# CNB-001 a Novel Curcumin Derivative, Guards Dopamine Neurons in MPTP Model of Parkinson’s Disease

Authors: Richard L. Jayaraj; Namasivayam Elangovan; Krishnan Manigandan; Sonu Singh; Shubha Shukla
Journal: BioMed Research International
 (2014)

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## Abstract

Copious experimental and postmortem studies have shown that oxidative stress mediated degeneration of nigrostriatal dopaminergic neurons underlies Parkinson’s disease (PD) pathology. CNB-001, a novel pyrazole derivative of curcumin, has recently been reported to possess various neuroprotective properties. This study was designed to investigate the neuroprotective mechanism of CNB-001 in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) rodent model of PD. Administration of MPTP (30 mg/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 ultrastructural changes such as distorted cristae and mitochondrial enlargement in substantia nigra and striatum region. Pretreatment with CNB-001 (24 mg/kg) not only ameliorated behavioral anomalies but also synergistically enhanced monoamine transporter expressions and cosseted mitochondria by virtue of its antioxidant action. These findings support the neuroprotective property of CNB-001 which may have strong therapeutic potential for treatment of PD.


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## Body

## 1. Introduction

Parkinson’s disease (PD), a progressive neurodegenerative disorder, is characterized by degeneration of dopaminergic (DA-ergic) neurons in substantia nigra pars compacta (SNpc) and resultant loss of dopamine (DA) in the striatum (motor loci). Clinical diagnosis reports have shown that cardinal behavioral features of PD include rigidity, dyskinesia, gait imbalance, and tremor at rest [1]. Though the etiology of PD remains obscure, various experimental studies have reported the involvement of oxidative stress, mitochondria dysfunction, apoptosis, and inflammation either separately or cooperatively to induce neurodegeneration [2]. DA, a vital neurotransmitter (important for motor coordination), is synthesized in nigrostriatal pathway and undergoes autooxidation to form toxic reactive oxygen species (ROS) and quinone molecules which in turn initiates pathological response [3]. Apart from DA depletion, a profound reduction in specific neurochemical markers such as tyrosine hydrolase (TH), DA transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) has been reported in PD [4, 5]. In idiopathic PD, mitochondrial complex I impairment leading to free radical generation which evokes respiratory 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 precise mechanisms underlying neurodegeneration in PD [7]. MPTP, a catecholamine neurotoxin, selectively degenerates dopaminergic neurons via monoamine oxidase-B- (MAO-B-) mediated conversion of MPTP to its toxic/active metabolite MPP+. Further MPP+ is taken into dopaminergic neurons via DA transporter and sequestered in mitochondria and inhibits complex I of electron transport chain resulting in enhanced ROS formation and decreased ATP production. Moreover, MPP+ is also taken into the cytosol through vesicular monoamine transporter 2 (VMAT2) and supports lethality [8]. Hence, assessment of DAT and VMAT2 reveals a greater picture regarding DA integrity and neuronal vulnerability [5]. For these reasons, antioxidant and antiapoptotic compounds are being explored for their therapeutic potential in treatment of PD. Current PD medications (predominantly L-Dopa, monoamine oxidase-B inhibitors) offer only symptomatic relief and could not counteract PD progression [9]. Various literature reports have shown that curcumin, a polyphenol, has excellent biological properties and is known to protect DA neurons [10]. In spite of its pharmacological properties, curcumin has poor bioavailability, fails to inhibit excitotoxicity, and does not 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 molecule synthesized 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 to also exhibit strong antioxidant and antiapoptotic properties [13]. Additionally, CNB-001 has been shown to possess lower EC50 values and superior anti-inflammatory properties compared to its parental compounds [11]. Since these results illuminate biological properties of CNB-001, the present study was aimed at investigating whether CNB-001 offers neuroprotection, by evaluating its effect on oxidative stress, behavioral impairments, expressions of TH, DAT, and VMAT2, and mitochondria ultrastructural analysis in a subacute MPTP model of PD.


## 2. Materials and Methods

### 2.1. Experimental Animals and Ethics Statement

Adult male C57BL/6 mice weighing between 25 and 27 g purchased from National Institute of Nutrition, Hyderabad, were used in this study. Since male C57BL/6 mice are more sensitive to MPTP intoxication than females and PD is observed frequently in males, female mice were avoided [14]. Animals were maintained at ambient conditions (22±1°C, 60% humidity, and 12 h diurnal cycle) and hadad libitum access to food and water. All experiments were performed in accordance with National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Academy, New Delhi, 2009) and approved by Institutional Animal Ethics Committee (1085/ac/07/PU-IAEC/2012/13).


### 2.2. Materials

MPTP was procured from Sigma-Aldrich, Bangalore, India. TH, DAT, and VMAT2 primary and corresponding secondary antibodies were purchased from Cell Signaling (USA). Enhanced chemiluminescence kit was obtained from GenScript, USA. All other chemicals used were 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 Biological Studies, La Jolla, CA).


### 2.3. Dose Fixation and Experimental Design

A dose dependent study was initially conducted with four different doses of CNB-001 (6, 12, 24, and 48 mg/kg) to analyze the effect of CNB-001 on DA content in MPTP intoxicated mice. After seven days of experimental period, it was observed that pretreatment with CNB-001 (6, 12, 24, and 48 mg/kg) significantly increased the levels of DA and its metabolites in MPTP intoxicated mice. Further, it was noted that CNB-001 at a dose of 24 and 48 mg/kg showed similar increase in DA content but markedly higher than 6 and 12 mg/kg. As a result, an optimum dose of 24 mg/kg was used for further studies.To evaluate the neuroprotective effect of CNB-001, we used a subacute MPTP paradigm which represents one of the most stable toxin based PD models (n=10). Group I received intraperitoneal (i.p) injection of saline and ethanol (10-fold dilution with 1% (v/v) Tween 80/saline) which were vehicles for MPTP and CNB-001, respectively, and served as control group. Groups II and III received MPTP injection (30 mg/kg; i.p) starting from 4th to 7th day of the experimental period. Guidelines for handling MPTP were strictly followed as described previously [15]. Groups III and IV received CNB-001 (24 mg/kg, i.p, 1 h prior to MPTP injection) for 7 consecutive days. CNB-001 was dissolved in 100% ethanol (40 μL for 1 mg) followed by 10-fold dilution with 1% (v/v) Tween 80/saline [16]. Two days after the treatment schedule, animals were subjected to behavioral tests. Since we were aware that behavioral tests might cause some kind of stress and alter biochemical actions, the animals used to analyze enzyme activities were not subjected to behavioral tests.


### 2.4. Behavioral Assessment

Behavioral tests were carried out in such a way that the order of tasks did not affect the outcome of our results. These tasks were carried out in two phases; in phase I we analyzed motor coordination by performing rotorod tests and in phase II we evaluated mental stress by open field test and hang test was performed to assess neuromuscular strength.


