research article cnb-001 a novel curcumin derivative, guards...

Research ArticleCNB-001 a Novel Curcumin Derivative, Guards DopamineNeurons in MPTP Model of Parkinson’s Disease

Richard L. Jayaraj,1 Namasivayam Elangovan,1 Krishnan Manigandan,1

Sonu Singh,2 and Shubha Shukla2

1 Department of Biotechnology, Periyar University, Salem, Tamilnadu 636011, India2 Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226031, India

Correspondence should be addressed to Namasivayam Elangovan; [email protected]

Received 27 February 2014; Accepted 14 May 2014; Published 17 June 2014

Academic Editor: Arianna Scuteri

Copyright © 2014 Richard L. Jayaraj et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Copious experimental and postmortem studies have shown that oxidative stress mediated degeneration of nigrostriataldopaminergic neurons underlies Parkinson’s disease (PD) pathology. CNB-001, a novel pyrazole derivative of curcumin, has recentlybeen reported to possess various neuroprotective properties.This study was designed to investigate the neuroprotectivemechanismof CNB-001 in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) rodent model of PD. Administration of MPTP(30mg/kg for four consecutive days) exacerbated oxidative stress and motor impairment and reduced tyrosine hydroxylase (TH),dopamine transporter, and vesicular monoamine transporter 2 (VMAT2) expressions. Moreover, MPTP induced ultrastructuralchanges such as distorted cristae and mitochondrial enlargement in substantia nigra and striatum region. Pretreatment with CNB-001 (24mg/kg) not only ameliorated behavioral anomalies but also synergistically enhanced monoamine transporter expressionsand cosseted mitochondria by virtue of its antioxidant action. These findings support the neuroprotective property of CNB-001which may have strong therapeutic potential for treatment of PD.

1. Introduction

Parkinson’s disease (PD), a progressive neurodegenerativedisorder, is characterized by degeneration of dopaminergic(DA-ergic) neurons in substantia nigra pars compacta (SNpc)and resultant loss of dopamine (DA) in the striatum (motorloci). Clinical diagnosis reports have shown that cardinalbehavioral features of PD include rigidity, dyskinesia, gaitimbalance, and tremor at rest [1]. Though the etiology ofPD remains obscure, various experimental studies havereported the involvement of oxidative stress, mitochondriadysfunction, apoptosis, and inflammation either separatelyor cooperatively to induce neurodegeneration [2]. DA, avital neurotransmitter (important for motor coordination),is synthesized in nigrostriatal pathway and undergoesautooxidation to form toxic reactive oxygen species (ROS)and quinone molecules which in turn initiates pathologicalresponse [3]. Apart fromDA depletion, a profound reductionin specific neurochemical markers such as tyrosinehydrolase (TH), DA transporter (DAT), and vesicular

monoamine transporter 2 (VMAT2) has been reportedin PD [4, 5]. In idiopathic PD, mitochondrial complex Iimpairment leading to free radical generation which evokesrespiratory chain deficits is very well documented [6]. Hence,various mitochondrial complex I inhibitors such as MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), rotenone,isoquinoline, paraquat, and 6-OHDA (6-hydroxydopamine)are used to create PD models to get insights into precisemechanisms underlying neurodegeneration in PD [7].MPTP, a catecholamine neurotoxin, selectively degeneratesdopaminergic neurons via monoamine oxidase-B- (MAO-B-) mediated conversion of MPTP to its toxic/active metaboliteMPP+. Further MPP+ is taken into dopaminergic neuronsvia DA transporter and sequestered in mitochondria andinhibits complex I of electron transport chain resulting inenhanced ROS formation and decreased ATP production.Moreover, MPP+ is also taken into the cytosol throughvesicular monoamine transporter 2 (VMAT2) and supportslethality [8]. Hence, assessment of DAT and VMAT2reveals a greater picture regarding DA integrity and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 236182, 11 pageshttp://dx.doi.org/10.1155/2014/236182

2 BioMed Research International

neuronal vulnerability [5]. For these reasons, antioxidantand antiapoptotic compounds are being explored for theirtherapeutic potential in treatment of PD. Current PDmedications (predominantly L-Dopa, monoamine oxidase-Binhibitors) offer only symptomatic relief and could notcounteract PD progression [9]. Various literature reportshave shown that curcumin, a polyphenol, has excellentbiological properties and is known to protect DA neurons[10]. In spite of its pharmacological properties, curcumin haspoor bioavailability, fails to inhibit excitotoxicity, and doesnot support neuronal growth during loss of trophic factors.These hindrances were surmounted by CNB-001[4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl) vinyl)-2-methoxy-phenol], a novel hybrid moleculesynthesized from curcumin, and cyclohexyl bisphenol A(CBA), a molecule with neurotrophic activity [11]. Further,CNB-001 was shown to protect against excitotoxicity,glucose-starvation assay [11], 𝛽-amyloid toxicity [12] and toalso exhibit strong antioxidant and antiapoptotic properties[13]. Additionally, CNB-001 has been shown to possess lowerEC50 values and superior anti-inflammatory propertiescompared to its parental compounds [11]. Since these resultsilluminate biological properties of CNB-001, the presentstudy was aimed at investigating whether CNB-001 offersneuroprotection, by evaluating its effect on oxidative stress,behavioral impairments, expressions of TH, DAT, andVMAT2, and mitochondria ultrastructural analysis in asubacute MPTP model of PD.

2. Materials and Methods

2.1. Experimental Animals and Ethics Statement. Adult maleC57BL/6 mice weighing between 25 and 27 g purchased fromNational Institute of Nutrition, Hyderabad, were used in thisstudy. Since male C57BL/6 mice are more sensitive to MPTPintoxication than females and PD is observed frequentlyin males, female mice were avoided [14]. Animals weremaintained at ambient conditions (22 ± 1∘C, 60% humidity,and 12 h diurnal cycle) and had ad libitum access to foodand water. All experiments were performed in accordancewith National Guidelines on the Proper Care and Use ofAnimals in Laboratory Research (Indian National Academy,New Delhi, 2009) and approved by Institutional AnimalEthics Committee (1085/ac/07/PU-IAEC/2012/13).

2.2. Materials. MPTP was procured from Sigma-Aldrich,Bangalore, India. TH, DAT, and VMAT2 primary andcorresponding secondary antibodies were purchased fromCell Signaling (USA). Enhanced chemiluminescence kit wasobtained from GenScript, USA. All other chemicals usedwere of analytical grade and procured from Merck. CNB-001 was obtained as a generous gift from Dr. Dave Schubert(Cellular Neurobiology Lab, Salk Institute for BiologicalStudies, La Jolla, CA).

