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Neuroscience xxx (2014) xxx–xxx

NEUROPROTECTIVE EFFECT OF HEMEOXYGENASE-1/GLYCOGEN SYNTHASE KINASE-3b MODULATORS IN 3-NITROPROPIONIC ACID-INDUCED NEUROTOXICITY IN RATS

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Q1 A. KHAN, a S. JAMWAL, a,b K. R. V. BIJJEM, a a Department of Pharmacology, I.S.F. College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga 142001, Punjab, India

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of HD-like symptoms. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.

A. PRAKASH a AND P. KUMAR a*

Q2

b

Research Scholar, Punjab Technical University, Jalandhar, India

Key words: hemeoxygenase-1, glycogen synthase kinase-3b, 3-nitropropionic acid, Huntington’s disease, oxidative stress. Q5

Abstract—The present study has been designed to explore the possible interaction between hemeoxygenase-1 (HO-1) and glycogen synthase kinase-3b (GSK-3b) pathway in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats. 3-NP produces neurotoxicity by inhibition of the mitochondrial complex II (enzyme succinate dehydrogenase) and by sensitizing the N-methyl-D-aspartate receptor. Recent studies have reported the therapeutic potential of HO-1/GSK-3b modulators in different neurodegenerative disorders. However, their exact role is yet to be explored. The present study is an attempt to investigate the effect of pharmacological modulation of HO-1/GSK-3b pathway against 3-NP-induced behavioral, biochemical and molecular alterations in rat. Behavioral observation, oxidative stress, pro-inflammatory [tumor necrosis factor-alpha (TNF-a) and interleukin-1 beta (IL-1b)], HO-1 and GSK-3b activity were evaluated post 3-NP treatment. Findings of the present study demonstrate a significant alteration in the locomotor activity, motor coordination, oxidative burden (increased lipid peroxidation, nitrite concentration and decreased endogenous antioxidants), pro-inflammatory mediators [TNF-a, IL-1b], HO-1 and GSK-3b activity in 3-NP-treated animals. Further, administration of hemin (10- and 30-mg/kg; i.p.) and lithium chloride (LiCl) (25- and 50-mg/kg; i.p.) prevented the alteration in body weight, motor impairments, oxidative stress and cellular markers. In addition, combined administration of hemin (10-mg/kg) and LiCl (25-mg/kg) showed synergistic effect on 3-NP-treated rats. Pretreatment with Tin (IV) protoporphyrin (40 lM/kg), HO-1 inhibitor reversed the beneficial effect of LiCl and hemin. Outcomes of the present study suggest that HO-1 and GSK-3b enzymes are involved in the pathophysiology of HD. The modulators of both the pathways might be used as adjuvants or prophylactic therapy for the treatment *Corresponding author. E-mail address: [email protected] (P. Kumar). Q3 Abbreviations: 3-NP, 3-nitropropionic acid; ANOVA, analysis of Q4 variance; AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; ARE, Antioxidant-responsive element; BDNF, Brain-derived neurotrophic factor; ELISA, Enzyme- linked immunosorbent assay; GABA, gamma amino butyric acid; GSH, glutathione; GSK-3b, glycogen synthase kinase-3b; H2O2, hydrogen peroxide; HD, Huntington’s disease; HO-1, hemeoxygenase-1; IL-1b, interleukin-1 beta; Keap1, Kelch-like ECH-associated protein 1; LPO, lipid peroxidation; MDA, malondialdehyde; NO, nitric oxide; Nrf2, nuclear factor erythroid-2-related factor 2; SnPP, Tin (IV) protoporphyrin; TNFa, tumor necrosis factor-alpha. http://dx.doi.org/10.1016/j.neuroscience.2014.12.018 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Huntington’s disease (HD) is an autosomal-dominant, inherited, progressive neurodegenerative and neuropsychiatric disease caused by a mutation in the huntingtin gene, resulting in an abnormally long polyglutamine cytosine adenine guanine (CAG > 40) repeat which gives rise to progressive motor, cognitive, and behavioral symptoms (Kumar et al., 2010a,b, 2012). HD has a worldwide prevalence of 5–8 per 100,000 peoples with no gender preponderance. Selective degeneration of GABAergic medium spiny neurons in the striatum is the prominent feature of HD pathology. The only available tetrabenazine therapy in clinical use is only palliative; leading to temporarily limited improvement of clinical symptoms. Therefore, new approaches are needed to find disease-modifying agents that may delay or stop the neuronal death. Recent reports clearly demonstrated the involvement of excitotoxicity, dysregulated energy metabolism and oxidative stress in striatal neuronal death observed in HD (Colle et al., 2012). An elevated level of oxidative damage products such as malondialdehyde (MDA), 8-hydroxy-deoxyguanosine, 3-nitrotyrosine and hemeoxygenase has been observed in the degenerated areas of HD brains (Johri and Beal, 2012).In physiological conditions, neuronal cells have a well-developed antioxidant system that includes a group of anti-xenobiotic genes termed as phase II detoxification genes to maintain redox homeostasis. Hemeoxygenase-1 (HO-1) is one among these genes and is the rate-limiting enzyme that degrades the pro-oxidant heme group and produces equimolecular quantities of carbon monoxide, iron, and biliverdin (Dal-Cim et al., 2012). HO-1 is induced in response to a variety of stress-inducing pathological conditions and has shown to be an important cytoprotective and antioxidant enzyme (Son et al., 2013). The induction of HO-1 is primarily regulated at the transcriptional level, secondary to nuclear translocation of nuclear factor erythroid 2-related factor (Nrf2) from the cytoplasm (Ryu et al.,

