Tea polyphenols alleviate motor impairments, dopaminergic neuronal injury, and cerebral α-synuclein aggregation in MPTP-intoxicated parkinsonian monkeys

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TEA POLYPHENOLS ALLEVIATE MOTOR IMPAIRMENTS, DOPAMINERGIC NEURONAL INJURY, AND CEREBRAL a-SYNUCLEIN AGGREGATION IN MPTP-INTOXICATED PARKINSONIAN MONKEYS Q1 M. CHEN, a,d  T. WANG, a  F. YUE, a,b X. LI, a,b P. WANG, a,b a Department of Neurobiology, Xuanwu Hospital of Capital Medical University, Beijing, China

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b Key Laboratory of Neurodegenerative Diseases (Capital Medical University), Ministry of Education, Beijing, China

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c Beijing Institute for Brain Disorders Parkinson’s Disease Center, Beijing, China

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injury, and a-syn aggregation in nonhuman primates. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.

Y. LI, a,b P. CHAN a,b,c AND S. YU a,b,c*

Key words: Parkinson’s disease, tea polyphenols, motor Q2 impairments, dopaminergic neuronal injury, a-synuclein aggregation. 16

Department of Human Anatomy, School of Basic Medical Sciences, Guilin Medical University, Guilin, China

Abstract—Tea polyphenols (TPs) are bioactive flavanolrelated catechins that have been shown to protect dopaminergic (DAergic) neurons against neurotoxin-induced injury in mouse Parkinson’s disease (PD) models. However, the neuroprotective efficacy of TP has not been investigated in nonhuman PD primates, which can more accurately model the neuropathology and motor impairments of human PD patients. Here, we show that oral administration of TP alleviates motor impairments and DAergic neuronal injury in the substantia nigra in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated PD monkeys, indicating an association between protection against motor deficits and preservation of DAergic neurons. We also show a significant inhibition of MPTP-induced accumulation of neurotoxic a-synuclein (a-syn) oligomers in the striatum and other brain regions, which may contribute to the neuroprotection and improved motor function conferred by TP. The association between reduced a-syn oligomerization and neuroprotection was confirmed in cultured DAergic cells. The most abundant and bioactive TP in the mixture used in vivo, (–)-epigallocatechin-3-gallate, reduced intracellular levels of a-syn oligomers in neurons treated with a-syn oligomers, 1-methyl-4 -phenylpyridiniumion, or both, accompanied by increased cell viability. The present study provides the first evidence that TP can alleviate motor impairments, DAergic neuronal

*Correspondence to: S. Yu, Department of Neurobiology, Xuanwu Hospital of China Capital Medical University, 45 Changchun Street, Beijing 100053, China. Tel: +86-10-8319-8890; fax: +86-10-83161294. E-mail address: [email protected] (S. Yu).   These authors contributed equally to the work. Abbreviations: a-syn, a-synuclein; DA, dopamine; DOPAC, 3,4dihydroxyphenylacetic acid; EC, (–)-epicatechin; ECG, (–)-epicatechin -3-gallate; EGC, (–)-epigal-locatechin; EGCG, (–)-epigallocatechin-3gallate; HVA, homovanillic acid; LBs, Lewy bodies; LNs, Lewy neurites; MPP+, 1-methyl-4-phenylpyridiniumion; MPTP, N-methyl-4-phenyl1,2,3,6-tetrahydropyridine; MTT, thiazolyl blue tetrazolium bromide; PD, Parkinson’s disease; SN, substantia nigra; TH, tyrosine hydroxylase; TPs, tea polyphenols. http://dx.doi.org/10.1016/j.neuroscience.2014.12.003 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting about 1% of adults older than 60 years. PD is characterized clinically by movement disorders, resulting from insufficient dopaminergic neurotransmission in the striatum due to loss of dopaminergic (DA) neurons in the substantia nigra (SN) (Samii et al., 2004). The pathological hallmark of PD is the accumulation of a-synuclein (a-syn)-immunoreactive intraneuronal inclusions called Lewy bodies (LBs) and Lewy neurites (LNs) (Spillantini et al., 1997; Baba et al., 1998; Wales et al., 2013), which are found not only in the SN but also in other regions of the central and peripheral nervous systems (Jellinger, 2009; Del Tredici and Braak, 2012; Gelpi et al., 2014). The presence of a-syn in LBs and LNs is compelling evidence for the involvement of a-syn in PD pathogenesis, and is further supported by genetic findings linking point mutations and multiplications in the a-syn gene to familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998; Singleton et al., 2003; Chartier-Harlin et al., 2004). However, the a-syn in LBs and LNs is fibrillated, while numerous studies indicate that only soluble a-syn aggregates (oligomers or protofibrils) are toxic to neurons (Brown, 2010; Winner et al., 2011; Celej et al., 2012). Therefore, interventions aimed at reducing soluble oligomeric a-syn in the brain may be among the most effective therapeutic approaches to PD (Gadad et al., 2011). A large number of epidemiological studies on PD and other diseases associated with cognitive impairments have found moderate risk reduction in consumers of black (fermented), oolong (semi-fermented), and green (non-fermented) teas compared to non-tea drinkers (Hellenbrand et al., 1996; Ng et al., 2008). These beneficial properties are believed to depend on tea polyphenols (TPs), particularly bioactive flavanol-related catechins such as (–)-epigallocatechin-3-gallate (EGCG), (–)-epicat echin-3-gallate (ECG), (–)-epigal-locatechin (EGC), and