#### 2.4.1. Rotorod Test

This test was performed to assess muscular coordination, strength, and balancing ability of animals using rotorod apparatus (Panlab). Initially all the animals were habituated and trained on accelerating rotating rod (5, 10, and 15 rpm) with a maximum cut-off time of 180 s for five consecutive days (twice/day) prior to treatment. After experimental period, the average retention time on the rod was noted as described previously [17].


#### 2.4.2. Hang Test

The effect of CNB-001 on neuromuscular strength was analyzed by grid hang test. Briefly, animals were placed on a horizontal grid and supported until they held the grid with all their four paws. The grid was then kept in an inverted position allowing the animals to hang upside down and the maximum hanging time was noted. Proper care was taken 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 in a wooden apparatus (W100 × D100 × H4 cm). The floor was covered with a rexin cloth and divided into25(5×5) equal squares. The animals were placed in the middle of apparatus and their behavior was observed for 5 min. Central and periphery movements were calculated based on number of central (nine) and peripheral (sixteen) squares crossed by the animals, respectively. One count was made only when the 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 5 min. The entire study was conducted in a blinded manner [19].


### 2.5. Perfusion and Tissue Processing

At the end of the experiments, animals were sacrificed by terminal anesthesia and perfused via intracardial infusion with 0.9% saline followed by 4% paraformaldehyde (pH 7.4). Brain was removed from the skull and postfixed in 4% paraformaldehyde for 24 h at 4°C. The brain tissue was then embedded in paraffin and sliced into 5μm coronal sections containing entire SN and ST (anteroposterior levels: bregma −2.92 to −3.64 mm and +0.02 to +0.86 mm) with the reference of mouse brain atlas [20] for immunohistochemical study. For immunoblotting, SN and ST were dissected out and rapidly frozen on dry ice and 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 previously described [21]. In brief, the tissue extracts were incubated in metabolic water bath shaker at 37°C with 0.2 mL phenyl methosulfate. After one hour, 0.4 mL of 0.67% thiobarbituric acid and 0.4 mL of 5% tricarboxylic acid were added. The reaction mixture was centrifuged at 4000 rpm for 15 min at room temp. (RT) and the resultant supernatant was boiled for 10 min. The samples were cooled and reading was taken at 535 nm. The rate of lipid peroxidation is expressed as nmol of TBARS formed per hour/g tissue.


#### 2.6.2. Estimation of Reduced Glutathione

The levels of reduced glutathione in brain tissue homogenate were measured as previously described [22]. Brain homogenate was centrifuged at 16,000 ×g for 15 min at 40°C. After centrifugation, 0.5 mL of the supernatant was added to 4 mL of ice-cold 0.1 mM 5,5-dithiobis[2-nitrobenzoic acid] solution in 1 M phosphate buffer (pH-8) and the absorbance was measured at 412 nm.


#### 2.6.3. Estimation of Glutathione Peroxidase (GPx)

The level of GPx in brain tissue homogenate was measured as previously described [23]. Briefly, the assay mixture consisted of 100 μL of 1 M Tris-Hcl (pH-8.0) containing 5 mM of EDTA, 20 μL of 0.1 M GSH, 100 μL of glutathione reductase solution (10 U/mL), 100 μL of 2 mM NADPH, 650 μL of distilled water, 10 μL of 7 mMtert-butyl hydroperoxidase, and 10 μL of brain homogenate. NADPH oxidation was determined spectrometrically at 340 nm. One unit of enzyme activity was defined as the amount of GPx required to oxidize 1 μmol of NADPH per min.


#### 2.6.4. Assay for Superoxide Dismutase (SOD) Activity

The activity of SOD was assessed following the method as previously described [24]. Xanthine and xanthine oxidase served as superoxide generator and nitro blue tetrazolium is used as superoxide indicator. Briefly, the reaction mixture consists of 960 μL of 50 mM sodium carbonate buffer (pH 10.2) containing 0.1 mM of EDTA, 0.1 mM of xanthine, 20 μL of xanthine oxidase, 0.025 mM of NBT, and 20 μL of brain supernatant. The absorbance is measured spectrometrically at 560 nm and the activity was expressed as units/min/mg protein.


#### 2.6.5. Determination of Catalase (CAT) Activity

Catalase activity was measured by determining the rate of decomposition of hydrogen peroxide (H2O2) at 240 nm. In brief, assay mixture consisted of 50 μL of 1 M Tris-Hcl buffer (pH 8.0) containing 5 mM of EDTA, 900 μL of 10 mM H2O2, 30 μL of water, and 20 μL of the supernatant. Rate of decomposition of hydrogen was measured at 240 nm spectrometrically. Enzyme activity is expressed as nmol of H2O2 decomposed/min/mg protein [25].


### 2.7. Immunohistochemical Studies

Immunohistochemistry was performed to investigate the presence of DA-ergic neurons with TH immunoreactivity. Substantia nigra (SN) and striatum (ST) region were serially sectioned and the slides were deparaffinized using xylene followed by rehydration in graded series of ethanol. The slides were boiled for 10 min in 10 mM of citrate buffer (pH-6) for antigen retrieval. The sections were then incubated in dark with H2O2 (0.3%) for 10 min at RT. The slides were placed in blocking buffer (10% normal goat serum (NGS) with 0.2% Triton X-100 in 0.01 M PBS) at 37°C for 30 min. In each treatment, slides were washed three times with 0.01 M PBS for 5 min. Sections were incubated primarily with anti-mouse TH (1 : 1000) in 2% normal goat serum, 0.2% Triton X-100, and 0.02% sodium azide in TBS for 24 h. After washing with 1% NGS in TBS, the sections were incubated with anti-mouse IgG-HRP conjugated secondary antibody (1 : 1000 in 1.5% NGS) for one hour followed by washing with PBS. The sections were then incubated with diaminobenzidine (DAB) to view and analyze TH immunoreactivity.The intensity of TH immunoreactivity in ST was measured using Micro Computer Imaging Device software and results are expressed as percentage of control. Mouse brain atlas was initially used to delineate SN at low magnification and the numbers of TH immunoreactive neurons in SN were counted at higher magnification (20x) by a person, blind to the treatment. Cell counts were determined in every sixth section (total of 8 to 10 sections) through SN corresponding to the bregma −2.92 to −3.64 mm from each animal. The analyses of TH positive neurons were restricted to SN and thus excluded the ventral tegmental area.