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2014). Pharmacological activation of HO-1 is shown to provide neuroprotection against oxidative stress (Li et al., 2013). Aberrant activation of another enzyme glycogen synthase kinase-3b (GSK-3b) has been reported in neurodegenerative disorders (Woodgett, 2005). GSK-3b is reported to exert a negative regulation on Nrf2 by controlling its sub cellular distribution (Kanninen et al., 2011; Q6 Chowdhry et al., 2012). The inhibition of GSK-3b results in nuclear accumulation and the elevation of transcriptional activity of Nrf2 indicating that GSK-3b is a fundamental element of Nrf2–ARE down regulation after oxidative injury (Kanninen et al., 2012). It can be concluded that Nrf2 is common link between HO-1 and GSK-3b, and inhibition of GSK-3b indirectly increases the expression of HO-1 or induction of HO-1 indirectly suppresses GSK-3b. But none of the previous studies or data reports the link between these two pathways against an experimental model of HD. 3-Nitropropionic acid (3-NP) is known to produce selective degeneration of GABAergic medium spiny neurons in the striatum and produces brain lesions in laboratory animals as observed in HD patients (Brouillet et al., 2005; Kumar et al., 2010a, 2011a, 2012). Hemin, a hemeoxygenase substrate analog that acts as a selective inducer of HO-1 and lithium chloride (LiCl), a selective GSK-3b inhibitor have been used in present study. Thus, modulation of the HO-1/GSK-3b system using suitable pharmacological agents may be a plausible therapeutic approach for HD. Therefore, the present study was designed to investigate whether pharmacological modulation of GSK-3b and HO-1 alone and/or in combination using suitable pharmacological agents would produce neuroprotection against 3-NP-induced neurotoxicity in rats or not.

EXPERIMENTAL PROCEDURES

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Experimental animals

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The experiments were carried out in male Wistar rats (200–250 g) obtained from central animal house of I.S.F. College of Pharmacy, Moga, Punjab (India). They were kept in groups of three in polyacrylic cages and maintained under standard husbandry conditions (room temperature 22 ± 1 °C and relative humidity of 60%) with a 12-h light/dark reverse cycle (lights turned on at 7 AM). The food in the form of dry pellets and water were made available ad libitum. All the behavioral assessments were carried between 9:00 and 17:00 h. The experimental protocol no 79, dated 13 Oct. 2012, was approved by the Institutional Animal Ethics Committee (IAEC) and was carried out in accordance with the guidelines of the Indian National Science Academy (INSA) for the use and care of experimental animals. All experiments for a given treatment were performed using age-matched animals in an attempt to avoid variability between experimental groups.

87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

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Drugs and chemicals

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3-NP (Sigma Aldrich, St. Louis, MO, USA); Tin (IV) protoporphyrin (SnPP) (Frontier scientific, Inc., Newark,

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DE, USA); LiCl and Hemin (Hi-Media, New Delhi, India); GSK-3b (product code: CSB-EL009963RA) and HO-1 (product code: CSB-E08267r) enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, Hubei, PR China); interleukin-1 beta (IL-1b) (product code: ELRIL1b) and tumor necrosis factor-alpha (TNF-a) (product code: ELR-TNFa) ELISA Kits (RayBiotech, Inc. Norcross, GA, USA). Unless stated, all other chemicals and biochemical reagents of highest analytical grade were used for the study. The experimental protocol was divided into 10 groups and each treatment group consisted of six animals (Total no. of animals = 60).