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(–)-epicatechin (EC) (Wiseman and Balentine 1997; Higdon and Frei, 2003; Mandel et al., 2005). The neuroprotective effects of TP and of EGCG alone have been demonstrated in both in vivo and in vitro PD models. For example, both green tea extracts and EGCG protected SN dopaminergic neurons in the N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) mouse PD model (Levites et al., 2001; Choi et al., 2002) and lipopolysaccharide (LPS)-induced PD rat model (Al-Amri et al., 2013). In vitro studies also demonstrated inhibition of 1-methyl -4-phenylpyridiniumion (MPP+)- and 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by TPs and EGCG (Nie et al., 2002; Pan et al., 2003; Wang et al., 2009). The neuroprotective effects of TP are likely mediated by several mechanisms, including free radical scavenging, ferric ion chelation, anti-inflammation, and activation of cell signaling pathways that attenuate neuronal death due to oxidative stress (Weinreb et al., 2009; Kim et al., 2014). In addition, TP or EGCG may reduce a-syn neurotoxicity since in vitro studies have shown that EGCG can inhibit the fibrillogenesis of a-syn (Ehrnhoefer et al., 2008) or convert large, mature a-syn fibrils into smaller, amorphous nontoxic protein aggregates (Bieschke et al., 2010). The association of TP- and EGCG-dependent neuroprotection with inhibition of a-syn aggregation has not been investigated in a nonhuman primate PD model. The MPTP-intoxicated nonhuman primate PD model reproduces most of the clinical and pathological hallmarks of PD (Gerlach and Riederer, 1996; Bezard et al., 1998) and thus allows for a comprehensive appraisal of both neuropathological and behavioral effects of pharmacological treatments (Langston et al., 1984; Kurlan et al., 1991; Starr, 1995; Kuno, 1997). In the present study we investigated the effects of TP on motor impairments and DA neuronal injury in MPTP-intoxicated cynomolgus PD monkeys. In addition, we examined the association between TP-mediated protection of cultured DAergic neurons against MPTP toxicity and prevention of a-syn aggregation into toxic oligomers.

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EXPERIMENTAL PROCEDURES

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animals were healthy, without any physical injury. Monkeys were divided into four groups: (1) untreated control group (n = 4), (2) MPTP group (n = 4), (3) TP group (n = 4), and (4) MPTP + TP group (n = 4). For MPTP administration, the animals were anesthetized with a mixture of 3% isoflurane and 97% O2 followed by continuous 1% isoflurane for maintenance. In MPTP and MPTP + TP groups, MPTP was injected intravenously as MPTP-HCl diluted in sterile saline at 0.2 mg/kg. Injection was performed once daily for 14 consecutive days. Starting on day 15, the TP and MPTP + TP groups were administered TP dissolved in purified water at 40 mg/kg by gastrogavage once daily for 80 consecutive days. The control and MPTP groups received the same volume of purified water by gavage.

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Ethics statement

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All animals were housed under a 12:12-h light/dark cycle in the animal care facility of Wincon TheraCells Biotechnologies Co., Ltd. The facility is certified by the Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animals Care (International). The ambient temperature was maintained at 24 ± 2 °C and relative humidity at 65 ± 4%. Reverse osmosis (RO) water was available ad libitum. Fresh fruit and vegetables were supplied twice daily. This study was approved by the Institutional Animal Care and Utilization Committee of Wincon TheraCells Biotechnologies (Permit Number: WD-0312010).