### 2.8. Levels of TH, DAT, and VMAT2 by Immunoblotting

Western blotting was performed to analyze the expression patterns of TH, DAT, and VMAT2 in SN and ST of experimental animals. In brief, the tissues were homogenized in ice-cold RIPA buffer (1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mM/L EDTA, 20 mM/L Tris (pH 7.4), 10 mM/L NAF, 150 mM/L NaCl, and 0.1 mM/L phenylmethylsulfonyl fluoride) and centrifuged (12,000 ×g for 15 min at 4°C) to remove cellular debris. Protein concentration in the samples was analyzed using Lowry method [26]. Proteins (50 μg) were separated by 10% SDS-polyacrylamide gel and electrotransferred onto a PVDF membrane by semidry transfer (BIO-RAD). After blocking the membrane for 1 hr in 5% nonfat dry milk in TBS at 25°C, the membranes were incubated with β-actin (rabbit polyclonal, 1 : 500 dilution in 5% BSA Tris-buffered saline and 0.05% Tween 20 (TBST), anti-mouse TH (1 : 1000), anti-mouse DAT (1 : 500), and anti-mouse VMAT2 (1 : 1000)) with gentle shaking at 4°C overnight. Corresponding secondary antibodies (anti-mouse or anti-rabbit IgG conjugated to HRP) were then added and incubated for 2 h at room temperature. The membranes were washed thrice with TBST (10 min/wash) and immunoreactive bands were visualized by chemiluminescence kit. Densitometry analysis was done using “Image J” analysis software. The blot intensities were normalized with that of β-actin as loading control.


### 2.9. Electron Microscopy

Brain regions (SN and ST) were fixed in 3% gluteraldehyde at 4°C for 24 h. After fixation, brain sections were cut into approximately 1 mm cubes and postfixed in 1% osmium tetraoxide for 2 h at 4°C. The cubes were then dehydrated in series of ethanol and treated twice with propylene oxide for 10 min at RT. The tissues were infiltrated 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. Ultra microtome was used to cut tissue into 0.6μm thick sections which were then stained with 0.5% toluidine blue to confirm the presence of neurons. Then the 60 nm ultrathin sections were cut and mounted on Nickel grids (300 mesh). The sections were double stained with uranyl acetate and lead citrate and then examined by Transmission Electron Microscope (Philips CM10) and photographed.


#### 2.9.1. Morphological Analysis of Mitochondria within SN and ST

Measurements were generated from three randomly acquired TEM images from SN and ST with a magnification of 16,000x and coded for blinded analysis. The perimeter length of each mitochondrion was analyzed by a single trained technician using MeasureIT software (Olympus Soft Imaging Solutions). Mitochondria were identified by presence of identifiable cristae and distinct double membrane; however, mitochondria with unclear morphology were avoided.


## 2.1. Experimental Animals and Ethics Statement

Adult male C57BL/6 mice weighing between 25 and 27 g purchased from National Institute of Nutrition, Hyderabad, were used in this study. Since male C57BL/6 mice are more sensitive to MPTP intoxication than females and PD is observed frequently in males, female mice were avoided [14]. Animals were maintained at ambient conditions (22±1°C, 60% humidity, and 12 h diurnal cycle) and hadad libitum access to food and water. All experiments were performed in accordance with National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Academy, New Delhi, 2009) and approved by Institutional Animal Ethics Committee (1085/ac/07/PU-IAEC/2012/13).


## 2.2. Materials

MPTP was procured from Sigma-Aldrich, Bangalore, India. TH, DAT, and VMAT2 primary and corresponding secondary antibodies were purchased from Cell Signaling (USA). Enhanced chemiluminescence kit was obtained from GenScript, USA. All other chemicals used were 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 Biological Studies, La Jolla, CA).


## 2.3. Dose Fixation and Experimental Design

A dose dependent study was initially conducted with four different doses of CNB-001 (6, 12, 24, and 48 mg/kg) to analyze the effect of CNB-001 on DA content in MPTP intoxicated mice. After seven days of experimental period, it was observed that pretreatment with CNB-001 (6, 12, 24, and 48 mg/kg) significantly increased the levels of DA and its metabolites in MPTP intoxicated mice. Further, it was noted that CNB-001 at a dose of 24 and 48 mg/kg showed similar increase in DA content but markedly higher than 6 and 12 mg/kg. As a result, an optimum dose of 24 mg/kg was used for further studies.To evaluate the neuroprotective effect of CNB-001, we used a subacute MPTP paradigm which represents one of the most stable toxin based PD models (n=10). Group I received intraperitoneal (i.p) injection of saline and ethanol (10-fold dilution with 1% (v/v) Tween 80/saline) which were vehicles for MPTP and CNB-001, respectively, and served as control group. Groups II and III received MPTP injection (30 mg/kg; i.p) starting from 4th to 7th day of the experimental period. Guidelines for handling MPTP were strictly followed as described previously [15]. Groups III and IV received CNB-001 (24 mg/kg, i.p, 1 h prior to MPTP injection) for 7 consecutive days. CNB-001 was dissolved in 100% ethanol (40 μL for 1 mg) followed by 10-fold dilution with 1% (v/v) Tween 80/saline [16]. Two days after the treatment schedule, animals were subjected to behavioral tests. Since we were aware that behavioral tests might cause some kind of stress and alter biochemical actions, the animals used to analyze enzyme activities were not subjected to behavioral tests.


## 2.4. Behavioral Assessment

Behavioral tests were carried out in such a way that the order of tasks did not affect the outcome of our results. These tasks were carried out in two phases; in phase I we analyzed motor coordination by performing rotorod tests and in phase II we evaluated mental stress by open field test and hang test was performed to assess neuromuscular strength.


### 2.4.1. Rotorod Test

This test was performed to assess muscular coordination, strength, and balancing ability of animals using rotorod apparatus (Panlab). Initially all the animals were habituated and trained on accelerating rotating rod (5, 10, and 15 rpm) with a maximum cut-off time of 180 s for five consecutive days (twice/day) prior to treatment. After experimental period, the average retention time on the rod was noted as described previously [17].


### 2.4.2. Hang Test

The effect of CNB-001 on neuromuscular strength was analyzed by grid hang test. Briefly, animals were placed on a horizontal grid and supported until they held the grid with all their four paws. The grid was then kept in an inverted position allowing the animals to hang upside down and the maximum hanging time was noted. Proper care was taken 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 in a wooden apparatus (W100 × D100 × H4 cm). The floor was covered with a rexin cloth and divided into25(5×5) equal squares. The animals were placed in the middle of apparatus and their behavior was observed for 5 min. Central and periphery movements were calculated based on number of central (nine) and peripheral (sixteen) squares crossed by the animals, respectively. One count was made only when the 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 5 min. The entire study was conducted in a blinded manner [19].


## 2.4.1. Rotorod Test

This test was performed to assess muscular coordination, strength, and balancing ability of animals using rotorod apparatus (Panlab). Initially all the animals were habituated and trained on accelerating rotating rod (5, 10, and 15 rpm) with a maximum cut-off time of 180 s for five consecutive days (twice/day) prior to treatment. After experimental period, the average retention time on the rod was noted as described previously [17].


## 2.4.2. Hang Test

The effect of CNB-001 on neuromuscular strength was analyzed by grid hang test. Briefly, animals were placed on a horizontal grid and supported until they held the grid with all their four paws. The grid was then kept in an inverted position allowing the animals to hang upside down and the maximum hanging time was noted. Proper care was taken 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 in a wooden apparatus (W100 × D100 × H4 cm). The floor was covered with a rexin cloth and divided into25(5×5) equal squares. The animals were placed in the middle of apparatus and their behavior was observed for 5 min. Central and periphery movements were calculated based on number of central (nine) and peripheral (sixteen) squares crossed by the animals, respectively. One count was made only when the 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 5 min. The entire study was conducted in a blinded manner [19].