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Treatment schedule

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3-NP was dissolved in normal saline (adjusted pH to 7.4) and administered intraperitoneally (i.p.) at a dose of 10-mg/kg for 14 days; Hemin and SnPP solution were prepared by dissolving in 0.1 N NaOH and diluted with phosphate-buffered saline (pH adjusted to 7.4). LiCl was prepared by dissolving in PBS (adjusted pH to 7.4). Hemin, LiCl and SnPP were administered half an hour prior to 3-NP treatment. All drugs or vehicle were administered once daily for 14 days by i.p. route in constant volume of 0.5 ml per 100 gm of body weight. On day 1st, 5th, 10th, and 15th behavioral parameters like grip strength, motor coordination and locomotor activity were assessed. Terminally on day 15th, animals were sacrificed and the striatum was separated to estimate biochemical parameters (LPO, nitrite, reduced glutathione (GSH), and catalase). The levels of inflammatory cytokines (IL-1b and TNF-a) and HO-1 and GSK-3b were estimated using ELISA kits. The experimental procedure is summarized in Table 1.

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Measurement of body weight

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Animal body weight was recorded on the first and last day of the experiment. Percent change in body weight was calculated-

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121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138

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Body weightð1st day  15th dayÞ=1st day body weight  100

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Behavioral assessments

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Assessment of gross behavioral activity (locomotor activity). The locomotor activity was monitored using an actophotometer. The motor activity was detected by infrared beams above the floor of the testing area. Animals were placed individually in the activity chamber for a 3-min acclimation period before starting actual activity tasks. Each animal was observed over a period of 5 min and activity was expressed as counts per 5 min (Kumar et al., 2011b, 2012).

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Rotarod activity. The motor coordination and grip performance of the animals were evaluated using the rotarod apparatus. Rats were exposed to a prior training session to acclimatize them to rotarod performance. Rats were placed on a rotating rod with a diameter of

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Table 1. The experimental protocol used was as follows Experimental groups

Treatment

GROUP GROUP GROUP GROUP GROUP GROUP GROUP GROUP GROUP GROUP

Normal control 3-NP-treated group-3 NP 10-mg/kg/i.p./day for 14 days 3-NP (10-mg/kg/i.p./day) + Hemin (10-mg/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + Hemin (30-mg/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + LiCl (25-mg/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + LiCl (50-mg/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + Hemin (10-mg/kg; i.p.) + LiCl (25-mg/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + Hemin (30-mg/kg) + SnPP (40 lM/kg; i.p.) 3-NP (10-mg/kg/i.p./day) + LiCl (50-mg/kg; i.p.) + SnPP (40 lM/kg; i.p.) 3-NP (10-mg/kg/i.p/day) + Hemin (10-mg/kg) + LiCl (25-mg/kg; i.p.) + SnPP (40 lM/kg; i.p.)

I II III IV V VI VII VIII IX X

3NP = 3-nitropropionic acid, LiCl = lithium chloride and SnPP = Tin (IV) protoporphyrin.

161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183

7 cm (speed 25 rpm). The cut-off time was 180 s and time of the fall was recorded (Kumar and Kumar, 2009a).

the chromophore (1.56  105 M1 cm1) and expressed as a percentage of the vehicle-treated group.

Grip strength measurement. Grip strength of the fore limbs was tested with a digital grip force meter (DFIS series, Chatillon, Greensboro, NC, USA). The rat was positioned to grab the grid with the fore limbs and was gently pulled so that the grip strength could be recorded (Kehl et al., 2000). The grip strength was recorded in Kgf (unit of measurement).

Estimation of nitrite. The accumulation of nitrite in the striatum supernatant, an indicator of the production of nitric oxide (NO), was determined by a colorimetric assay with Griess reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) as described by Green et al., 1982. Equal volumes of supernatant and Griess reagent were mixed, and this mixture was incubated for 10 min at room temperature in the dark. Absorbance at 540 nm was measured with a Perkin Elmer lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as a percentage of the vehicle-treated group.

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Estimation of GSH levels. Reduced GSH in the striatum was estimated according to the method described by Ellman (1959). Results were calculated using molar extinction coefficient of chromophore (1.36  104 M1 cm1) and expressed as percentage of vehicle.

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Catalase estimation. Catalase activity was assayed by the method of Luck, 1971, in which the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 12.5 mM H2O2 in Phosphate buffer (50 mM of pH 7.0) and 0.05 ml of supernatant from the striatum tissue homogenate (10%), and the change in absorbance was recorded at 240 nm. Results were expressed as mM of H2O2 decomposed per milligram of protein/min.

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Beam-crossing task. This task requires an animal to walk on across a narrow wooden beam, measuring its motor coordination ability. The beam consisted of two platforms (8 cm in diameter) connected by a wooden beam (0.5 mm in thickness, 2.0 cm in width, and 120 cm in length). The beam was elevated 50 cm above the ground. A box filled with sawdust was placed below the beam, serving as protection for a falling rat. In order to adapt to the elevated beam, a rat was allowed to explore it for 5 min before training. A training trial started by placing the rat on the platform at one end. When a rat walked across the beam from one end to the other end, the time taken to cross the beam was recorded (Wang et al., 2006; Kalonia et al., 2010).