2.3. Dose Fixation and Experimental Design. A dose depen-dent study was initially conducted with four different dosesof CNB-001 (6, 12, 24, and 48mg/kg) to analyze the effect

of CNB-001 on DA content in MPTP intoxicated mice.After seven days of experimental period, it was observedthat pretreatment with CNB-001 (6, 12, 24, and 48mg/kg)significantly increased the levels of DA and its metabolites inMPTP intoxicated mice. Further, it was noted that CNB-001at a dose of 24 and 48mg/kg showed similar increase in DAcontent but markedly higher than 6 and 12mg/kg. As a result,an optimum dose of 24mg/kg was used for further studies.

To evaluate the neuroprotective effect of CNB-001, weused a subacuteMPTP paradigmwhich represents one of themost stable toxin based PDmodels (𝑛 = 10). Group I receivedintraperitoneal (i.p) injection of saline and ethanol (10-folddilution with 1% (v/v) Tween 80/saline) which were vehiclesfor MPTP and CNB-001, respectively, and served as controlgroup. Groups II and III receivedMPTP injection (30mg/kg;i.p) starting from 4th to 7th day of the experimental period.Guidelines for handling MPTP were strictly followed asdescribed previously [15]. Groups III and IV received CNB-001 (24mg/kg, i.p, 1 h prior to MPTP injection) for 7 consec-utive days. CNB-001 was dissolved in 100% ethanol (40 𝜇Lfor 1mg) followed by 10-fold dilution with 1% (v/v) Tween80/saline [16]. Two days after the treatment schedule, animalswere subjected to behavioral tests. Since we were aware thatbehavioral tests might cause some kind of stress and alterbiochemical actions, the animals used to analyze enzymeactivities were not subjected to behavioral tests.

2.4. Behavioral Assessment. Behavioral tests were carried outin such away that the order of tasks did not affect the outcomeof our results. These tasks were carried out in two phases;in phase I we analyzed motor coordination by performingrotorod tests and in phase II we evaluated mental stressby open field test and hang test was performed to assessneuromuscular strength.

2.4.1. Rotorod Test. This test was performed to assess muscu-lar coordination, strength, and balancing ability of animalsusing rotorod apparatus (Panlab). Initially all the animalswere habituated and trained on accelerating rotating rod (5,10, and 15 rpm) with a maximum cut-off time of 180 s forfive consecutive days (twice/day) prior to treatment. Afterexperimental period, the average retention time on the rodwas noted as described previously [17].

2.4.2. Hang Test. The effect of CNB-001 on neuromuscularstrength was analyzed by grid hang test. Briefly, animals wereplaced on a horizontal grid and supported until they held thegrid with all their four paws. The grid was then kept in aninverted position allowing the animals to hang upside downand the maximum hanging time was noted. Proper care wastaken to prevent injury/damage to animals in case of falling.Maximum latency time was fixed as 300 seconds [18].

2.4.3. Open Field Test. Open field test was carried out ina wooden apparatus (W100 × D100 × H4 cm). The floorwas covered with a rexin cloth and divided into 25(5 × 5)equal squares. The animals were placed in the middle of

BioMed Research International 3

apparatus and their behavior was observed for 5min. Centraland periphery movements were calculated based on numberof central (nine) and peripheral (sixteen) squares crossed bythe animals, respectively. One count was made only whenthe animal enters a square with both its forelimbs. Further,number of grooming (licking the fur, scratching behavior,or wiping face) and rearing activities (exploratory behavior)were also manually scored in the open field for 5min. Theentire study was conducted in a blinded manner [19].

2.5. Perfusion and Tissue Processing. At the end of the exper-iments, animals were sacrificed by terminal anesthesia andperfused via intracardial infusion with 0.9% saline followedby 4% paraformaldehyde (pH 7.4). Brain was removed fromthe skull and postfixed in 4% paraformaldehyde for 24 h at4∘C. The brain tissue was then embedded in paraffin andsliced into 5𝜇m coronal sections containing entire SN andST (anteroposterior levels: bregma −2.92 to −3.64mm and+0.02 to +0.86mm) with the reference of mouse brain atlas[20] for immunohistochemical study. For immunoblotting,SN and ST were dissected out and rapidly frozen on dry iceand stored at −80∘C until used.

2.6. Biochemical Analysis

2.6.1. Estimation of Thiobarbituric Acid Reactive Substances(TBARS). TBARS content was analyzed as previouslydescribed [21]. In brief, the tissue extracts were incubatedin metabolic water bath shaker at 37∘C with 0.2mL phenylmethosulfate. After one hour, 0.4mL of 0.67% thiobarbituricacid and 0.4mL of 5% tricarboxylic acid were added. Thereaction mixture was centrifuged at 4000 rpm for 15min atroom temp. (RT) and the resultant supernatant was boiledfor 10min. The samples were cooled and reading was takenat 535 nm.The rate of lipid peroxidation is expressed as nmolof TBARS formed per hour/g tissue.

2.6.2. Estimation of Reduced Glutathione. The levels ofreduced glutathione in brain tissue homogenate were mea-sured as previously described [22]. Brain homogenate wascentrifuged at 16,000×g for 15min at 40∘C. After centrifuga-tion, 0.5mL of the supernatant was added to 4mL of ice-cold0.1mM 5,5-dithiobis[2-nitrobenzoic acid] solution in 1Mphosphate buffer (pH-8) and the absorbance was measuredat 412 nm.

2.6.3. Estimation of Glutathione Peroxidase (GPx). The levelof GPx in brain tissue homogenate was measured as previ-ously described [23]. Briefly, the assay mixture consisted of100 𝜇L of 1M Tris-Hcl (pH-8.0) containing 5mM of EDTA,20𝜇L of 0.1M GSH, 100 𝜇L of glutathione reductase solution(10U/mL), 100𝜇L of 2mM NADPH, 650𝜇L of distilledwater, 10 𝜇L of 7mM tert-butyl hydroperoxidase, and 10 𝜇Lof brain homogenate. NADPH oxidation was determinedspectrometrically at 340 nm. One unit of enzyme activity wasdefined as the amount of GPx required to oxidize 1 𝜇mol ofNADPH per min.