## 2.5. Perfusion and Tissue Processing

At the end of the experiments, animals were sacrificed by terminal anesthesia and perfused via intracardial infusion with 0.9% saline followed by 4% paraformaldehyde (pH 7.4). Brain was removed from the skull and postfixed in 4% paraformaldehyde for 24 h at 4°C. The brain tissue was then embedded in paraffin and sliced into 5μm coronal sections containing entire SN and ST (anteroposterior levels: bregma −2.92 to −3.64 mm and +0.02 to +0.86 mm) with the reference of mouse brain atlas [20] for immunohistochemical study. For immunoblotting, SN and ST were dissected out and rapidly frozen on dry ice and 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 previously described [21]. In brief, the tissue extracts were incubated in metabolic water bath shaker at 37°C with 0.2 mL phenyl methosulfate. After one hour, 0.4 mL of 0.67% thiobarbituric acid and 0.4 mL of 5% tricarboxylic acid were added. The reaction mixture was centrifuged at 4000 rpm for 15 min at room temp. (RT) and the resultant supernatant was boiled for 10 min. The samples were cooled and reading was taken at 535 nm. The rate of lipid peroxidation is expressed as nmol of TBARS formed per hour/g tissue.


### 2.6.2. Estimation of Reduced Glutathione

The levels of reduced glutathione in brain tissue homogenate were measured as previously described [22]. Brain homogenate was centrifuged at 16,000 ×g for 15 min at 40°C. After centrifugation, 0.5 mL of the supernatant was added to 4 mL of ice-cold 0.1 mM 5,5-dithiobis[2-nitrobenzoic acid] solution in 1 M phosphate buffer (pH-8) and the absorbance was measured at 412 nm.


### 2.6.3. Estimation of Glutathione Peroxidase (GPx)

The level of GPx in brain tissue homogenate was measured as previously described [23]. Briefly, the assay mixture consisted of 100 μL of 1 M Tris-Hcl (pH-8.0) containing 5 mM of EDTA, 20 μL of 0.1 M GSH, 100 μL of glutathione reductase solution (10 U/mL), 100 μL of 2 mM NADPH, 650 μL of distilled water, 10 μL of 7 mMtert-butyl hydroperoxidase, and 10 μL of brain homogenate. NADPH oxidation was determined spectrometrically at 340 nm. One unit of enzyme activity was defined as the amount of GPx required to oxidize 1 μmol of NADPH per min.


### 2.6.4. Assay for Superoxide Dismutase (SOD) Activity

The activity of SOD was assessed following the method as previously described [24]. Xanthine and xanthine oxidase served as superoxide generator and nitro blue tetrazolium is used as superoxide indicator. Briefly, the reaction mixture consists of 960 μL of 50 mM sodium carbonate buffer (pH 10.2) containing 0.1 mM of EDTA, 0.1 mM of xanthine, 20 μL of xanthine oxidase, 0.025 mM of NBT, and 20 μL of brain supernatant. The absorbance is measured spectrometrically at 560 nm and the activity was expressed as units/min/mg protein.


### 2.6.5. Determination of Catalase (CAT) Activity

Catalase activity was measured by determining the rate of decomposition of hydrogen peroxide (H2O2) at 240 nm. In brief, assay mixture consisted of 50 μL of 1 M Tris-Hcl buffer (pH 8.0) containing 5 mM of EDTA, 900 μL of 10 mM H2O2, 30 μL of water, and 20 μL of the supernatant. Rate of decomposition of hydrogen was measured at 240 nm spectrometrically. Enzyme activity is expressed as nmol of H2O2 decomposed/min/mg protein [25].


## 2.6.1. Estimation of Thiobarbituric Acid Reactive Substances (TBARS)

TBARS content was analyzed as previously described [21]. In brief, the tissue extracts were incubated in metabolic water bath shaker at 37°C with 0.2 mL phenyl methosulfate. After one hour, 0.4 mL of 0.67% thiobarbituric acid and 0.4 mL of 5% tricarboxylic acid were added. The reaction mixture was centrifuged at 4000 rpm for 15 min at room temp. (RT) and the resultant supernatant was boiled for 10 min. The samples were cooled and reading was taken at 535 nm. The rate of lipid peroxidation is expressed as nmol of TBARS formed per hour/g tissue.


## 2.6.2. Estimation of Reduced Glutathione

The levels of reduced glutathione in brain tissue homogenate were measured as previously described [22]. Brain homogenate was centrifuged at 16,000 ×g for 15 min at 40°C. After centrifugation, 0.5 mL of the supernatant was added to 4 mL of ice-cold 0.1 mM 5,5-dithiobis[2-nitrobenzoic acid] solution in 1 M phosphate buffer (pH-8) and the absorbance was measured at 412 nm.


## 2.6.3. Estimation of Glutathione Peroxidase (GPx)

The level of GPx in brain tissue homogenate was measured as previously described [23]. Briefly, the assay mixture consisted of 100 μL of 1 M Tris-Hcl (pH-8.0) containing 5 mM of EDTA, 20 μL of 0.1 M GSH, 100 μL of glutathione reductase solution (10 U/mL), 100 μL of 2 mM NADPH, 650 μL of distilled water, 10 μL of 7 mMtert-butyl hydroperoxidase, and 10 μL of brain homogenate. NADPH oxidation was determined spectrometrically at 340 nm. One unit of enzyme activity was defined as the amount of GPx required to oxidize 1 μmol of NADPH per min.


## 2.6.4. Assay for Superoxide Dismutase (SOD) Activity

The activity of SOD was assessed following the method as previously described [24]. Xanthine and xanthine oxidase served as superoxide generator and nitro blue tetrazolium is used as superoxide indicator. Briefly, the reaction mixture consists of 960 μL of 50 mM sodium carbonate buffer (pH 10.2) containing 0.1 mM of EDTA, 0.1 mM of xanthine, 20 μL of xanthine oxidase, 0.025 mM of NBT, and 20 μL of brain supernatant. The absorbance is measured spectrometrically at 560 nm and the activity was expressed as units/min/mg protein.


## 2.6.5. Determination of Catalase (CAT) Activity

Catalase activity was measured by determining the rate of decomposition of hydrogen peroxide (H2O2) at 240 nm. In brief, assay mixture consisted of 50 μL of 1 M Tris-Hcl buffer (pH 8.0) containing 5 mM of EDTA, 900 μL of 10 mM H2O2, 30 μL of water, and 20 μL of the supernatant. Rate of decomposition of hydrogen was measured at 240 nm spectrometrically. Enzyme activity is expressed as nmol of H2O2 decomposed/min/mg protein [25].