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Dissection and homogenization. On the 15th day, animals were randomly divided into two groups, one for biochemical estimations and the other for cellular marker estimations immediately after the behavioral assessments. Brains were dissected out. The striatum was separated by placing on ice. A 10% (w v1) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000g for 15 min. The supernatant was separated and used for biochemical estimations.

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Measurement of oxidative stress parameters

Protein estimation. The protein was measured by the Biuret method using bovine serum albumin as a standard (Gornall et al., 1949).

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Measurement of lipid peroxidation (LPO). The quantitative measurement of LPO in the brain striatum was performed according to the method of Wills (Wills, 1966). The amount of MDA, a measure of LPO, was measured by reaction with thiobarbituric acid at 532 nm using a Perkin Elmer lambda 20 spectrophotometer. Values were calculated using the molar extinction coefficient of

Estimation of tumor necrosis factor-alpha (TNF-a) and IL-1b in striatum. Quantifications of TNF-a and IL-1b were done by rat TNF-a and IL-1b immunoassay kit (R&D Systems, Minneapolis, MN, USA). The Quantikine rat TNF-a and IL-1b immunoassay is a 4.5-h solid phase ELISA designed to measure rat TNF-a and IL-1b levels. It is a solid-phase sandwich ELISA using a

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microtitre plate reader. Concentrations of TNF-a were calculated from the plotted standard curves. Estimation of is GSK-3b levels. GSK-3b level was estimated by using rat GSK-3b kit (Cusabio, GSK-3b ELISA kit protocol). It is a solid phase sandwich ELISA, which uses a microtitre plate reader read at 450 nm. Concentrations of GSK-3b were calculated from plotted standard curve.

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Estimation of HO-1 levels. HO-1 level was estimated by using rat HO-1 (Cusabio, HO-1 ELISA kit protocol). It is a solid-phase sandwich ELISA, which uses a microtitre plate reader read at 450 nm. Concentrations of HO-1 were calculated from the plotted standard curve.

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STATISTICAL ANALYSIS

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Data obtained were expressed as mean ± S.E.M. The behavioral data were analyzed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for multiple comparison. P < 0.05 was considered statistically significant. For biochemical parameters a one-way ANOVA followed by Tukey’s post hoc test was used for comparison. P < 0.05 was considered statistically significant.

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RESULTS

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Effect of HO-1 and GSK-3b modulators on 3-NPinduced decrease in body weight of rats There was no significant change in the initial and final body weights of vehicle-treated animals. However, administration of 3-NP caused a significant decrease in body weight on the last day (15th day), as compared to the vehicle-treated group. Treatment with HO-1 inducer

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Effect of HO-1 and GSK-3b modulators on 3-NPinduced changes in locomotor activity, rotarod and grip strength performance of rats

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Systemic 3-NP (10 mg/kg) administration significantly decreased grip strength (on rotarod and grip strength meter) and locomotor activity on day 10th and 15th as compared to the vehicle-treated group. Hemin (10- and 30-mg/kg) and LiCl (50-mg/kg) significantly ameliorated the impairment in grip strength and locomotor activity. (Figs. 2–4) (P < 0.05), while the combined administration of hemin (10-mg/kg) and LiCl (25-mg/kg) produced a synergistic effect as compared to their effect alone on grip strength and locomotor activity (Figs. 2–4). Pretreatment with HO-1 inhibitor SnPP (40-lg/kg) with hemin (30-mg/kg) or LiCl (50-mg/kg) or a combination of [hemin (10-mg/kg) + LiCl (25-mg/kg)] significantly reversed their beneficial effects as compared to their effect alone.

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Effect of HO-1 and GSK-3b modulators on narrow beam walk parameters in 3-NP-treated rats

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In the present set of experiments, 3-NP administration significantly increased transfer latency to cross the

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10

g

BODY WEIGHT 0

% Change in body weight

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hemin (10- and 30-mg/kg; i.p.) and LiCl (50-mg/kg) significantly restored the body weight as compared to the 3-NP-treated groups (Fig. 1), while a combined administration of low doses of hemin (10-mg/kg) and LiCl (25-mg/kg) produced a synergistic effect as compared to their effect alone. However, pre-treatment Q7 of HO-1 inhibitor SnPP (40 lg/kg) with hemin (30 mg/ kg) or LiCl (50-mg/kg) or [hemin (10-mg/kg) + LiCl (25 mg/kg)] groups reversed their beneficial effect as compared to their effect alone.