2.6.4. Assay for Superoxide Dismutase (SOD) Activity. Theactivity of SOD was assessed following the method aspreviously described [24]. Xanthine and xanthine oxidaseserved as superoxide generator and nitro blue tetrazoliumis used as superoxide indicator. Briefly, the reaction mixtureconsists of 960𝜇L of 50mM sodium carbonate buffer (pH10.2) containing 0.1mM of EDTA, 0.1mM of xanthine, 20 𝜇Lof xanthine oxidase, 0.025mM of NBT, and 20 𝜇L of brainsupernatant. The absorbance is measured spectrometricallyat 560 nm and the activity was expressed as units/min/mgprotein.

2.6.5. Determination of Catalase (CAT) Activity. Catalaseactivity was measured by determining the rate of decom-position of hydrogen peroxide (H

2O2) at 240 nm. In brief,

assay mixture consisted of 50 𝜇L of 1M Tris-Hcl buffer(pH 8.0) containing 5mM of EDTA, 900𝜇L of 10mMH2O2, 30 𝜇L of water, and 20 𝜇L of the supernatant. Rate

of decomposition of hydrogen was measured at 240 nmspectrometrically. Enzyme activity is expressed as nmol ofH2O2decomposed/min/mg protein [25].

2.7. Immunohistochemical Studies. Immunohistochemistrywas performed to investigate the presence of DA-ergic neu-rons with TH immunoreactivity. Substantia nigra (SN) andstriatum (ST) region were serially sectioned and the slideswere deparaffinized using xylene followed by rehydration ingraded series of ethanol. The slides were boiled for 10minin 10mM of citrate buffer (pH-6) for antigen retrieval. Thesections were then incubated in dark with H

2O2(0.3%)

for 10min at RT. The slides were placed in blocking buffer(10% normal goat serum (NGS) with 0.2% Triton X-100 in0.01M PBS) at 37∘C for 30min. In each treatment, slideswere washed three times with 0.01M PBS for 5min. Sectionswere incubated primarily with anti-mouse TH (1 : 1000) in 2%normal goat serum, 0.2% Triton X-100, and 0.02% sodiumazide in TBS for 24 h. After washing with 1% NGS inTBS, the sections were incubated with anti-mouse IgG-HRPconjugated secondary antibody (1 : 1000 in 1.5%NGS) for onehour followed by washing with PBS. The sections were thenincubated with diaminobenzidine (DAB) to view and analyzeTH immunoreactivity.

The intensity of TH immunoreactivity in ST was mea-sured using Micro Computer Imaging Device software andresults are expressed as percentage of control. Mouse brainatlas was initially used to delineate SN at low magnificationand the numbers of TH immunoreactive neurons in SN werecounted at higher magnification (20x) by a person, blind tothe treatment. Cell counts were determined in every sixthsection (total of 8 to 10 sections) through SN correspondingto the bregma −2.92 to −3.64mm from each animal. Theanalyses of TH positive neurons were restricted to SN andthus excluded the ventral tegmental area.

2.8. Levels of TH, DAT, and VMAT2 by Immunoblotting.Western blotting was performed to analyze the expressionpatterns of TH, DAT, and VMAT2 in SN and ST of exper-imental animals. In brief, the tissues were homogenized in


ice-cold RIPA buffer (1% Triton, 0.1% SDS, 0.5% deoxy-cholate, 1mM/L EDTA, 20mM/L Tris (pH 7.4), 10mM/LNAF, 150mM/L NaCl, and 0.1mM/L phenylmethylsulfonylfluoride) and centrifuged (12,000×g for 15min at 4∘C) toremove cellular debris. Protein concentration in the sampleswas analyzed using Lowrymethod [26]. Proteins (50𝜇g)wereseparated by 10% SDS-polyacrylamide gel and electrotrans-ferred onto a PVDF membrane by semidry transfer (BIO-RAD). After blocking the membrane for 1 hr in 5% nonfatdry milk in TBS at 25∘C, the membranes were incubatedwith 𝛽-actin (rabbit polyclonal, 1 : 500 dilution in 5% BSATris-buffered saline and 0.05% Tween 20 (TBST), anti-mouseTH (1 : 1000), anti-mouse DAT (1 : 500), and anti-mouseVMAT2 (1 : 1000)) with gentle shaking at 4∘C overnight. Cor-responding secondary antibodies (anti-mouse or anti-rabbitIgG conjugated to HRP) were then added and incubatedfor 2 h at room temperature. The membranes were washedthrice with TBST (10min/wash) and immunoreactive bandswere visualized by chemiluminescence kit. Densitometryanalysis was done using “Image J” analysis software. The blotintensities were normalized with that of 𝛽-actin as loadingcontrol.

2.9. Electron Microscopy. Brain regions (SN and ST) werefixed in 3% gluteraldehyde at 4∘C for 24 h. After fixation,brain sections were cut into approximately 1mm cubes andpostfixed in 1% osmium tetraoxide for 2 h at 4∘C. The cubeswere then dehydrated in series of ethanol and treated twicewith propylene oxide for 10min at RT. The tissues were infil-trated with EPON mixture and propylene oxide ((1 : 1), 2 h,RT) and embedded in EPON mixture containing Taab/812,followed by polymerization at 60∘C for 24 h. Ultramicrotomewas used to cut tissue into 0.6 𝜇m thick sections whichwere then stained with 0.5% toluidine blue to confirm thepresence of neurons. Then the 60 nm ultrathin sections werecut and mounted on Nickel grids (300 mesh). The sectionswere double stained with uranyl acetate and lead citrateand then examined by Transmission Electron Microscope(Philips CM10) and photographed.

2.9.1. Morphological Analysis of Mitochondria within SN andST. Measurements were generated from three randomlyacquired TEM images from SN and ST with a magnificationof 16,000x and coded for blinded analysis. The perime-ter length of each mitochondrion was analyzed by a sin-gle trained technician using MeasureIT software (OlympusSoft Imaging Solutions). Mitochondria were identified bypresence of identifiable cristae and distinct double mem-brane; however,mitochondriawith unclearmorphologywereavoided.

3. Results

3.1. CNB-001 Conspicuously Mitigates MPTP Induced Behav-ioral Impairments. Motor coordination ability of experimen-tal animals was evaluated by performing rotorod test, asshown in Figure 1. MPTP treated group showed neuromus-cular incoordination and significant reduction in retention

250

200

150

100

50

0

Rete

ntio

n tim

e in

roto

rod

(s)

CtrlMPTP CNB + MPTP

CNB

5 10 15

Groups (rpm)

Rotorod

∗∗

∗

# # #

Figure 1: Rotorod performance of control and experimental mice.Administration ofMPTP significantly reduced the ability of animalsto stay on the rotating rod (5, 10, and 15 rpm) when compared tocontrol. Retention time was significantly improved as compared toMPTP group when animals were pretreated with CNB-001. Valuesare expressed as mean ± SD for six animals in each group. ∗𝑃 < 0.05compared to control, #

𝑃 < 0.05 compared to MPTP.