## 2.7. Immunohistochemical Studies

Immunohistochemistry was performed to investigate the presence of DA-ergic neurons with TH immunoreactivity. Substantia nigra (SN) and striatum (ST) region were serially sectioned and the slides were deparaffinized using xylene followed by rehydration in graded series of ethanol. The slides were boiled for 10 min in 10 mM of citrate buffer (pH-6) for antigen retrieval. The sections were then incubated in dark with H2O2 (0.3%) for 10 min at RT. The slides were placed in blocking buffer (10% normal goat serum (NGS) with 0.2% Triton X-100 in 0.01 M PBS) at 37°C for 30 min. In each treatment, slides were washed three times with 0.01 M PBS for 5 min. Sections were incubated primarily with anti-mouse TH (1 : 1000) in 2% normal goat serum, 0.2% Triton X-100, and 0.02% sodium azide in TBS for 24 h. After washing with 1% NGS in TBS, the sections were incubated with anti-mouse IgG-HRP conjugated secondary antibody (1 : 1000 in 1.5% NGS) for one hour followed by washing with PBS. The sections were then incubated with diaminobenzidine (DAB) to view and analyze TH immunoreactivity.The intensity of TH immunoreactivity in ST was measured using Micro Computer Imaging Device software and results are expressed as percentage of control. Mouse brain atlas was initially used to delineate SN at low magnification and the numbers of TH immunoreactive neurons in SN were counted at higher magnification (20x) by a person, blind to the treatment. Cell counts were determined in every sixth section (total of 8 to 10 sections) through SN corresponding to the bregma −2.92 to −3.64 mm from each animal. The analyses of TH positive neurons were restricted to SN and thus excluded the ventral tegmental area.


## 2.8. Levels of TH, DAT, and VMAT2 by Immunoblotting

Western blotting was performed to analyze the expression patterns of TH, DAT, and VMAT2 in SN and ST of experimental animals. In brief, the tissues were homogenized in ice-cold RIPA buffer (1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mM/L EDTA, 20 mM/L Tris (pH 7.4), 10 mM/L NAF, 150 mM/L NaCl, and 0.1 mM/L phenylmethylsulfonyl fluoride) and centrifuged (12,000 ×g for 15 min at 4°C) to remove cellular debris. Protein concentration in the samples was analyzed using Lowry method [26]. Proteins (50 μg) were separated by 10% SDS-polyacrylamide gel and electrotransferred onto a PVDF membrane by semidry transfer (BIO-RAD). After blocking the membrane for 1 hr in 5% nonfat dry milk in TBS at 25°C, the membranes were incubated with β-actin (rabbit polyclonal, 1 : 500 dilution in 5% BSA Tris-buffered saline and 0.05% Tween 20 (TBST), anti-mouse TH (1 : 1000), anti-mouse DAT (1 : 500), and anti-mouse VMAT2 (1 : 1000)) with gentle shaking at 4°C overnight. Corresponding secondary antibodies (anti-mouse or anti-rabbit IgG conjugated to HRP) were then added and incubated for 2 h at room temperature. The membranes were washed thrice with TBST (10 min/wash) and immunoreactive bands were visualized by chemiluminescence kit. Densitometry analysis was done using “Image J” analysis software. The blot intensities were normalized with that of β-actin as loading control.


## 2.9. Electron Microscopy

Brain regions (SN and ST) were fixed in 3% gluteraldehyde at 4°C for 24 h. After fixation, brain sections were cut into approximately 1 mm cubes and postfixed in 1% osmium tetraoxide for 2 h at 4°C. The cubes were then dehydrated in series of ethanol and treated twice with propylene oxide for 10 min at RT. The tissues were infiltrated 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. Ultra microtome was used to cut tissue into 0.6μm thick sections which were then stained with 0.5% toluidine blue to confirm the presence of neurons. Then the 60 nm ultrathin sections were cut and mounted on Nickel grids (300 mesh). The sections were double stained with uranyl acetate and lead citrate and then examined by Transmission Electron Microscope (Philips CM10) and photographed.


### 2.9.1. Morphological Analysis of Mitochondria within SN and ST

Measurements were generated from three randomly acquired TEM images from SN and ST with a magnification of 16,000x and coded for blinded analysis. The perimeter length of each mitochondrion was analyzed by a single trained technician using MeasureIT software (Olympus Soft Imaging Solutions). Mitochondria were identified by presence of identifiable cristae and distinct double membrane; however, mitochondria with unclear morphology were avoided.


## 2.9.1. Morphological Analysis of Mitochondria within SN and ST

Measurements were generated from three randomly acquired TEM images from SN and ST with a magnification of 16,000x and coded for blinded analysis. The perimeter length of each mitochondrion was analyzed by a single trained technician using MeasureIT software (Olympus Soft Imaging Solutions). Mitochondria were identified by presence of identifiable cristae and distinct double membrane; however, mitochondria with unclear morphology were avoided.


## 3. Results

### 3.1. CNB-001 Conspicuously Mitigates MPTP Induced Behavioral Impairments

Motor coordination ability of experimental animals was evaluated by performing rotorod test, as shown in Figure1. MPTP treated group showed neuromuscular incoordination and significant reduction in retention time as compared to control (P<0.05). Moreover none of the MPTP treated animals could balance themselves on the rotating rod for full cut-off time (180 s). Pretreatment with CNB-001 distinctly enhanced balancing ability and retention time and inhibited disorientation as compared to MPTP treated mice (P<0.05).Figure 1
Rotorod performance of control and experimental mice. Administration of MPTP significantly reduced the ability of animals to stay on the rotating rod (5, 10, and 15 rpm) when compared to control. Retention time was significantly improved as compared to MPTP group when animals were pretreated with CNB-001. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.Hang test was performed to analyze the neuromuscular strength of control and experimental animals. Compared to control group, the average hanging time of MPTP intoxicated mice was significantly lower (P<0.05) (Figure 2). CNB-001 treatment distinctly enhanced neuromuscular strength compared to MPTP group as evinced by improved hanging time.Figure 2
Effect of CNB-001 on hanging performance. MPTP administration resulted in a decrease in neuromuscular strength as evidenced by reduction in hanging time as compared to control. Pretreatment with CNB-001 to MPTP treated mice distinctly enhanced hanging time as compared to MPTP group. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.Locomotor and exploratory behavior, an intricate feature of physiological and behavioral functions in experimental animals, was assessed by open field test. MPTP intoxication caused a significant decline in peripheral and central movements and rearing and grooming activities when compared to control group (P<0.05) (Figures 3(a)–3(d)). However, these impairments were reduced in animals pretreated with CNB-001 as compared to MPTP group (P<0.05).Open field test of control and experimental mice. MPTP injection resulted in significant decrease in central and peripheral movements and grooming and rearing activities when compared to control. However, these motor impairments were reduced when pretreated with CNB-001. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)
(c)
(d)


### 3.2. Effect of CNB-001 on Lipid Peroxidation and Antioxidants

MPTP administration caused a significant increase in the levels of TBARS and activities of SOD and CAT and decreased the levels of GSH and activity of GPx when compared to control group (P<0.05). Alternatively, CNB-001 inhibited MPTP induced oxidative stress significantly by attenuating enhanced TBARS, SOD, and CAT activities and increased the levels of GSH and GPX activities as compared to MPTP group (P<0.05) (Table 1).Table 1
In vivo antioxidant status of control and experimental mice.
Groups
Control
MPTP
CNB-001 + MPTP
CNB-001
TBARS (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 otherP<0.05.
Anmol NADPH oxidized/minute/mg protein.
BAmount of enzyme required to inhibit 50% of NBT reduction.
Cnmol H2O2 consumed/minute/mg protein.