c,d -10

b -20

b f

-30

a -40

Normal control 3NP 3NP + Hemin 10 3NP+Hemin 30 3NP + LiCl 25

3NP + LiCl 50 3NP+ Hemin 10 + LiCl 25 3NP+ Hemin 30+ SnPP 40 3NP+ LiCl 50+ SnPP 40 3NP+ Hemin 10+LiCl 25+ SnPP 40

Fig. 1. Effect of HO-1 and GSK-3b modulators on body weight in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.01 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30-mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg. Please cite this article in press as: Khan A et al. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3b modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.018

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Locomotor activity g

bcd

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b

b

Counts/5min

bb 200

a

b b

b e

b a

100

15 th

10 th

1s t

0

Days 3NP + LiCl 50 3NP + Hemin 10 + LiCl 25 3NP + Hemin 30 + SnPP 40 3NP +LiCl 50 + SnPP 40 3NP+Hemin10+LiCl25+SnPP 40

Vehicle control 3NP 3NP + Hemin 10 3NP + Hemin 30 3NP + LiCl 25

Fig. 2. Effect of HO-1 and GSK-3b modulators on locomotor activity in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.01 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

Rotarod 200

g

bcd bcd

b

Fall off Time

150

bc

b b

e

a

b

b b

100

e a 50

15 th

10 th

1s t

0

Days Vehicle control

3NP + LiCl 50

3NP

3NP + Hemin 10 + LiCl 25

3NP + Hemin 10

3NP + Hemin 30 + SnPP 40

3NP + Hemin 30

3NP +LiCl 50 + SnPP 40

3NP + LiCl 25

3NP+Hemin10+LiCl25+SnPP 40

Fig. 3. Effect of HO-1 and GSK-3b modulators on Rotarod activity in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.01 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

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straight runway and foot errors on the narrow beam walk on day 14 as compared to the normal control group (Fig. 5). Induction of HO-1 via hemin treatment (30-mg/ kg), and LiCl (50-mg/kg) significantly improved the

latency and decreased foot errors on the narrow beam walk apparatus in 3-NP-treated rats. Low-dose Hemin (10-mg/kg) and LiCl (25-mg/kg) did not produce any significant effect, while the combined administration of

Please cite this article in press as: Khan A et al. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3b modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.018

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Grip strength bcd b b

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Days

15

3NP + LiCl 50 3NP+ Hemin 10 + LiCl 25 3NP+ Hemin 30+ SnPP 3NP+ LiCl 50+ SnPP 3NP+ LiCl 25+Hemin 10+ SnPP 40

Fig. 4. Effect of HO-1 and GSK-3b modulators on Grip strength in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.05 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

Fig. 5. Effect of HO-1 and GSK-3b modulators on Narrow beam walk performance in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.05 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30-mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg. Please cite this article in press as: Khan A et al. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3b modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.018

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A. Khan et al. / Neuroscience xxx (2014) xxx–xxx Table 2. Effect of Hemin, Lithium Chloride and their modulator on 3-NP treatment-induced biochemical changes in the striatum Treatment (mg/kg)

Vehicle 3-NP (10) Hemin (10) + 3-NP Hemin (30) + 3-NP LiCl (25) + 3-NP LiCl (50) + 3-NP Hemin (10) + LiCl (25) + 3-NP SnPP (40 lM/kg) + Hemin (30) + 3-NP SnPP (40 lM/kg) + LiCl (50) + 3-NP SnPP (40 lM/kg) + Hemin (10) + LiCl (25) + 3-NP

MDA nmol/mg protein (% of vehicle)

Nitrite level (% of vehicle)

100 ± 5.2 256 ± 4.7a 201 ± 6.1b 176 ± 6.2b,c 221 ± 8.4b 195 ± 4.7b,d 127 ± 5.7NS 237 ± 6.1b 202 ± 6.4NS 210 ± 7.8b

100 ± 5.7 195 ± 4.3a 156 ± 4.9b 135 ± 5.9b,c 172 ± 8.1b 163 ± 4.6b,d 118 ± 5.3NS 185 ± 4.7b 152 ± 4.9NS 138 ± 6.5b

lmol/mg protein

GSH (% of vehicle)

Catalase u mole of H2O2 decomposed/min/mg protein (% of vehicle)

100 ± 6.1 45 ± 6.8 a 64 ± 5.7b 73 ± 8.1b,c 58 ± 7.4b 65 ± 5.4b,d 92 ± 6.1NS 53 ± 6.9NS 61 ± 6.7NS 63 ± 6.1NS

100 ± 5.5 45 ± 5.4 a 69 ± 5.2b 78 ± 5.4b 60 ± 4.7b 71 ± 5.4b 93 ± 3.9NS 54 ± 4.3b 63 ± 6.4NS 77 ± 5.9b

Values expressed as % of vehicle-treated group.