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Clinical rating scores

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Motor deficits were quantified twice a week by a previously validated parkinsonian monkey clinical rating scale (Kurlan et al., 1991). Animal behaviors in testing cages were recorded by a videotape system. The scale rated nine items: bradykinesia (0–5), posture (0–2), rigidity (0–2), gait (0–5), balance (0–2), resting tremor on each side (0–3 for each side), gross motor skill (0–4) for each upper limb, gross motor skill (0–4) for each lower limb, and defensive reaction (0–2). The minimum score (0) corresponds to normal and the maximum total score (32) to severe disability. The scores were rated by an experienced neurologist and a technician, both blinded to the study protocol.

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Tissue processing

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After TP or water gavage for 80 consecutive days, all animals were euthanized and the brains rapidly removed. The striatum, midbrain, cerebellum, hippocampus, and occipital cortex were isolated from each animal. All brain regions except the midbrain were frozen in liquid nitrogen and stored in 80 °C for later use. The midbrain section containing the SN was fixed with 4% paraformaldehyde for 24 h to prepare paraffin-embedded tissue sections.

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Reagents

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A mixture of green TPs was purchased from Zelang Medical Technology Co. Ltd (Nanjing, China). MPTP, MPP+ and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma–Aldrich Chemical (St Louis, MO, USA). The 3D5 monoclonal mouse antihuman a-syn antibody was produced by the Department of Neurobiology, Xuanwu Hospital of Capital Medical University (AB_2315782; Yu et al., 2007). A rabbit polyclonal anti-tyrosine hydroxylase (anti-TH) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

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Animals

HPLC and UPLC analysis

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Sixteen female cynomolgus monkeys from 10 to 12 years old were purchased from Grandforest Co. (Nanning, China), a local primate-breeding company. All the

To measure the levels of dopamine and its metabolites in the striatum, fresh-frozen striatal tissues were homogenized and ultrasonicated on ice in 0.3 N

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perchloric acid at 50-mg wet tissue weigh per mL. The homogenate was then centrifuged at 15,000g for 15 min at 4 °C. The supernatant was diluted (1:2) in mobile phase containing 75 mM sodium dihydrogen phosphate, 1.7 mM 1-octanesulfonic acid sodium salt, 100 lL/L triethylamine, 25 lM EDTA, and 10% (v/v) acetonitrile. The pH of the mixture was adjusted to 3.00 using phosphoric acid. Dopamine and its metabolites were measured by high-performance liquid chroma tography (HPLC) with coulometric electrochemical detection (Lane et al., 2013). The concentration of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were calculated and expressed as ng/mg tissue. The constituents of the green TPs were analyzed by an ultra performance liquid chromatography (UPLC) system (Waters, Milford, MA) using an OSTC18 column (1.8 lm, 2.1  150 mm; Waters, Milford, MA). A binary gradient was employed for elution of the polyphenols and metabolites where acetonitrile (A) and 1% phosphoric acid solution (B) served as the mobile phase. The elution program started at 90% B (0–3 min), then 90%80% B (3–8 min), and then 80% B (8–14 min). The flow rate was held constant at 0.25 mL/min. The temperature of the column was maintained at 4 °C. The detection wavelength was 210 nm.

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Immunohistochemistry and stereology

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All quantitative histochemical analyses were performed by an investigator blinded to the animal treatment. Stereological cell counting was performed as previously described (Mackey et al., 2013). Briefly, every twelfth section through the SN (8 or 9 sections per hemisphere) was immunostained with anti-TH using the avidin–biotin immunoperoxidase method (Vector Laboratories Inc., Burlingame, CA, USA) and 3,30 -diaminobenzidine (DAB) as the chromagen. Dopaminergic neurons were identified by the presence of TH immunoreactivity and/or neuromelanin pigmentation.

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Preparation of a-syn oligomers

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Recombinant human a-syn was solubilized in sterilized phosphate-buffered saline (PBS, pH 7.0) at 100 lM in an Eppendorf tube. The tube was sealed with Parafilm and incubated at 37 °C for 7 days with continuous shaking (650 rpm) on an Eppendorf Thermomixer comfort (Eppendorf AG, Hamburg, Germany). The resultant a-syn oligomer mixture was examined by Western blot, aliquoted, and stored at 80 °C until use.

