAP kinases was significantly reduced by MPP� exposure (*). The reduced ratio was significantlyelevated by the co-administration of 1.0 �M lactacystin (#). *, p � 0.001 compared with control; #, p � 0.001 compared with MPP� alone by ANOVA,n � 3. B, immunoblotting analysis of total and phosphorylated MAPK. Exposure to 10 �M MPP� for 24 h slightly reduced the protein level ofphosphorylated MAPK, which was elevated by 1.0 �M lactacystin, 100 nM MG-132, and 100 nM PSI; and this elevation was blocked by 30 �M

PD098059. C, effect of PD098059 on neuroprotection provided by lactacystin and PSI. Neuroprotection provided by lactacystin (0.1–1.0 �M) or PSI(0.01–0.1 �M) was significantly blocked by PD098059, an inhibitor of MEK1/2, a MAP kinase kinase. 30 �M PD098059 alone had no effect on thesurvival rate of the dopaminergic neurons (data not shown). *, p � 0.01 compared with MPP� alone; #, p � 0.01 compared with MPP� � lactacystin(Lacta) or MPP� � PSI by ANOVA, n � 4 coverslips/experiment.

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The proteasome inhibitors, lactacystin, PSI, and MG-132,blocked MPP�-induced dopaminergic neuronal death in vitroand in vivo. A temporal profile of neuroprotection by lactacys-tin revealed that its administration within 12 h after the be-ginning of MPP� exposure provided neuroprotection. It did notprovide neuroprotection when administered after 24 h whencytoplasmic proteins were ubiquitinated. Taken together, theseresults show that the proteasome, which catalyzes and elimi-nates ubiquitinated proteins within 24–48 h after exposure toMPP�, and the protein degradation process by the proteasomecould play an important role in dopaminergic neuronal death.

The proteasome inhibitors caused the appearance of round-shaped inclusions in dopaminergic neurons. These data arepartly consistent with previous studies (10, 11), in which pro-teasome inhibitors caused �-synuclein inclusions. However, in

the present study, neither MPP� exposure alone nor protea-some inhibitors alone led to �-synuclein-positive inclusion bodyformation. One of the reasons for this discrepancy is concen-trations of proteasome inhibitors because sub-lethal doses ofproteasome inhibitors were used in the present study. In con-trast to proteasome inhibitors alone, combination treatment ofrotenone and proteasome inhibitors caused the formation of�-synuclein-positive inclusions. Therefore, both proteasomesuppression and mitochondrial complex I inhibition were re-quired for inclusion body formation in dopaminergic neurons.

Previous studies show that proteasome inhibitors block celldeath in some paradigms (32–34) and enhance it in otherparadigms (35–37). One of the possible reasons for this discrep-ancy may be related to the mitotic ability of the cells. Theproteasome plays an important role in the regulation of pro-

FIG. 9. Effect of lactacystin on caspase-3. A and B, immunostaining with anti-TH antibody revealed that MPP�-induced dopaminergicneuronal death was blocked by a caspase-3 inhibitor, Ac-DMQD-CHO. In the control experiment dopaminergic neurons had long neurites.Exposure to MPP� reduced the number of surviving dopaminergic neurons and shortened the neurites. Co-administration of 10 �M Ac-DMQD-CHO with MPP� blocked the neurotoxicity (A). Quantitative analysis revealed that MPP� caused significant neuronal death (*), which was blockedby Ac-DMQD-CHO (#) (B). *, p � 0.001 compared with control. #, p � 0.001 compared with MPP� alone. n � 4 coverslips/experiment; C and D,immunostaining with anti-cleaved caspase-3 antibody revealed that lactacystin blocked caspase-3 activation by MPP�. Cleaved caspase-3 was notdetected in the control experiment (C, boxed area in the lower column at a higher magnification). After exposure to MPP� for 16 h cells werepositively stained with anti-cleaved caspase-3 antibody. Lactacystin (0.1 �M) blocked the cleavage of caspase-3 by MPP�. Quantitative analysisshowed that lactacystin significantly blocked the cleavage of caspase-3 (D). *, p � 0.001 compared with control. #, p � 0.001 compared with MPP�

alone. n � 6 coverslips/experiment. Bars represent 100 �m (A) or 10 �m (C).

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teins that are related to cell cycle progression such as cyclin-dependent kinases. Therefore, the blockade of proteasome ac-tivity could be toxic to cells with mitotic ability but would not betoxic to primary cultured neurons or in vivo brain neurons,which are post-mitotic. Recently Hoglinger et al. (38) demon-strated that MPP� or rotenone neurotoxicity is enhanced by100 nM epoximicin, a proteasome inhibitor which suppressesthe proteasome activity to less than 10%. Therefore, overallinhibition of proteasome activity may enhance toxicity, andneuroprotection may require partial suppression of theactivity.

The ubiquitin/proteasome system degrades short-lived pro-teins including proteins mediating signal transduction (39).Among them MAPK is of particular interest because MKK6,the MAPK kinase that activates c-Jun N-terminal kinase, isactivated by the polyubiquitination of lysine 63 of MKK6 ki-nase (40), and p42 MAPK is expressed in Lewy bodies (29, 30).We found that the protein level of total and phosphorylatedMAPK was slightly reduced, and the relative ratio of phospho-rylated MAPK to total MAPK was significantly reduced byMPP� treatment, and that this was reversed by proteasomeinhibitors. These data are supported by a previous report dem-onstrating that phosphorylation of p42 and p44 MAPK is stim-ulated by lactacystin (41). Stimulation of MAPK phosphoryla-tion by proteasome inhibitors was blocked by PD098059, aselective inhibitor of MEK1/2, p42 and p44 MAPK kinase, andthe neuroprotection provided by proteasome inhibitors was alsoblocked by PD098059 in the present study. MAPK can beregulated by the degradation of MAPK itself directly by theproteasome (42), but these data suggest that MAPK phospho-rylation is mediated by MEK1/2 activation. Because Raf, akinase of MEK1/2, is degraded by the proteasome (43, 44),MEK1/2 might be activated by the stabilization of Raf by pro-teasome inhibitors. Activation of p42 and p44 MAPK providesan antiapoptotic effect (45–50), and therefore, the neuro-protection resulting from proteasome inhibition was thoughtto be mediated by the reversion of p42 and p44 MAPKdephosphorylation.

Previous studies on autopsied brains showed that caspase-3is elevated and activated in the substantia nigra of PD patientscompared with that in control patients (51), indicating thatcaspase-3 activation is involved in the pathogenesis of PD (52).We revealed that MPP� caused caspase-3 activation, whichmediated dopaminergic neuronal death, and that lactacystinblocked the caspase-3 activation. The results of the presentstudy indicate that the proteasome plays an important roleboth in inclusion body formation and dopaminergic neuronaldeath. In this PD model, inclusion body formation and neuro-nal death may form opposite sides on the proteasome activity.

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Takashi Taniguchi and Shun ShimohamaShibasaki, Noriyuki Yamamoto, Akinori Akaike, Masatoshi Inden, Yoshihisa Kitamura,

Masakazu Ibi, Miki Nakanishi, Tomoki Nakamizo, Kentarou Yamakawa, Hiroshi Hideyuki Sawada, Ryuichi Kohno, Takeshi Kihara, Yasuhiko Izumi, Noriko Sakka,

-Synuclein InclusionsαCauses Proteasome Mediates Dopaminergic Neuronal Degeneration, and Its Inhibition

doi: 10.1074/jbc.M308434200 originally published online December 12, 20032004, 279:10710-10719.J. Biol. Chem. 

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