313 314 315 316 317 318 319

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hemin (10-mg/kg) and LiCl (25-mg/kg) produced a synergistic effect as compared to their effect alone on Q8 these parameters. Similarly pre-treatment of HO-1 inhibitor SnPP (40-lg/kg) with hemin (30-mg/kg) or LiCl (50-mg/kg) or a combination of hemin (10-mg/kg) + LiCl (25-mg/kg) also significantly reversed the effect of these drugs alone on transfer latency and foot error count. Effect of HO-1 and GSK-3b modulators on LPO and nitrite levels in rats administered with 3-NP Systemic administration of 3-NP significantly increased oxido-nitrosative stress parameters, i.e., MDA and nitrite level in the striatum as compared to the vehicle-treated groups (Table 2). Pretreatment with hemin (10- and 30-mg/kg) and LiCl (50-mg/kg) significantly restored the levels of altered oxido-nitrosative stress in 3-NP administered rats as compared to the group receiving 3-NP only. However, a combination of hemin (10-mg/kg) and LiCl (25-mg/kg) produced most significant results in lowering MDA and nitrite levels in the striatum. On the other hand, concurrent administration of SnPP (40 lM/ kg) along with hemin (30-mg/kg), or LiCl (50-mg/kg) and a low-dose combination (hemin 10-mg/kg and LiCl 25-mg/kg), significantly reversed their beneficial effect on oxido-nitrosative stress, as compared to hemin, LiCl and a low-dose combination of alone-treated groups. Effect of HO-1 and GSK-3b modulators on reduced GSH and catalase levels in 3-NP-treated rats Administration of 3-NP for 14 days significantly depleted reduced GSH and catalase enzyme concentration in the brain striatum (Table 2). Treatment with HO-1 inducer hemin (10- and 30-mg/kg; i.p.) and LiCl (50-mg/kg) significantly restored the levels of GSH and catalase as compared to the 3-NP-treated groups. Further combined administration of hemin (10-mg/kg) and LiCl (25-mg/kg) produced most effective restoration of these enzyme markers of oxidative stress as compared to alonetreated groups. However, pretreatment of HO-1 inhibitor SnPP (40 lM/kg) either with hemin (30-mg/kg) or LiCl (50-mg/kg) reversed the beneficial effect of these drugs as compared to their effect alone. Similarly, SnPP (40 lM/kg) pretreatment also significantly reversed the

beneficial effect of hemin (10-mg/kg) + LiCl (25-mg/kg) on these enzymes.

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Effect of HO-1 and GSK-3b modulators on TNF-a and IL-1b levels in 3-NP-treated rats

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TNF-a and IL-1b are important pro-inflammatory markers in HD and other disorders. Systemic administration of 3-NP significantly increased the level of TNF-a and IL-1b levels in the brain striatum as compared to the normal control group (Fig. 6). Hemin (10- and 30-mg/kg; i.p.) and LiCl (50-mg/kg) significantly reduced the levels of TNF-a and IL-1b as compared to the 3-NP-treated groups. Further, concomitant administration of HO-1 inhibitor SnPP (40 lM/kg) treatment with Hemin (30-mg/ kg), LiCl (50-mg/kg) and a combination (hemin 10-mg/ kg + LiCl 25-mg/kg) increased the level of TNF-a and IL-1b, thus showing its antagonizing characteristics to HO-1 induction.

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Effect of various pharmacological interventions on HO-1 levels in rats administered with 3-NP

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HO-1 is an inducible enzyme that is upregulated in response to cellular stress and toxic insults. Treatment with HO-1 inducer hemin (10- and 30-mg/kg) and LiCl (25- and 50-mg/kg) significantly elevated HO-1 levels in the striatum as compared to the 3-NP-treated group (Fig. 7). Further, a combination of hemin 10-mg/kg and LiCl 25-mg/kg produced a synergistic effect by elevating the level of the cytoprotective enzyme. In antagonistic study groups, SnPP that is a HO-1 inhibitor significantly attenuated HO-1 levels in the striatum when co administered with hemin (30-mg/kg), LiCl (50-mg/kg) and a combination of hemin 10-mg/kg + LiCl 25-mg/kg.