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incubated with 100 lL of ExtrAvidin-Alkaline phosphatase (E-2636; Sigma–Aldrich, MO, USA) di luted 1:20,000 in blocking buffer, and reacted with the enzyme substrate p-nitrophenyl phosphate (N1891; Sigma–Aldrich). The reaction was allowed to proceed for 30 min at room temperature, and the absorbance was read at 405 nm using a microplate ELISA reader (Mutiskan MK3, Thermo Scientific, USA).

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Cell culture

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MES23.5 dopaminergic cells (a gift from Dr. Weidong Le, Baylor College of Medicine) (Crawford et al., 1992) were cultured in DMEM/F12 medium (Gibco, NY, USA) supplemented with 5% fetal calf serum and Sato’s ingredients as described previously (Yu et al., 2004). The cells were plated on 35-mm dishes for immunofluorescent cytochemistry and a-syn oligomer detection and on 96-well plates for cell viability assays. All the dishes and plates were precoated with poly-L-lysine as described previously (Yu et al., 2004).

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Cell viability assay

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Cell viability was estimated using the MTT formazan colorimetric assay. Briefly, 20 lL of MTT solution (5 mg/ mL in PBS) was added to each well of 96-well plates and incubated in a humidified incubator with 5% CO2 at 37 °C for 4 h. The medium was removed followed by addition of 100 lL dimethyl sulfoxide (DMSO). The plates were centrifuged at 40,000 rpm for 10 min. Optical density of the formazan product in solution was measured at 490 nm using a microplate reader (Mutiskan MK3, Thermo Scientific, USA).

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Data analysis

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All data are expressed as mean ± SD. GraphPad Prism 6 Software was applied for data analysis. A one-way ANOVA followed by Tukey’s post hoc test was used to compare the differences in the concentration of DA and its metabolites and count of TH-positive neurons between different groups. The difference of integrated clinical rating scores between MPTP and MPTP + TP groups was compared using Student’s T test. The data from all other experiments were analyzed by a two-way ANOVA, followed by either Sidak’s or Tukey’s post hoc test for multiple comparisons. P < 0.05 is regarded as statistically significant.

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RESULTS

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Detection of oligomeric a-syn

Constituents of TPs

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The a-syn oligomers in brain tissue and cell lysates were Q4 measured using the ELISA method described previously

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(El-Agnaf et al., 2006) but with non-biotinylated and biotinylated 3D5 mouse monoclonal antibodies (Yu et al., 2007) as the capture and detection antibodies, respectively. A series of concentrations of the prepared a-syn oligomer mixture was used as the standard. After completion of the immunoreaction, each well of the ELISA plate was

Ultra performance liquid chromatography (UPLC) revealed that the TP extract used in the present study contained several catechins, the most abundant being EGCG. The second most abundant TPs were EGC and ECG, both present at around 1/4 the concentration of EGCG. Other catechins were detected at lower levels, including EC, gallocatechin (GC), and gallocatechingallate (GCG) (Fig. 1).

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TPs improved MPTP-induced motor impairments in cynomolgus monkeys Daily intravenous injection of MPTP for 14 days induced a variety of motor symptoms, which were collectively expressed by a clinical rating score (0, normal; 32, severely disabled). In the MPTP group, mean clinical rating score increased over the first 24 days and then stabilized (Fig. 2A). Administration of TP by gastric gavage to MPTP-treated monkeys (MPTP + TP group) decreased the mean clinical rating score [MPTP/ MPTP + TP, F(8,54) = 3.06, P < 0.01], an effect that was detectable on day 8 after the last MPTP injection and statistically significant from day 48 to day 72–80 [F(1,54) = 64.65, P < 0.0001] (Fig. 2A). No interaction was found between factors of treatment (MPTP/ MPTP + TP) and time (F(8,54) = 1.03, P > 0.5]. This improved motor function was also noticeable by integrated clinical rating scores (P < 0.01) (Fig. 2B).