### 3.3. CNB-001 Abrogates MPTP Induced Loss of TH-Positive Neurons

Acute administration of MPTP resulted in a significant decrease in TH density and TH-positive neurons in ST and SN, respectively, when compared to control group (P<0.05). However, treatment with CNB-001 prior to MPTP administration distinctly cosseted neurons by increasing TH expression in ST (Figure 4) and SN (Figure 5) compared to MPTP group (P<0.05). Western blotting was performed subsequently to confirm the immunohistochemical finding.Photomicrographs of ST sections depicting TH-immunoreactive (TH-ir) fibers. (a) (4X) Administration of MPTP (B) significantly depleted TH-positivity in striatum and this depletion was inhibited by pretreatment with CNB-001 (C). (b) TH immunoreactivity was analyzed by measuring the optical density in ST. Mean value of TH-ir was determined for each group and expressed as percentage control. Values are expressed as mean ± SD for three animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)Photomicrograph of SN sections illustrating TH-immunoreactive (TH-ir) neurons. (a) (4X) MPTP administration (F) significantly degenerated TH-ir neurons. Pretreatment with CNB-001 (G) significantly increased TH positivity as compared to MPTP group. (b) Quantification of TH positive neurons was performed by counting number of TH-ir neurons and values are expressed as mean ± SD of three animals per group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)


### 3.4. Effect of CNB-001 on TH, DAT, and VMAT2 Expressions

To determine the protective effect of CNB-001 against MPTP induced neurodegeneration, the expression pattern of phenotypic markers (TH, DAT, and VMAT2) in SN and ST was analyzed by western blotting. As depicted in Figure6, MPTP significantly alleviated the expression of TH, DAT, and VMAT2 in both SN and ST compared to control group (P<0.05). Meanwhile, treatment with CNB-001 reinstated these protein expressions distinctly as compared to MPTP group (P<0.05). There were, however, no significant changes between control and CNB-001 treated groups.Immunoblotting of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) in substantia nigra (SN) and striatum (ST) of experimental animals. Injection of MPTP significantly reduced TH, DAT, and VMAT2 expressions in SN and ST. Pretreatment with CNB-001 significantly increased TH, DAT, and VMAT2 expressions ((a) and (c)). Protein expressions were quantified usingβ-actin as an internal standard and values are expressed as arbitrary units and given as mean ± SD ((b) and (d)). P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)
(c)
(d)


### 3.5. CNB-001 Cosseted Mitochondria from MPTP Induced Ultrastructural Changes

Electron microscopic studies were performed to analyze whether the antioxidant potential of CNB-001 was strong enough to protect mitochondria morphology against MPTP toxicity. Striking structural changes were observed in mitochondria of ST (Figure7) and SN (Figure 8) in MPTP treated mice. MPTP intoxication resulted in distortion of cristae, permeabilization, and enlargement of mitochondria in SN and ST (788 and 1032 nm resp.) (Figure 9). In contrast, pretreatment with CNB-001 protected mitochondria from MPTP toxicity by maintaining normal mitochondrial morphology and size (660 and 825 nm) with distinct nucleus and cristae compared to MPTP group (P<0.05).Photomicrographs of striatum viewed under transmission electron microscope. All panels demonstrate representative striata at 2000x. Control (a) and CNB-001 (d) alone treated groups showed normal ultrastructural morphology with enhanced mitochondrial population with membrane stability and cytoplasmic contents. Strikingly, MPTP treated group (b) showed mitochondria greatly enlarged and swollen rather than normal rod shaped. Moreover, there were also distinct loss of mitochondrial cristae and permeabilization of mitochondrial membrane. These toxicity changes were distinctly diminished upon CNB-001 treatment (c) showing normal nuclear morphology with increased mitochondrial population with structural stability.
(a)
(b)
(c)
(d)Transmission electron micrographs of SN showing ultrastructural changes in control and experimental mice. All panels demonstrate SN neurons at 2000x. In control (a) and CNB-001 groups (d), increased mitochondrial population with prominent cristae were seen. In MPTP group (b), mitochondria showed abnormal structure with distorted cristae and the number of mitochondria was greatly reduced. In contrast CNB-001 treatment (c) dampened these apoptotic changes and improved mitochondrial morphology and population as compared to MPTP group.
(a)
(b)
(c)
(d)Figure 9
Analysis of mitochondrial perimeter length in SN and ST of experimental animals. Mitochondrial perimeter length/mitochondrion was significantly more increased in MPTP group than in control. Pretreatment with CNB-001 significantly maintained mitochondrial size as compared to MPTP group. No significant changes were observed in control and CNB-001 treated groups.P*<0.05 compared to control, P#<0.05 compared to MPTP.


## 3.1. CNB-001 Conspicuously Mitigates MPTP Induced Behavioral Impairments

Motor coordination ability of experimental animals was evaluated by performing rotorod test, as shown in Figure1. MPTP treated group showed neuromuscular incoordination and significant reduction in retention time as compared to control (P<0.05). Moreover none of the MPTP treated animals could balance themselves on the rotating rod for full cut-off time (180 s). Pretreatment with CNB-001 distinctly enhanced balancing ability and retention time and inhibited disorientation as compared to MPTP treated mice (P<0.05).Figure 1
Rotorod performance of control and experimental mice. Administration of MPTP significantly reduced the ability of animals to stay on the rotating rod (5, 10, and 15 rpm) when compared to control. Retention time was significantly improved as compared to MPTP group when animals were pretreated with CNB-001. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.Hang test was performed to analyze the neuromuscular strength of control and experimental animals. Compared to control group, the average hanging time of MPTP intoxicated mice was significantly lower (P<0.05) (Figure 2). CNB-001 treatment distinctly enhanced neuromuscular strength compared to MPTP group as evinced by improved hanging time.Figure 2
Effect of CNB-001 on hanging performance. MPTP administration resulted in a decrease in neuromuscular strength as evidenced by reduction in hanging time as compared to control. Pretreatment with CNB-001 to MPTP treated mice distinctly enhanced hanging time as compared to MPTP group. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.Locomotor and exploratory behavior, an intricate feature of physiological and behavioral functions in experimental animals, was assessed by open field test. MPTP intoxication caused a significant decline in peripheral and central movements and rearing and grooming activities when compared to control group (P<0.05) (Figures 3(a)–3(d)). However, these impairments were reduced in animals pretreated with CNB-001 as compared to MPTP group (P<0.05).Open field test of control and experimental mice. MPTP injection resulted in significant decrease in central and peripheral movements and grooming and rearing activities when compared to control. However, these motor impairments were reduced when pretreated with CNB-001. Values are expressed as mean ± SD for six animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)
(c)
(d)