050

100150200250300350

Ctrl MPTP CNBGroups

Hang test

CNB + MPTP

Han

ging

tim

e (s)

∗

#

Figure 2: Effect of CNB-001 on hanging performance. MPTPadministration resulted in a decrease in neuromuscular strength asevidenced by reduction in hanging time as compared to control. Pre-treatment with CNB-001 to MPTP treated mice distinctly enhancedhanging time as compared to MPTP group. Values are expressed asmean ± SD for six animals in each group. ∗𝑃 < 0.05 compared tocontrol, #


time as compared to control (𝑃 < 0.05). Moreover none ofthe MPTP treated animals could balance themselves on therotating rod for full cut-off time (180 s). Pretreatment withCNB-001 distinctly enhanced balancing ability and retentiontime and inhibited disorientation as compared to MPTPtreated mice (𝑃 < 0.05).

Hang test was performed to analyze the neuromuscularstrength of control and experimental animals. Compared tocontrol group, the average hanging time ofMPTP intoxicatedmice was significantly lower (𝑃 < 0.05) (Figure 2). CNB-001 treatment distinctly enhanced neuromuscular strengthcompared to MPTP group as evinced by improved hangingtime.

Locomotor and exploratory behavior, an intricate featureof physiological and behavioral functions in experimentalanimals, was assessed by open field test. MPTP intoxicationcaused a significant decline in peripheral and central move-ments and rearing and grooming activities when comparedto control group (𝑃 < 0.05) (Figures 3(a)–3(d)). However,


Ctrl MPTP CNB + MPTP CNBGroups

7

6

5

4

3

2

1

0

Central

∗

#

Num

ber o

f cen

tral

squa

res c

ross

ed/5min

(a)

0102030405060708090

Ctrl MPTP CNBGroups

Peripheral

CNB + MPTP

∗

#

Num

ber o

f squ

ares

cros

sed/

5min

(b)

0123456789

Ctrl MPTP CNBGroups

Grooming

CNB + MPTP

∗

#

Num

ber o

f act

iviti

es/

5min

(c)

02468

1012

Ctrl MPTP CNBGroups

Rearing

CNB + MPTP

∗

#

Num

ber o

f act

iviti

es/

5min

(d)

Figure 3: Open field test of control and experimental mice. MPTP injection resulted in significant decrease in central and peripheralmovements and grooming and rearing activities when compared to control. However, thesemotor impairmentswere reducedwhen pretreatedwith CNB-001. Values are expressed as mean ± SD for six animals in each group. ∗𝑃 < 0.05 compared to control, #

𝑃 < 0.05 compared toMPTP.

Table 1: In vivo antioxidant status of control and experimental mice.

Groups Control MPTP CNB-001 + MPTP CNB-001TBARS (nmoles/g tissue) 0.86 ± 0.90a 2.54 ± 0.20b 1.79 ± 0.13c 0.82 ± 0.09a

GSH (mg/g of tissue) 9.31 ± 1.05a 4.9 ± 0.39b 7.92 ± 0.71c 10.1 ± 1.02a

GPx (UA/mg protein) 9.6 ± 0.94a 4.31 ± 0.32b 7.36 ± 0.75c 9.9 ± 0.90a

SOD (UB/mg protein) 1.32 ± 0.19a 4.63 ± 0.50b 3.36 ± 0.23c 1.27 ± 0.21a

Catalase (UC/mg protein) 1.98 ± 0.17a 3.96 ± 0.34b 2.82 ± 0.14c 1.94 ± 0.11a

Values not sharing common superscript are significant with each other 𝑃 < 0.05.Anmol NADPH oxidized/minute/mg protein.BAmount of enzyme required to inhibit 50% of NBT reduction.Cnmol H2O2 consumed/minute/mg protein.

these impairments were reduced in animals pretreated withCNB-001 as compared to MPTP group (𝑃 < 0.05).

3.2. Effect of CNB-001 on Lipid Peroxidation and Antioxidants.MPTP administration caused a significant increase in thelevels of TBARS and activities of SODandCATanddecreasedthe levels of GSH and activity of GPx when compared tocontrol group (𝑃 < 0.05). Alternatively, CNB-001 inhibitedMPTP induced oxidative stress significantly by attenuatingenhanced TBARS, SOD, andCAT activities and increased thelevels ofGSHandGPX activities as compared toMPTP group(𝑃 < 0.05) (Table 1).

3.3. CNB-001 Abrogates MPTP Induced Loss of TH-PositiveNeurons. Acute administration of MPTP resulted in a sig-nificant decrease in TH density and TH-positive neurons inST and SN, respectively, when compared to control group

(𝑃 < 0.05). However, treatmentwithCNB-001 prior toMPTPadministration distinctly cosseted neurons by increasing THexpression in ST (Figure 4) and SN (Figure 5) compared toMPTP group (𝑃 < 0.05). Western blotting was performedsubsequently to confirm the immunohistochemical finding.

3.4. Effect of CNB-001 on TH, DAT, and VMAT2 Expres-sions. To determine the protective effect of CNB-001 againstMPTP induced neurodegeneration, the expression patternof phenotypic markers (TH, DAT, and VMAT2) in SNand ST was analyzed by western blotting. As depicted inFigure 6, MPTP significantly alleviated the expression of TH,DAT, and VMAT2 in both SN and ST compared to controlgroup (𝑃 < 0.05). Meanwhile, treatment with CNB-001reinstated these protein expressions distinctly as compared toMPTP group (𝑃 < 0.05). There were, however, no significantchanges between control and CNB-001 treated groups.


50𝜇m

50𝜇m50𝜇m

50𝜇m

(A)

(C) (D)

(B)

(a)

020406080

100120

Ctrl MPTP CNB

Den

sity

(% o

f con

trol)

GroupsCNB + MPTP

∗

#

(b)

Figure 4: Photomicrographs of ST sections depicting TH-immunoreactive (TH-ir) fibers. (a) (4X) Administration of MPTP(B) significantly depleted TH-positivity in striatum and thisdepletion was inhibited by pretreatment with CNB-001 (C). (b) THimmunoreactivity was analyzed by measuring the optical densityin ST. Mean value of TH-ir was determined for each group andexpressed as percentage control. Values are expressed as mean ± SDfor three animals in each group. ∗𝑃 < 0.05 compared to control,#𝑃 < 0.05 compared to MPTP.