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TPs alleviated dopaminergic neuronal injury

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To determine if these parkinsonian-like motor deficits were associated with reduced striatal DA levels due to MPTP-induced injury of nigral DA neurons, we measured DA and metabolites (DOPAC and HVA) in striatal homogenates by HPLC and counted the number of TH-positive neurons in histological sections of the SN. In MPTP-treated monkeys, the levels of striatal DA [F(3,8) = 14.20, P < 0.01], DOPAC [F(3,8) = 9.88, P < 0.01], and HVA [F(3,8) = 8.19, P < 0.01] were significantly reduced compared to untreated controls and monkeys treated with TP alone. In addition, the ratio of (DOPAC + HVA)/DA [F(3,8) = 7.30, P < 0.05] was elevated, indicating reduced DA turnover in dopaminergic terminals (Fig. 3A–D). The number of TH-positive neurons in the SN was also significantly reduced in the MPTP group [F(3,68) = 63.90, P < 0.001] (Fig. 4A, B), suggesting injury and death of DAergic neurons. This MPTP-induced DAergic neuronal injury

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was significantly alleviated in monkeys receiving therapeutic treatment with TP (Fig. 4A, B), an effect accompanied by partial recovery of striatal DA levels (Fig. 3A) and reversal of the decrease in TH-positive neuron counts (Fig. 4A, B).

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TPs reduced the level of -syn oligomers in the brain of MPTP-treated monkeys

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Interventions aimed at reducing brain a-syn oligomers are promising therapies for PD (Gadad et al., 2011), so we examined if TP administration could reduce a-syn oligomers in the brain of MPTP-treated monkeys. In normal monkey brain, the striatum and hippocampus contained higher levels of a-syn oligomers than the cerebellum and occipital cortex. MPTP treatment increased the levels of a-syn oligomers in all brain regions investigated, but this increase was larger in the cerebellum and occipital cortex than in the striatum and hippocampus. Tea phenols significantly reduced the levels of a-syn oligomers in the striatum and hippocampus, both when delivered alone (TP group) and when co-administered with MPTP (MPTP + TP group) [F(3,220) = 520.74, P < 0.0001]. However, in TP group brains, significant reductions in a-syn oligomers were only observed in the striatum and hippocampus [F(4,220) = 531.41, P < 0.0001] (Fig. 5). In addition, an interaction between factors of treatments (TP/MPTP/MPTP + TP) and brain regions was observed [F(12,220) = 12.11, P < 0.0001].

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EGCG reduced the levels of pre-formed and MPP+induced -syn oligomers in cultured dopaminergic cells

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The capacity of TP to reduce a-syn oligomerization was further examined in cultured MES23.5 DAergic neurons treated with a-syn monomers or oligomers (5 lM) in the presence and absence of MPP+ (0.1 mM), the neurotoxic metabolite of MPTP, followed by treatment with vehicle or EGCG. According to our previous study,

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Fig. 1. Ultra performance liquid chromatography analysis of TP constituents. The most abundant bioactive catechin was EGCG, followed by EGC and ECG in about equal concentration, and lower levels of EC. Please cite this article in press as: Chen M et al. Tea polyphenols alleviate motor impairments, dopaminergic neuronal injury, and cerebral a-synuclein aggregation in MPTP-intoxicated parkinsonian monkeys. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.003

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Fig. 2. Animal behavior assessed by clinical rating scores. (A) Time-dependent increase in clinical rating scores in MPTP-intoxicated monkeys (black column), indicating progressive motor impairment. Scores were stable until completion of the experiment. Clinical rating scores were markedly reduced by TP (gray column), and the reduction reached statistical significance by 48 days. Sidak’s post hoc test after two-way ANOVA, ⁄ P < 0.05 and ⁄⁄P < 0.01, compared to MPTP-treated monkeys. (B) Integrated clinical rating scores for MPTP- and MPTP + TP-treated monkeys. Student T test, ⁄⁄P < 0.01, compared to MPTP-treated monkeys. n = 4/group.

Fig. 3. Analysis of DA and DA metabolites in striatum. The levels of DA (A), DOPAC (B), and HAV (C) were significantly reduced in MPTP-treated monkeys and the reduction partially reversed by TP treatment. (D) The ratio of (DOPAC + HAV)/DA. Tukey’s post hoc test after One-way ANOVA, ⁄P < 0.05 and ⁄⁄P < 0.01, compared to control group; #P < 0.05, compared to MPTP group. n = 4/group.