## 3.2. Effect of CNB-001 on Lipid Peroxidation and Antioxidants

MPTP administration caused a significant increase in the levels of TBARS and activities of SOD and CAT and decreased the levels of GSH and activity of GPx when compared to control group (P<0.05). Alternatively, CNB-001 inhibited MPTP induced oxidative stress significantly by attenuating enhanced TBARS, SOD, and CAT activities and increased the levels of GSH and GPX activities as compared to MPTP group (P<0.05) (Table 1).Table 1
In vivo antioxidant status of control and experimental mice.
Groups
Control
MPTP
CNB-001 + MPTP
CNB-001
TBARS (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 otherP<0.05.
Anmol NADPH oxidized/minute/mg protein.
BAmount of enzyme required to inhibit 50% of NBT reduction.
Cnmol H2O2 consumed/minute/mg protein.


## 3.3. CNB-001 Abrogates MPTP Induced Loss of TH-Positive Neurons

Acute administration of MPTP resulted in a significant decrease in TH density and TH-positive neurons in ST and SN, respectively, when compared to control group (P<0.05). However, treatment with CNB-001 prior to MPTP administration distinctly cosseted neurons by increasing TH expression in ST (Figure 4) and SN (Figure 5) compared to MPTP group (P<0.05). Western blotting was performed subsequently to confirm the immunohistochemical finding.Photomicrographs of ST sections depicting TH-immunoreactive (TH-ir) fibers. (a) (4X) Administration of MPTP (B) significantly depleted TH-positivity in striatum and this depletion was inhibited by pretreatment with CNB-001 (C). (b) TH immunoreactivity was analyzed by measuring the optical density in ST. Mean value of TH-ir was determined for each group and expressed as percentage control. Values are expressed as mean ± SD for three animals in each group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)Photomicrograph of SN sections illustrating TH-immunoreactive (TH-ir) neurons. (a) (4X) MPTP administration (F) significantly degenerated TH-ir neurons. Pretreatment with CNB-001 (G) significantly increased TH positivity as compared to MPTP group. (b) Quantification of TH positive neurons was performed by counting number of TH-ir neurons and values are expressed as mean ± SD of three animals per group.P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)


## 3.4. Effect of CNB-001 on TH, DAT, and VMAT2 Expressions

To determine the protective effect of CNB-001 against MPTP induced neurodegeneration, the expression pattern of phenotypic markers (TH, DAT, and VMAT2) in SN and ST was analyzed by western blotting. As depicted in Figure6, MPTP significantly alleviated the expression of TH, DAT, and VMAT2 in both SN and ST compared to control group (P<0.05). Meanwhile, treatment with CNB-001 reinstated these protein expressions distinctly as compared to MPTP group (P<0.05). There were, however, no significant changes between control and CNB-001 treated groups.Immunoblotting of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) in substantia nigra (SN) and striatum (ST) of experimental animals. Injection of MPTP significantly reduced TH, DAT, and VMAT2 expressions in SN and ST. Pretreatment with CNB-001 significantly increased TH, DAT, and VMAT2 expressions ((a) and (c)). Protein expressions were quantified usingβ-actin as an internal standard and values are expressed as arbitrary units and given as mean ± SD ((b) and (d)). P*<0.05 compared to control, P#<0.05 compared to MPTP.
(a)
(b)
(c)
(d)


## 3.5. CNB-001 Cosseted Mitochondria from MPTP Induced Ultrastructural Changes

Electron microscopic studies were performed to analyze whether the antioxidant potential of CNB-001 was strong enough to protect mitochondria morphology against MPTP toxicity. Striking structural changes were observed in mitochondria of ST (Figure7) and SN (Figure 8) in MPTP treated mice. MPTP intoxication resulted in distortion of cristae, permeabilization, and enlargement of mitochondria in SN and ST (788 and 1032 nm resp.) (Figure 9). In contrast, pretreatment with CNB-001 protected mitochondria from MPTP toxicity by maintaining normal mitochondrial morphology and size (660 and 825 nm) with distinct nucleus and cristae compared to MPTP group (P<0.05).Photomicrographs of striatum viewed under transmission electron microscope. All panels demonstrate representative striata at 2000x. Control (a) and CNB-001 (d) alone treated groups showed normal ultrastructural morphology with enhanced mitochondrial population with membrane stability and cytoplasmic contents. Strikingly, MPTP treated group (b) showed mitochondria greatly enlarged and swollen rather than normal rod shaped. Moreover, there were also distinct loss of mitochondrial cristae and permeabilization of mitochondrial membrane. These toxicity changes were distinctly diminished upon CNB-001 treatment (c) showing normal nuclear morphology with increased mitochondrial population with structural stability.
(a)
(b)
(c)
(d)Transmission electron micrographs of SN showing ultrastructural changes in control and experimental mice. All panels demonstrate SN neurons at 2000x. In control (a) and CNB-001 groups (d), increased mitochondrial population with prominent cristae were seen. In MPTP group (b), mitochondria showed abnormal structure with distorted cristae and the number of mitochondria was greatly reduced. In contrast CNB-001 treatment (c) dampened these apoptotic changes and improved mitochondrial morphology and population as compared to MPTP group.
(a)
(b)
(c)
(d)Figure 9
Analysis of mitochondrial perimeter length in SN and ST of experimental animals. Mitochondrial perimeter length/mitochondrion was significantly more increased in MPTP group than in control. Pretreatment with CNB-001 significantly maintained mitochondrial size as compared to MPTP group. No significant changes were observed in control and CNB-001 treated groups.P*<0.05 compared to control, P#<0.05 compared to MPTP.