3.5. CNB-001 Cosseted Mitochondria from MPTP InducedUltrastructural Changes. Electron microscopic studies wereperformed to analyze whether the antioxidant potential ofCNB-001 was strong enough to protect mitochondria mor-phology against MPTP toxicity. Striking structural changeswere observed in mitochondria of ST (Figure 7) and SN(Figure 8) inMPTP treatedmice.MPTP intoxication resultedin distortion of cristae, permeabilization, and enlargementof mitochondria in SN and ST (788 and 1032 nm resp.)(Figure 9). In contrast, pretreatment with CNB-001 protectedmitochondria from MPTP toxicity by maintaining normalmitochondrial morphology and size (660 and 825 nm) withdistinct nucleus and cristae compared to MPTP group (𝑃 <0.05).

50𝜇m

50𝜇m50𝜇m

50𝜇m(E)

(G) (H)

(F)

(a)

0100020003000400050006000700080009000

10000

Ctrl MPTP CNBGroups

Num

ber o

f TH+

neur

ons

CNB + MPTP

∗

#

(b)

Figure 5: Photomicrograph of SN sections illustrating TH-immunoreactive (TH-ir) neurons. (a) (4X) MPTP administration(F) significantly degenerated TH-ir neurons. Pretreatment withCNB-001 (G) significantly increased TH positivity as comparedto MPTP group. (b) Quantification of TH positive neurons wasperformed by counting number of TH-ir neurons and values areexpressed as mean ± SD of three animals per group. ∗𝑃 < 0.05compared to control, #


4. Discussion

In the present study, we substantiate that neuroprotectiveaction of CNB-001 against MPTP induced neurodegen-eration is by (i) attenuation of characteristic Parkinso-nian impairments, (ii) inhibition of lipid peroxidation andenhancement of antioxidant response, (iii) prevention of DA-ergic neuronal loss by reinstating expression of TH, DAT, andVMAT2 in SN and ST, and (iv) protection of mitochondrialmorphology in SN and ST againstMPTP toxicity.Though theetiology of PD is unknown, various biochemical alterationsinitiated by oxidative stress and mitochondrial dysfunctionare major factors which self-propel neurodegeneration [6].Thus antioxidant remains to be the Holy Grail for PD ther-apeutics. Postmortem and biochemical studies provide directevidence for involvement of hyperoxidation phenomenonin PD. Catecholaminergic neurons are particularly vulner-able to oxidative and nitrosative stress due to the presence


TH

DAT

VMAT2

𝛽-Actin

Ctrl MPTP CNBCNB + MPTP

(a)

00.20.40.60.8

11.21.4

TH DAT VMAT2Groups

CtrlMPTP CNB

TH, DAT, VMAT2 western blot densitometry in SN

∗

#

∗

#

∗

#

Rela

tive i

nten

sity

(fold

of c

ontro

l)

CNB + MPTP

(b)

TH

DAT

VMAT2

𝛽-Actin

Ctrl MPTP CNBCNB + MPTP

(c)

00.20.40.60.8

11.21.4

TH DAT VMAT2Groups

∗

#

∗

#

∗

#

TH, DAT, VMAT2 western blot densitometry in ST

Rela

tive i

nten

sity

(fold

of c

ontro

l)

CtrlMPTP CNB

CNB + MPTP

(d)

Figure 6: Immunoblotting of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) insubstantia nigra (SN) and striatum (ST) of experimental animals. Injection ofMPTP significantly reduced TH, DAT, andVMAT2 expressionsin SN and ST. Pretreatment with CNB-001 significantly increased TH, DAT, and VMAT2 expressions ((a) and (c)). Protein expressions werequantified using 𝛽-actin as an internal standard and values are expressed as arbitrary units and given as mean ± SD ((b) and (d)). ∗𝑃 < 0.05compared to control, #


of 6-hydroxydopamine resulting in enhanced formation ofintramitochondrial and cytosolic peroxynitrite. These toxicradicals initiate mitochondrial deficits by S-nitrosylation andFe-nitrosation of complex I subunit resulting in ATP deple-tion and neuronal death [27]. A recent study conducted inour lab showed that CNB-001 possessed stronger antioxidantactivity than curcumin by scavenging various free radicalssuch as DPPH, ABTS, and superoxide anions [28]. Hencethis study was aimed at finding profound effect of CNB-001 against PD. This study utilizes an MPTP (mitochondrialcomplex I inhibitor) model of PD which selectively interfereswith nigrostriatal pathway that connects SN with ST whichresults in DA depletion and subsequently leads to behavioralabnormalities as seen in PD [29, 30]. Ultimately, all neu-ropathological changes and neuroprotective action of drugsare reflected in animal behavior. Rotorod tests arewidely usedto analyze motor learning and motor-coordination ability byallowing the animals to walk on a rotating rod. Hang test wasperformed to analyze the neuromuscular strength [18] andopenfield test indicates acclimatization activity,mental stress,andmotor activity of mice [31]. In our experiments, we foundthat MPTP administration induced behavioral impairmentsas evidenced by rotorod, open field, and hang tests which are

ultimately linked to dopaminergic degeneration and deteri-oration of motor performance. Intraperitoneal administra-tion of CNB-001 significantly improved behavioral deficitssupporting the neuroprotective effect of CNB-001 againstMPTP toxicity. Antioxidant activity of CNB-001 might bethe possible mechanism involved in neuroprotection dueto the presence of electron donating styryl groups at 3,5-position of pyrazole and methoxyphenol group accountablefor iron chelation. To strengthen this property, our resultsrevealed that administration of MPTP increased the levelsof TBARS, due to lipid peroxidation, and reduced GSHand GPx levels along with increased activities of SOD andCAT. Pretreatment with CNB-001 modulated free radicaltoxicity by alleviating TBARS levels along with increasedactivities of antioxidant enzymes. Increase in SOD and CATin MPTP animals is due to adaptive response since MPTPmediated mitochondrial impairment results in leakage ofsuperoxide anions [32]. Moreover depletion of GSH andGPx might be an early mechanism involved in initiationof oxidative stress and possibly provides sensitivity to DA-ergic neurons against toxin [33]. Previous reports from ourlab showed that CNB-001 protected SK-N-SH cells againstrotenone toxicity by inhibiting accumulation of intracellular


873.18nm

530.41nm

561.47nm

(a)

919.95nm

1142.44nm

(b)

1154.82nm

1023.72nm

889.68nm

(c)

381.19 nm444.17nm

393.17nm

329.22nm

(d)

Figure 7: Photomicrographs of striatum viewed under transmission electron microscope. All panels demonstrate representative striataat 2000x. Control (a) and CNB-001 (d) alone treated groups showed normal ultrastructural morphology with enhanced mitochondrialpopulation withmembrane stability and cytoplasmic contents. Strikingly,MPTP treated group (b) showedmitochondria greatly enlarged andswollen rather than normal rod shaped.Moreover, therewere also distinct loss ofmitochondrial cristae and permeabilization ofmitochondrialmembrane. These toxicity changes were distinctly diminished upon CNB-001 treatment (c) showing normal nuclear morphology withincreased mitochondrial population with structural stability.