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this concentration of a-syn can rapidly enter MES23.5 cells, reaching an intracellular concentration equivalent to that in a-syn-overexpressing cells (Yin et al., 2011). Cultures were treated with MPP + 1 h after the addition of 5 lM a-syn monomers or oligomers. After 24 h, cells were lysed and intracellular levels of a-syn oligomers measured by ELISA (Fig. 6). No or very low levels of asyn oligomers were detected in cells treated with PBS (untreated) or a-syn monomers, while cells treated with a-syn oligomers exhibited significantly increased intracellular levels of a-syn oligomers, indicating that the a-syn oligomers detected in cells were predominantly derived from the medium. Treatment with 0.1 mM MPP + for 24 h led to a dramatic elevation of a-syn oligomers in both untreated and a-syn monomer-treated neurons, and to a further elevation in a-syn oligomer-treated neurons [F(3,48) = 113.22, P < 0.0001]. Application of EGCG (20 lM) 2 h after the addition of a-syn oligomers and/or 1 h after MPP + administration reduced the level of

intracellular a-syn oligomers in cultures treated with asyn oligomers alone, MPP + alone, or a-syn oligomers plus MPP+ [F(2,48) = 322.17, P < 0.0001]. An interactive effect on intracellular a-syn oligomer levels was detected between treatments with a-syn (monomers and oligomers) and drugs (MPP+/EGCG/MPP+ +EGCG) [F(6,48) = 12.27, P < 0.0001].

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EGCG alleviated the -syn oligomer-induced reduction in cell viability in both control and MPP+-treated DAergic neurons

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Since both TP and its predominant bioactive component EGCG can reduce the levels of a-syn oligomers (the toxic form of the protein) in the brain and cultured cells, it is possible that this inhibition of a-syn aggregation is involved in neuroprotection. To test this, cell viability was measured in MES23.5 cultures treated with either a-syn monomers or oligomers in the presence or

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Fig. 4. Count of TH-positive neurons in the substantia nigra. (A) Photomicrographs showing the TH-positive neurons in the substantia nigra. A significant reduction of TH-positive neurons was found in MPTP-treated monkeys, which was partially prevented by TP treatment. Control: no treatment; TP: TP only MPTP: MPTP only; MPTP + TP: treatment with MPTP and then TP. Bar = 100 lM. (B) Bar graphs showing the number of TH-positive neurons/mm2. A significant reduction in TH-positive neurons was observed in monkeys treated with only MPTP. TP prevented the reduction of TH-positive neurons in MPTP-treated monkeys. Tukey’s post hoc test after One-way ANOVA, ⁄⁄⁄P < 0.001, compared to control group; ##P < 0.01, compared to MPTP group. n = 4/group.

Fig. 5. Effect of TP on levels of a-syn oligomers in normal and MPTP-intoxicated monkey brains. Therapeutic TP treatment reduced the levels of a-syn oligomers in the striatum (STR) and hippocampus (HIP) of control monkeys and in all regions of the MPTP-intoxicated monkeys. Tukey’s post hoc test after Two-way ANOVA, ⁄⁄P < 0.01 compared to the same brain region in control monkeys; ##P < 0.01 compared to the same brain region in MPTP-treated monkeys. n = 4/ group.

Fig. 6. Effects of EGCG on a-syn oligomerization. The levels of intracellular a-syn oligomers were significantly higher in a-syn oligomer-treated cells than in control or a-syn monomer-treated cells. The elevation in a-syn oligomers was reduced by EGCG. MPP + dramatically elevated the levels of a-syn oligomers in control cells as well as cells treated with a-syn monomers or a-syn oligomers. Accumulation in all treatment groups was reduced by EGCG. Tukey’s post hoc test after Two-way ANOVA, ⁄⁄P < 0.01 compared to control cells; #P < 0.05 and ##P < 0.01 compared to EGCG-untreated cells. n = 5 cultures/group.

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absence of MPP+ and EGCG (Fig. 7) Treatment with asyn oligomers alone (but not monomers) significantly reduced cell viability. Application of MPP + decreased cell viability in untreated cells and cells treated with either a-syn oligomers or monomers, with lowest viability in cells treated with MPP + plus a-syn oligomers [F(3,60) = 848.73, P < 0.0001]. Application of EGCG partially mitigated the reductions in cell viability induced by a-syn oligomers, MPP+, and a-syn oligomers plus MPP+ [F(2,60) = 153.93, P < 0.0001]. An interactive effect on cell viability was also detected between treatments with a-syn (monomers and oligomers) and drugs (MPP+/EGCG/MPP++EGCG) [F(6,60) = 13.92, P < 0.0001]. These results strongly suggest that EGCG protected against cell death mediated by accumulation of a-syn oligomers and that mediated directly by MPP+ (in the absence of a-syn oligomer accumulation).