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Effect of various pharmacological interventions on GSK-3b levels in rats administered with 3-NP

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Systemic administration of 3-NP significantly increased GSK-3b levels as compared to normal control animals (Fig. 8). Treatment with LiCl (25- and 50-mg/kg; i.p.) and hemin (30-mg/kg) significantly reduced GSK-3b levels in the striatum as compared to the 3-NP-treated group. Further, SnPP upon concomitant treatment with

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Please cite this article in press as: Khan A et al. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3b modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.018

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TNF- alpha & IL - 1β b

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LiCl 50 Hemin 10 + LiCl 25 Hemin 30+SnPP 40 LiCl 50+SnPP 40 LiCl 25+Hemin 10+SnPP 40

Fig. 6. Effect of HO-1 and GSK-3b modulators on TNF-a and IL-1b levels in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.05 vs. normal control, b = p < 0.05 vs. 3NP-treated group, c = p < 0.05 vs. Hemin 10-mg/kg, d = p < 0.05 LiCl 25-mg/kg, e = p < 0.05 vs. hemin 30-mg/kg, f = p < 0.05 vs. hemin 30-mg/kg + SnPP 40 lM/kg, g = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

Hemeoxygenase - 1 level 3NP+ LiCl 25+Hemin 10+ SnPP 40

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Fig. 7. Effect of HO-1 and GSK-3b modulators on HO-1 levels in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.05 vs. 3NPtreated group, b = p < 0.05 vs. Hemin 10-mg/kg, c = p < 0.05 LiCl 25-mg/kg, d = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

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hemin (30-mg/kg), LiCl (50-mg/kg) and a combination group (hemin 10-mg/kg + LiCl 25-mg/kg) produced significantly elevated levels of GSK-3b in the rat striatum when compared to alone-treated groups.

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DISCUSSION

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The present study demonstrates the potential role of HO-1/GSK-3b pathway in 3-NP-induced neurotoxicity. 3-NP model has been commonly used to explore the various behavioral, biochemical and cellular alterations involved in the pathogenesis of HD. Systemic administration of 3-NP is reported to produce HD-like symptoms in animals and non-human primates (Shear et al., 2000; Keene et al., 2001; Kumar et al., 2010a,b,c, 2011a, 2012; Kumar and Kumar, 2009b,c,d). 3-NP has

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been reported to induce motor impairment and striatal toxicity by causing the degeneration of GABAergic MSN in the striatum in a pattern similar to the neuronal cell death as seen in HD patients (Beal et al., 1993). 3-NP administration significantly caused a loss in body weight that could be due to the involvement of peripheral and central effects. The striatal lesions and bradykinesia could be partly responsible for reduced appetite and food intake in rats (Guyot et al., 1997). In addition, in support of the present study, observations of patients with late-stage HD showed that they also exhibit dysphagia and loss of body weight (Saydoff et al., 2003). Loss in body weight and hypoactivity could be simply because of depressed energy metabolism after 3-NP treatment. 3-NP administration for 14 days significantly impaired locomotor activity, rotarod performance, grip strength and

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GSK-3β levels 3NP+ LiCl 25+Hemin 10+ SnPP 40

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Fig. 8. Effect of HO-1 and GSK-3b modulators on GSK-3beta levels in 3-NP-treated rats: Data are expressed as mean ± SEM. a = p < 0.05 vs. 3NP-treated group, b = p < 0.05 vs. Hemin 10-mg/kg, c = p < 0.05 LiCl 25-mg/kg, d = p < 0.05 vs. hemin 10-mg/kg + LiCl 25-mg/kg.

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narrow beam walk performance in rats as compared to the vehicle-treated group. The present findings are in tune with earlier reports following 3-NP administration (Shear et al., 2000; Keene et al., 2001, 2010a,b,c, 2011b, 2012; Kumar and Kumar 2009). Pretreatment with hemin, a selective HO-1 activator and LiCl, a GSK-3b inhibitor significantly reversed the decline in body weight in the 3-NP-treated animals. Further this beneficial effect was more profound when hemin and lithium were concomitantly administered. On the other hand, the beneficial effect of hemin was abolished by co-administration of SnPP (HO-1 inhibitor). In addition, the observed beneficial effects of LiCl were also partly, but significantly reversed by concurrent treatment with SnPP. Although, it seems that 3-NP produces an alteration in behavior by motor signal influencing the striatum, the possibility of involvement of other brain areas such as the cortex cannot be ignored in 3-NP action (Rodrı´ guez-Martinez et al., 2004; Silva Q9 et al., 2007). However, the beneficial effect of hemin and LiCl were abolished with SnPP treatment. This indicates the beneficial effect of hemin may be mediated through upregulated activity of HO-1. Promising therapeutic effects of increased brain HO-1 levels have also been reported in various experimental models of neurodegenerative disorders including Alzheimer disease (AD) and Parkinson disease (PD) (Cuadrado and Rojo, 2008). Oxidative stress is considered to be one of the major key determinants in 3-NP-induced neurotoxicity. Impaired energy metabolism and oxidative stress have been reported to occur after the inhibition of complex-II succinate dehydrogenase enzyme (SDH) in 3-NPtreated animals (Villara´n et al., 2008). 3-NP treatment has been shown to increase oxidative stress markers and induce protein oxidation in synaptosomes (Teunissen et al., 2001). Indeed, free radical formation and oxidant injury play a critical role in excitotoxic damage as evidenced by rise in MDA and nitrite level in the brain tissues of HD patients (Boll et al., 2008). In the present study, 3-NP significantly increased oxido-nitrosative stress (MDA and nitrite levels), depleted endogenous antioxidant defense enzymes (reduced GSH and catalase) in the striatum, which is consistent with previous