534.57nm 510.9nm

609.33nm 940.91nm

455.4nm

(a)

702.94nm

1185.9nm

473.1nm

(b)

662.43nm

732.48nm635.84nm

622.13 nm

(c)

473.5nm

578.58nm

640.42nm

615.8nm

(d)

Figure 8: Transmission electron micrographs of SN showing ultrastructural changes in control and experimental mice. All panelsdemonstrate SN neurons at 2000x. In control (a) and CNB-001 groups (d), increased mitochondrial population with prominent cristae wereseen. In MPTP group (b), mitochondria showed abnormal structure with distorted cristae and the number of mitochondria was greatlyreduced. In contrast CNB-001 treatment (c) dampened these apoptotic changes and improved mitochondrial morphology and population ascompared to MPTP group.


0

200

400

600

800

1000

1200

SN ST

Mitochondrial perimeter length

CtrlMPTP CNB

Mea

n pe

rimet

er le

ngth

(nm

)

CNB + MPTP

∗

#

∗

#

Figure 9: Analysis of mitochondrial perimeter length in SNand ST of experimental animals. Mitochondrial perimeterlength/mitochondrion was significantly more increased in MPTPgroup than in control. Pretreatment with CNB-001 significantlymaintained mitochondrial size as compared to MPTP group. Nosignificant changes were observed in control and CNB-001 treatedgroups. ∗𝑃 < 0.05 compared to control, #

𝑃 < 0.05 compared toMPTP.

ROS formation [13]. Thus, neuroprotective activity of CNB-001 may be partially due to the regulation of antioxidantenzymes which is depleted upon MPTP treatment. Further,the neuroprotective mechanism of CNB-001 may correlatewell with previous study where CNB-001 protected HT-22cells against glutamate induced oxidative stress [11].

Antioxidant mediated neuroprotection by CNB-001 isfurther strongly supported by immunohistochemical andwestern blotting studies. Administration of MPTP signif-icantly reduced the expression of TH, DAT, and VMAT2in SN and ST of mice. These proteins play a vital role innigrostriatal pathway and are involved in DA biosynthesis.TH is a rate-limiting enzyme responsible for the conversionof tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Fur-ther, aromatic amino acid decarboxylase removes carboxylicacid moiety from L-DOPA to form DA. Synthesized DAis sequestered by VMAT2 and released in synaptic spaceand further transmitted. VMAT2 also plays an importantrole in sequestering of neurotoxins and prevents toxicity[34]. Nontransmitted DA is further recycled into presynapticneurons via DA transporter (DAT). Previous reports showedthat diminished expressions of TH, DAT [4], and VMAT2 [5]in SN and ST are thought to underlie PD pathogenesis. Ourresults were concordant with various reports which showedthat MPTP intoxication selectively targets and destroys DA-ergic neurons as evidenced by reduced TH immunoreactivityand resultant DA depletion [35, 36]. Apart from the roleof DAT in DA biosynthesis, it also acts as a gateway forseveral neurotoxins including MPTP for DA-ergic toxicity[2]. Hence, immunoblotting results in our study showeddecrease in DAT expression in SN and ST of MPTP treatedmice might be due to loss of nigrostriatal neurons and fibers,respectively. Similar to TH and DAT expressions, MPTPinjection significantly depleted the expression of VMAT2

in brain. VMAT2 plays an important role in inhibitingdopamine mediated toxicity by sequestering cytosolic DAand it has also been reported that VMAT2 deletion causesmice lethality [37]. Accumulation of cytosolic DA supportsformation of oxyradical stress and excess cytosolic DA causesproteotoxicity to DA-ergic neurons and autophagy block[38, 39]. Therefore enhanced expression of VMAT2 couldpotentially contribute to neuroprotection. Consistent to thesereports TH, DAT, and VMAT2 protein levels were reduceddistinctly in patients affected by PD [40]. Pretreatment withCNB-001 enhanced TH-ir and expressions of TH, DAT, andVMAT2 which might contribute to enhanced DA synthesisand mitigation of behavioral impairments caused by MPTP.

Several clinical reports have demonstratedmodest reduc-tion in mitochondrial complex I activity in PD patients[41, 42]. Further, MPP+ selectively blocks complex I ofmitochondria and inhibits flow of electrons across electrontransport chain and resultant ATP depletion with increasedROS production. Electronmicroscopic results from our stud-ies showed that MPTP intoxication enhanced mitochondrialpathology in SN and STwhich reflect the sequelae of injury tomitochondria. Furthermore mitochondria from PD patientsand MPTP treated animals have also been described to beswollen and enlarged, possessing distorted mitochondrialcristae with discontinuous outer membrane rather thanordered cristae with normal rod shaped morphology, whichare similar to our results. To confirm the toxicity and toaddress difference in mitochondria structure, we measuredmitochondrial perimeter length of experimental animalsbecause increase inmitochondrial perimeter length as a resultof mitochondrial swelling is a well-accepted hallmark inorganelle dysfunction [43]. MPTP treatment increased themitochondrial size when compared to the control group.Pretreatment with CNB-001 protected the mitochondria andthe ultrastructural changes were less prominent as comparedto MPTP group. Our findings are also similar to thosereported during apoptosis mediated cell death in SN ofPD [44]. Moreover CNB-001 protected SK-N-SH neuroblas-toma against rotenone toxicity bymaintainingmitochondrialmembrane potential through inhibition of proapoptotic fac-tors [13]. These results confirm the protective action of CNB-001 on mitochondria, thus alleviating neuronal pathology.Our qualitative structural study showed that dendritic (ST)mitochondria are larger than axonal (SN) mitochondriawhich are similar to previous reports [45]. We have alsoobserved that CNB-001 prevented DA-ergic neuronal loss byattenuating inflammatory and apoptotic response in MPTPmodel of PD (data not shown). Further results of the presentstudy are substantiated by previous reports, where CNB-001has been shown to possess neuroprotective effects againstintracellular amyloid toxicity, trophic factor withdrawal, andexcitotoxicity [11].