## 4. Discussion

In the present study, we substantiate that neuroprotective action of CNB-001 against MPTP induced neurodegeneration is by (i) attenuation of characteristic Parkinsonian impairments, (ii) inhibition of lipid peroxidation and enhancement of antioxidant response, (iii) prevention of DA-ergic neuronal loss by reinstating expression of TH, DAT, and VMAT2 in SN and ST, and (iv) protection of mitochondrial morphology in SN and ST against MPTP toxicity. Though the etiology of PD is unknown, various biochemical alterations initiated by oxidative stress and mitochondrial dysfunction are major factors which self-propel neurodegeneration [6]. Thus antioxidant remains to be the Holy Grail for PD therapeutics. Postmortem and biochemical studies provide direct evidence for involvement of hyperoxidation phenomenon in PD. Catecholaminergic neurons are particularly vulnerable to oxidative and nitrosative stress due to the presence of 6-hydroxydopamine resulting in enhanced formation of intramitochondrial and cytosolic peroxynitrite. These toxic radicals initiate mitochondrial deficits by S-nitrosylation and Fe-nitrosation of complex I subunit resulting in ATP depletion and neuronal death [27]. A recent study conducted in our lab showed that CNB-001 possessed stronger antioxidant activity than curcumin by scavenging various free radicals such as DPPH, ABTS, and superoxide anions [28]. Hence this study was aimed at finding profound effect of CNB-001 against PD. This study utilizes an MPTP (mitochondrial complex I inhibitor) model of PD which selectively interferes with nigrostriatal pathway that connects SN with ST which results in DA depletion and subsequently leads to behavioral abnormalities as seen in PD [29, 30]. Ultimately, all neuropathological changes and neuroprotective action of drugs are reflected in animal behavior. Rotorod tests are widely used to analyze motor learning and motor-coordination ability by allowing the animals to walk on a rotating rod. Hang test was performed to analyze the neuromuscular strength [18] and open field test indicates acclimatization activity, mental stress, and motor activity of mice [31]. In our experiments, we found that MPTP administration induced behavioral impairments as evidenced by rotorod, open field, and hang tests which are ultimately linked to dopaminergic degeneration and deterioration of motor performance. Intraperitoneal administration of CNB-001 significantly improved behavioral deficits supporting the neuroprotective effect of CNB-001 against MPTP toxicity. Antioxidant activity of CNB-001 might be the possible mechanism involved in neuroprotection due to the presence of electron donating styryl groups at 3,5-position of pyrazole and methoxyphenol group accountable for iron chelation. To strengthen this property, our results revealed that administration of MPTP increased the levels of TBARS, due to lipid peroxidation, and reduced GSH and GPx levels along with increased activities of SOD and CAT. Pretreatment with CNB-001 modulated free radical toxicity by alleviating TBARS levels along with increased activities of antioxidant enzymes. Increase in SOD and CAT in MPTP animals is due to adaptive response since MPTP mediated mitochondrial impairment results in leakage of superoxide anions [32]. Moreover depletion of GSH and GPx might be an early mechanism involved in initiation of oxidative stress and possibly provides sensitivity to DA-ergic neurons against toxin [33]. Previous reports from our lab showed that CNB-001 protected SK-N-SH cells against rotenone toxicity by inhibiting accumulation of intracellular ROS formation [13]. Thus, neuroprotective activity of CNB-001 may be partially due to the regulation of antioxidant enzymes which is depleted upon MPTP treatment. Further, the neuroprotective mechanism of CNB-001 may correlate well with previous study where CNB-001 protected HT-22 cells against glutamate induced oxidative stress [11].Antioxidant mediated neuroprotection by CNB-001 is further strongly supported by immunohistochemical and western blotting studies. Administration of MPTP significantly reduced the expression of TH, DAT, and VMAT2 in SN and ST of mice. These proteins play a vital role in nigrostriatal pathway and are involved in DA biosynthesis. TH is a rate-limiting enzyme responsible for the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Further, aromatic amino acid decarboxylase removes carboxylic acid moiety from L-DOPA to form DA. Synthesized DA is sequestered by VMAT2 and released in synaptic space and further transmitted. VMAT2 also plays an important role in sequestering of neurotoxins and prevents toxicity [34]. Nontransmitted DA is further recycled into presynaptic neurons via DA transporter (DAT). Previous reports showed that diminished expressions of TH, DAT [4], and VMAT2 [5] in SN and ST are thought to underlie PD pathogenesis. Our results were concordant with various reports which showed that MPTP intoxication selectively targets and destroys DA-ergic neurons as evidenced by reduced TH immunoreactivity and resultant DA depletion [35, 36]. Apart from the role of DAT in DA biosynthesis, it also acts as a gateway for several neurotoxins including MPTP for DA-ergic toxicity [2]. Hence, immunoblotting results in our study showed decrease in DAT expression in SN and ST of MPTP treated mice might be due to loss of nigrostriatal neurons and fibers, respectively. Similar to TH and DAT expressions, MPTP injection significantly depleted the expression of VMAT2 in brain. VMAT2 plays an important role in inhibiting dopamine mediated toxicity by sequestering cytosolic DA and it has also been reported that VMAT2 deletion causes mice lethality [37]. Accumulation of cytosolic DA supports formation of oxyradical stress and excess cytosolic DA causes proteotoxicity to DA-ergic neurons and autophagy block [38, 39]. Therefore enhanced expression of VMAT2 could potentially contribute to neuroprotection. Consistent to these reports TH, DAT, and VMAT2 protein levels were reduced distinctly in patients affected by PD [40]. Pretreatment with CNB-001 enhanced TH-ir and expressions of TH, DAT, and VMAT2 which might contribute to enhanced DA synthesis and mitigation of behavioral impairments caused by MPTP.Several clinical reports have demonstrated modest reduction in mitochondrial complex I activity in PD patients [41, 42]. Further, MPP+ selectively blocks complex I of mitochondria and inhibits flow of electrons across electron transport chain and resultant ATP depletion with increased ROS production. Electron microscopic results from our studies showed that MPTP intoxication enhanced mitochondrial pathology in SN and ST which reflect the sequelae of injury to mitochondria. Furthermore mitochondria from PD patients and MPTP treated animals have also been described to be swollen and enlarged, possessing distorted mitochondrial cristae with discontinuous outer membrane rather than ordered cristae with normal rod shaped morphology, which are similar to our results. To confirm the toxicity and to address difference in mitochondria structure, we measured mitochondrial perimeter length of experimental animals because increase in mitochondrial perimeter length as a result of mitochondrial swelling is a well-accepted hallmark in organelle dysfunction [43]. MPTP treatment increased the mitochondrial size when compared to the control group. Pretreatment with CNB-001 protected the mitochondria and the ultrastructural changes were less prominent as compared to MPTP group. Our findings are also similar to those reported during apoptosis mediated cell death in SN of PD [44]. Moreover CNB-001 protected SK-N-SH neuroblastoma against rotenone toxicity by maintaining mitochondrial membrane potential through inhibition of proapoptotic factors [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) mitochondria which are similar to previous reports [45]. We have also observed that CNB-001 prevented DA-ergic neuronal loss by attenuating inflammatory and apoptotic response in MPTP model of PD (data not shown). Further results of the present study are substantiated by previous reports, where CNB-001 has been shown to possess neuroprotective effects against intracellular amyloid toxicity, trophic factor withdrawal, and excitotoxicity [11].


## 5. Conclusion

In summary, CNB-001 protects DA-ergic neurons against MPTP toxicity by regulating various molecular and cellular events. The therapeutic potential of CNB-001 is supported by its ability to reduce behavioral impairments, oxidative stress, and mitochondrial deficits and by enhancing expressions of TH, DAT, and VMAT2 in animal model of PD. Hence, based on these results we speculate that further investigation on CNB-001 would be worthwhile to bring out a potent therapeutic contender in the treatment of PD.



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*Source: 236182-2014-06-17.xml*