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DISCUSSION

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In the present study, we provide evidence that the administration of TP can alleviate motor impairments and dopaminergic neuronal injury in monkeys treated with the neurotoxin MPTP. Improved motor function in TP-treated animals was measureable in the early stages after MPTP intoxication and reached statistical significance compared to untreated animals within about 2 months. Additional studies are required to further improve the dosing and administration regimens to provide greater and more rapid amelioration of motor deficits in this model. Nonetheless, the results clearly indicate that daily TP can improve motor impairments in PD model monkeys. Subsequent neurochemical, immunohistochemical, and cell viability studies strongly suggested that this therapeutic effect was mediated by rescue of nigral DAergic neurons from both MPP+-induced metabolic toxicity (Langston et al., 1983; Beal, 2001; Watanabe et al., 2005; Tieu, 2011; Porras et al., 2012) and from the

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Fig. 7. Effect of EGCG on cell viability. Cell viability was significantly decreased in a-syn oligomer-treated cells compared to control and asyn monomers-treated cells. This reduced cell viability was partially reversed by EGCG. MPP + dramatically reduced cell viability in control cells as well as cells treated with a-syn monomers and a-syn oligomers, effects partially reversed by EGCG. Tukey’s post hoc test after Two-way ANOVA, ⁄⁄P < 0.01 compared to control cells; # P < 0.05 compared to EGCG-untreated cells. n = 6 cultures/group.

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cytotoxic effects of a-syn oligomer accumulation, thereby maintaining striatal DAergic neurotransmission. Rescue of DAergic neurons in vivo was indicated by preservation of TH immunoreactivity. Since MPTPinduced loss of TH expression may result from the death of DAergic neurons and (or) a decrease in TH expression without loss of individual neurons (Kozina et al., 2014), improved motor function in TP-treated monkey could involve preservation of DA neurons and (or) prevention of TH downregulation. Further in vitro evidence indicated that TP reduced MPP + neurotoxicity. TPs may protect neurons by enhancing cellular antioxidant capacity and by chelating (pro-oxidant-generating) ferric ions as both oxidative stress and iron accumulation have been observed in the SN of MPTP-treated animals (Hare et al., 2013; Mandel et al., 2004). Moreover, TPs appear to activate signaling pathways linked to cell survival, including anti-apoptotic, anti-autophagic, and metabolic pathways (Weinreb et al., 2009; Kim et al., 2014), In addition to these mechanisms, our results suggest that TP may also protect DAergic neurons from MPTP toxicity by preventing the accumulation of a-syn oligomers. Levels of a-syn oligomers in the striatum were significantly increased in MPTP-treated monkeys. Since the striatum receives projections from DAergic neurons in the SN, the increase in striatal oligomeric a-syn may reflect the accumulation of a-syn oligomers, which are known to be toxic to neurons (Anichtchik and Calo, 2013; Danzer et al., 2007; van Rooijen et al., 2010; Winner et al., 2011; Tsigelny et al., 2012; Fellner et al., 2013; Kim et al., 2013). This MPTP-induced accumulation of a-syn oligomers in the nigrostrial DAergic system likely exacerbated MPTP-induced DAergic neuron damage in the SN. Indeed, overexpression of mutant human a-syn A30P, which has a higher propensity to form toxic oligomers, increased the vulnerability of DAergic neurons to MPTP (Nieto et al., 2006), and mice lacking a-syn exhibited an attenuated loss of striatal DA following prolonged chronic MPTP administration (Schlu¨ter et al., 2003). In addition, MPP+ accelerated a-syn aggregation even in the absence of mitochondrial complex I components, the molecular target of MPP+ (Jethva et al., 2011). If increased accumulation of a-syn oligomers is an additional mechanism for MPTP-induced DAergic neuronal damage, reducing oligomer formation should protect neurons from MPTP/ MPP+. Indeed, we show that TP significantly reduced the accumulation of a-syn oligomers in the striatum of MPTP-intoxicated PD monkeys and DAergic neurons in culture, and reversed the reduction in DAergic cell viability. Multiple mechanisms may be involved in the inhibitory effect of TP on a-syn aggregation. As mentioned, TP may reduce oligomeric a-syn levels indirectly by antioxidation and (or) iron chelation, since oxidative stress (Borza, 2014) and ferric ions (Nu¨bling et al., 2012) promote a-syn aggregation. In addition, TP may directly inhibit a-syn toxicity by inhibiting aggregation as demonstrated in vitro (Ehrnhoefer et al., 2008; Bieschke et al., 2010). To confirm that the neuroprotection conferred by TP was mediated, at least in part, by inhibition of MPTP-induced a-syn oligomerization, we conducted in vitro experiments on MES23.5 DAergic neuronal