5. Conclusion

In summary, CNB-001 protects DA-ergic neurons againstMPTP toxicity by regulating various molecular and cellularevents.The therapeutic potential of CNB-001 is supported by


its ability to reduce behavioral impairments, oxidative stress,and mitochondrial deficits and by enhancing expressionsof TH, DAT, and VMAT2 in animal model of PD. Hence,based on these results we speculate that further investigationon CNB-001 would be worthwhile to bring out a potenttherapeutic contender in the treatment of PD.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

The authors are very grateful to Dr. Dave Schubert (CellularNeurobiology Lab) at Salk Institute for Biological Studies forhis constant advice and generosity in providing CNB-001.This work is supported in part by CSIR Network ProjectGrant, miND (BSC0115), to Shubha Shukla. Sonu Singh isthankful to ICMR, NewDelhi, India, for research fellowship.

References

[1] J. Jankovic, “Parkinson's disease: clinical features and diagno-sis,” Journal of Neurology, Neurosurgery and Psychiatry, vol. 79,no. 4, pp. 368–376, 2008.

[2] W.Dauer and S. Przedborski, “Parkinson's disease:mechanismsand models,” Neuron, vol. 39, no. 6, pp. 889–909, 2003.

[3] M. B. Spina and G. Cohen, “Dopamine turnover and glu-tathione oxidation: implications for Parkinson disease,” Pro-ceedings of the National Academy of Sciences of the United Statesof America, vol. 86, no. 4, pp. 1398–1400, 1989.

[4] R. E. Heikkila and P. K. Sonsalla, “The MTPT-treated mouseas a model of Parkinsonism: how good is it?” NeurochemistryInternational, vol. 20, supplement 1, pp. 299S–303S, 1992.

[5] G. W. Miller, J. D. Erickson, J. T. Perez et al., “Immunochemicalanalysis of vesicular monoamine transporter (VMAT2) proteinin Parkinson's disease,” Experimental Neurology, vol. 156, no. 1,pp. 138–148, 1999.

[6] A. H. Schapira, “Mitochondria in the aetiology and pathogen-esis of Parkinson's disease,” The Lancet Neurology, vol. 7, no. 1,pp. 97–109, 2008.

[7] N. Li, K. Ragheb, G. Lawler et al., “Mitochondrial complexI inhibitor rotenone induces apoptosis through enhancingmitochondrial reactive oxygen species production,”The Journalof Biological Chemistry, vol. 278, no. 10, pp. 8516–8525, 2003.

[8] M. del Zompo, M. P. Piccardi, S. Ruiu, M. Quartu, G. L. Gessa,andA. Vaccari, “SelectiveMPP+ uptake into synaptic dopaminevesicles: possible involvement in MPTP neurotoxicity,” BritishJournal of Pharmacology, vol. 109, no. 2, pp. 411–414, 1993.

[9] R. Tintner and J. Jankovic, “Treatment options for Parkinson'sdisease,” Current Opinion in Neurology, vol. 15, no. 4, pp. 467–476, 2002.

[10] G. M. Cole, B. Teter, and S. A. Frautschy, “Neuroprotectiveeffects of curcumin,” Advances in Experimental Medicine andBiology, vol. 595, pp. 197–212, 2007.

[11] Y. Liu, R. Dargusch, P. Maher, and D. Schubert, “A broadly neu-roprotective derivative of curcumin,” Journal of Neurochemistry,vol. 105, no. 4, pp. 1336–1345, 2008.

[12] E. Valera, R. Dargusch, P. A. Maher, and D. Schubert, “Mod-ulation of 5-lipoxygenase in proteotoxicity and Alzheimer'sdisease,” Journal of Neuroscience, vol. 33, no. 25, pp. 10512–10525,2013.

[13] R. L. Jayaraj, K. Tamilselvam, T. Manivasagam, and N. Elan-govan, “Neuroprotective effect of CNB-001, a novel pyrazolederivative of curcumin on biochemical and apoptotic markersagainst rotenone-induced SK-N-SH cellular model of Parkin-son’s disease,” Journal of Molecular Neuroscience, vol. 51, no. 3,pp. 863–870, 2013.

[14] E. Antzoulatos, M. W. Jakowec, G. M. Petzinger, and R. I.Wood, “Sex differences in motor behavior in the MPTP mousemodel of Parkinson's disease,” Pharmacology Biochemistry andBehavior, vol. 95, no. 4, pp. 466–472, 2010.

[15] Y.-S. Lau, L. Novikova, and C. Roels, “MPTP treatment inmice does not transmit and cause Parkinsonian neurotoxicityin non-treated cagemates through close contact,” NeuroscienceResearch, vol. 52, no. 4, pp. 371–378, 2005.

[16] O. Narumoto, Y. Matsuo, M. Sakaguchi et al., “Suppressiveeffects of a pyrazole derivative of curcumin on airway inflam-mation and remodeling,” Experimental and Molecular Pathol-ogy, vol. 93, no. 1, pp. 18–25, 2012.

[17] G. Rozas, E. Lopez-Martın, M. J. Guerra, and J. L. Labandeira-Garcıa, “The overall rod performance test in the MPTP-treated-mouse model of Parkinsonism,” Journal of NeuroscienceMethods, vol. 83, no. 2, pp. 165–175, 1998.

[18] J. L. Tillerson and G. W. Miller, “Grid performance test tomeasure behavioral impairment in the MPTP-treated-mousemodel of parkinsonism,” Journal of Neuroscience Methods, vol.123, no. 2, pp. 189–200, 2003.

[19] S. RajaSankar, T. Manivasagam, and S. Surendran, “Ashwa-gandha leaf extract: a potential agent in treating oxidativedamage and physiological abnormalities seen in amousemodelof Parkinson's disease,” Neuroscience Letters, vol. 454, no. 1, pp.11–15, 2009.

[20] K. B. J. Franklin and G. Paxinos,TheMouse Brain in StereotaxicCoordinates, Academic Press, London, UK, 2007.

[21] A. Bhattacharya, S. Ghosal, and S. K. Bhattacharya, “Anti-oxidant effect of Withania somnifera glycowithanolides inchronic footshock stress-induced perturbations of oxidativefree radical scavenging enzymes and lipid peroxidation in ratfrontal cortex and striatum,” Journal of Ethnopharmacology, vol.74, no. 1, pp. 1–6, 2001.

[22] D. J. Jollow, J. R. Mitchell, N. Zampaglione, and J. R. Gillette,“Bromobenzene induced liver necrosis. Protective role of glu-tathione and evidence for 3,4 bromobenzene oxide as thehepatotoxic metabolite,” Pharmacology, vol. 11, no. 3, pp. 151–169, 1974.

[23] Y. Yamamoto and K. Takahashi, “Glutathione peroxidaseisolated from plasma reduces phospholipid hydroperoxides,”Archives of Biochemistry and Biophysics, vol. 305, no. 2, pp. 541–545, 1993.

[24] L. W. Oberley and D. R. Spitz, “Assay of superoxide dismutaseactivity in tumor tissue,” Methods in Enzymology, vol. 105, pp.457–464, 1984.

[25] H. Aebi, “Catalase in vitro,”Methods in Enzymology, vol. 105, pp.121–126, 1984.

[26] O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,“Protein measurement with the Folin phenol reagent,” TheJournal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.


[27] G. C. Brown and V. Borutaite, “Inhibition of mitochondrialrespiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols,” Biochimica et Biophysica Acta: Bioenergetics, vol.1658, no. 1-2, pp. 44–49, 2004.

[28] R. L. Jayaraj and N. Elangovan, “In vitro antioxidant potentialand DNA protecting activity of CNB-001, a novel pyrazolederivative of curcumin,” Chronicles of Young Scientists, vol. 5,pp. 44–52, 2014.

[29] T. Hanakawa, Y. Katsumi, H. Fukuyama et al., “Mechanismsunderlying gait disturbance in Parkinson's disease. A singlephoton emission computed tomography study,” Brain, vol. 122,no. 7, pp. 1271–1282, 1999.

[30] S. J. Crocker, P. Liston,H.Anisman et al., “Attenuation ofMPTP-induced neurotoxicity and behavioural impairment in NSE-XIAP transgenic mice,” Neurobiology of Disease, vol. 12, no. 2,pp. 150–161, 2003.

[31] A. Fredriksson and T. Archer, “MPTP-induced behavioural andbiochemical deficits: a parametric analysis,” Journal of NeuralTransmission: Parkinson's Disease and Dementia Section, vol. 7,no. 2, pp. 123–132, 1994.

[32] C. Thiffault, N. Aumont, R. Quirion, and J. Poirier, “Effectof MPTP and L-deprenyl on antioxidant enzymes and lipidperoxidation levels in mouse brain,” Journal of Neurochemistry,vol. 65, no. 6, pp. 2725–2733, 1995.

[33] S. Toffa, G. M. Kunikowska, B.-Y. Zeng, P. Jenner, and C.D. Marsden, “Glutathione depletion in rat brain does notcause nigrostriatal pathway degeneration,” Journal of NeuralTransmission, vol. 104, no. 1, pp. 67–75, 1997.

[34] E. A. Fon, E. N. Pothos, B.-C. Sun, N. Killeen, D. Sulzer,and R. H. Edwards, “Vesicular transport regulates monoaminestorage and release but is not essential for amphetamine action,”Neuron, vol. 19, no. 6, pp. 1271–1283, 1997.

[35] A. Anandhan, U. Janakiraman, and T. Manivasagam,“Theaflavin ameliorates behavioral deficits, biochemical indicesand monoamine transporters expression against subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-inducedmouse model of Parkinson's disease,”Neuroscience, vol. 218, pp.257–267, 2012.

[36] K. R. Rekha, G. P. Selvakumar, S. Sethupathy, K. Santha, and R.I. Sivakamasundari, “Geraniol ameliorates the motor behaviorand neurotrophic factors inadequacy in MPTP-induced micemodel of Parkinson’s disease,” Journal of Molecular Neuro-science, vol. 51, no. 3, pp. 851–862, 2013.

[37] Y.-M.Wang, R. R. Gainetdinov, F. Fumagalli et al., “Knockout ofthe vesicular monoamine transporter 2 gene results in neonataldeath and supersensitivity to cocaine and amphetamine,” Neu-ron, vol. 19, no. 6, pp. 1285–1296, 1997.

[38] M.Martinez-Vicente, Z. Talloczy, S. Kaushik et al., “Dopamine-modified 𝛼-synuclein blocks chaperone-mediated autophagy,”Journal of Clinical Investigation, vol. 118, no. 2, pp. 777–778,2008.

[39] E. V. Mosharov, K. E. Larsen, E. Kanter et al., “Interplaybetween cytosolic dopamine, calcium, and alpha-synucleincauses selective death of substantia nigra neurons,”Neuron, vol.62, no. 2, pp. 218–229, 2009.

[40] J. M. Wilson, K. S. Kalasinsky, A. I. Levey et al., “Striataldopamine nerve terminal markers in human, chronic metham-phetamine users,” Nature Medicine, vol. 2, no. 6, pp. 699–703,1996.

[41] Y. Mizuno, S. Ohta, M. Tanaka et al., “Deficiencies in ComplexI subunits of the respiratory chain in Parkinson's disease,”

Biochemical and Biophysical Research Communications, vol. 163,no. 3, pp. 1450–1455, 1989.

[42] W. D. Parker Jr., S. J. Boyson, and J. K. Parks, “Abnormalities ofthe electron transport chain in idiopathic Parkinson's disease,”Annals of Neurology, vol. 26, no. 6, pp. 719–723, 1989.

[43] P. A. Trimmer, R. H. Swerdlow, J. K. Parks et al., “Abnor-mal mitochondrial morphology in sporadic Parkinson's andAlzheimer's disease cybrid cell lines,” Experimental Neurology,vol. 162, no. 1, pp. 37–50, 2000.

[44] P. Anglade, “Apoptosis in dopaminergic neurons of thehuman substantia nigra during normal aging,” Histology andHistopathology, vol. 12, no. 3, pp. 603–610, 1997.

[45] B. K. Binukumar, A. Bal, R. J. L. Kandimalla, and K. D.Gill, “Nigrostriatal neuronal death following chronic dichlor-vos exposure: crosstalk between mitochondrial impairments,𝛼 synuclein aggregation, oxidative damage and behavioralchanges,”Molecular Brain, vol. 3, no. 1, article 35, 2010.

Submit your manuscripts athttp://www.hindawi.com

Neurology Research International

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Alzheimer’s DiseaseHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


BioMed Research International


Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014


Neural Plasticity


Parkinson’s Disease


Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Psychiatry Journal


Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Neurodegenerative Diseases


Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

research article cnb-001 a novel curcumin derivative, guards...

Documents