Please cite this article in press as: Chen M et al. Tea polyphenols alleviate motor impairments, dopaminergic neuronal injury, and cerebral a-synuclein aggregation in MPTP-intoxicated parkinsonian monkeys. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.003

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cells. Since these cells express no or very low levels of endogenous a-syn under normal conditions (Yin et al., 485 2011), we added a-syn monomers and oligomers to the 486 medium. Significantly, increased a-syn oligomers were 487 detected in cells treated with a-syn oligomers but not in 488 cells treated with a-syn monomers, indicating that the 489 intracellular a-syn oligomers mainly came from the pre490 formed a-syn oligomers added to the medium. Addition 491 of MPP+ significantly elevated a-syn oligomers in both 492 untreated cells and cells treated with a-syn oligomers or 493 a-syn monomers. Thus, the capacity of MPP+ to 494 increase a-syn expression and promote a-syn aggrega495 tion (Go´mez-Santos et al., 2002; Jethva et al., 2011) likely 496 stems from two distinct mechanisms: (1) induced expres497 sion and aggregation of endogenous a-syn, and (2) 498 induced aggregation of a-syn monomers entering the 499 cells. A cell viability assay revealed that MPP+ treatment 500 in both the presence and absence of a-syn accumulation 501 increased cell death, while EGCG protected against cell 502 death induced by a-syn accumulation alone (cultures 503 treated with a-syn oligomers but not MPP+) and 504 MPP+ alone, consistent with multimodal protective 505 effects of TPs against a-syn oligomer aggregation and 506 metabolic inhibition (Kim et al., 2014). 507 In addition to nigral DAergic neurons, MPTP is 508 neurotoxic to neurons in many other brain regions 509 (Porras et al., 2012), so we investigated possible a-syn 510 oligomer accumulation in the hippocampus, cortex, and 511 cerebellum of MPTP-intoxicated PD monkeys. The hippo512 campus is particularly vulnerable in the late stage of PD, 513 leading to PD-related cognitive impairments (Braak et al., 514 2003; Jellinger, 2009), while other regions examined, the 515 cerebellum and occipital cortex, are relatively resistant to 516 PD-related neurodegeneration (Braak et al., 2003; 517 Jellinger, 2009). In untreated monkeys, the hippocampus 518 contained higher levels of a-syn oligomers than the cere519 bellum and occipital cortex. MPTP-intoxication increased 520 a-syn oligomers in all regions examined, suggesting that 521 the effect of MPTP on a-syn oligomerization is not confined 522 to the nigrostriatal DAergic system. This finding may 523 explain the variable neurological and neuropathological 524 effects of MPTP in different animal models (Porras et al., 525 Q5 2014). Elevation of a-syn oligomers in multiple regions of 526 the MPTP-intoxicated monkey brain is also in accord with 527 neuropathological studies of PD patients, where patho528 logic LB-like and LN-like a-syn aggregates are extensively 529 distributed, with highest levels found in particularly vulner530 able regions such as the striatum and hippocampus. We 531 showed that TP prevented a-syn oligomerization in 532 extra-nigrostriatal regions, suggesting that TP is broadly 533 neuroprotective and hence may ameliorate multiple PD534 like symptoms, such as PD-associated cognitive decline, 535 in addition to motor dysfunction. 483 484

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CONCLUSIONS

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The present study demonstrates for the first time the therapeutic effect of TP in an MPTP-intoxicated nonhuman primate PD model. In addition, the present study provides the first in vivo evidence for inhibition of a-syn oligomerization by TP in multiple brain regions of

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normal and MPTP-intoxicated monkeys. The multiple potential benefits of TP make these compounds promising neuroprotective reagents for PD patients.

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CONFLICT OF INTEREST STATEMENT

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The authors declare that they have no competing interests.

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UNCITED REFERENCE

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Liu et al. (2013).

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Acknowledgments—This work was supported by grants from the National Basic Research Program (‘‘973’’ Program) of China (2011CB504101), Natural Science Foundation of China (81071014, 81371200), National Science and Technology Support Program (2012BAI10B03), Funding Project for Academic Human Resources Development in the Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (PHR200907113), National High Technology Research and Development Program (‘‘863’’ Program) of China (2006AA02A408), the Natural Science Foundation of Beijing (7122035), and The Open Fund of Laboratory of Brain Disorders, Ministry of Science and Technology (2013NZDJ05).

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(Accepted 3 December 2014) (Available online xxxx)

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