reported studies (Kumar et al., 2010b,d) Hemin and LiCl pretreatment significantly attenuated the abnormal levels of free radicals in 3-NP-treated rats. Further a low-dose combination of hemin and lithium pretreatment much more significantly improved the altered biochemical marker levels whereas SnPP showed increased biochemical toxicity in the hemin-treated group, suggesting its antagonistic potential. A study by Guan et al. (2009), showed, hemin pretreatment significantly reduced the levels of MDA, a marker of oxidative stress; this reduction was abolished by SnPP, suggesting that the anti-oxidant properties of HO-1 may contribute to its protective effects. As mentioned above, neuronal cells can respond to toxic oxidative insult by upregulating intracellular compensatory responses, among them induction of the heat-shock responses and upregulation of HSP chaperons, particularly highly inducible Hsp-70 and HO-1 provides a promising therapeutic approach for neurodegenerative diseases, it is ideal to pharmacologically induce endogenous molecular chaperons by the administration of small chemical compounds, instead of expressing exogenous genes (Eftekharzadeh et al., 2010). This is further comprehended by recent evidences that overexpression of HO-1 by pharmacological modulation or gene transfer may represent a novel strategy for therapeutic or preventative intervention against stress and toxicity (Immenschuh and Ramadori, 2000; Cuadrado and Rojo, 2008). Although many complex signaling cascades and metabolic events play a role in regulating cell death it is now well recognized that antioxidant response element (ARE)-mediated expression and coordinated induction of antioxidant enzymes are critical mechanisms of protection against chemically induced oxidative/ electrophilic stress (Johnson et al., 2008; Mate´s et al., 2008). Under basal conditions, Nrf2 is sequestered in the cytoplasm by its repressor Kelch-like ECH-associated protein 1 (Keap1) (Itoh et al., 1997). Electrophiles and other inducers liberate Nrf2 from Keap1 repression, and allow its translocation into the nucleus to associate with other transcription factors and bind to the ARE (Kobayashi and Yamamoto, 2006).

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In this study, Lithium by inhibiting GSK-3b might have facilitated Nrf2 nuclear translocation, as well as HO-1 509 expression. HO-1 is one of the antioxidant proteins in 510 response to oxidative stress which provides 511 cytoprotection in various animal models (Lee et al., 512 1996). HO-1 expression is predominantly regulated at 513 transcriptional level by ARE located at its promoter 514 region. In fact, long-term oxidative stress causes GSK515 3b activation and reduces nuclear Nrf2, suggestive of 516 downregulation of the Nrf2-ARE pathway (Rojo et al., 517 2008). This hypothesis is further supported by the finding 518 that pharmacological inhibition of GSK-3b, has been 519 reported to reduce Ab pathology and cognitive impairment 520 in AD mice (Phiel et al., 2003). Moreover, lithium, a GSK521 3b inhibitor, has been shown to promote the transcrip522 tional activity of Nrf2 insults (Aztatzi-Santilla´n et al., 523 2010). GSK-3b regulates Nrf2, thus making this kinase 524 a potential target for therapeutic intervention aiming to 525 boost the protective activation of Nrf2 (Kanninen et al., 526 2011). Recent study reports that GSK-3b modulation 527 shows a protective effect against oxidative stress in AD 528 by involvement of the Nrf2 pathway (Kanninen et al., 529 2011). HO-1 expression is a sensitive marker in response 530 to cellular oxidative stress. The administration of exoge531 nous HO-1 in the MPP+-treated parkinsonian rat model 532 is beneficial in reducing dopaminergic neuronal degener533 ation, attenuating behavior asymmetry induced by 534 MPP+, and up-regulation of brain-derived neurotrophic 535 factor (BDNF) and glial cell-derived neurotrophic factor 536 (GDNF) expression both in vitro and in vivo (Hung et al., 537 2008). 538 Neuroinflammation also plays a major role in the 539 pathogenesis of CNS disorders (Tansey et al., 2008; 540 Frank-Cannon et al., 2009). Cytokines such as IL-1b 541 and TNF-a have been implicated in the pathogenesis of 542 various neurodegenerative diseases (Meffert and 543 Baltimore 2005). In the present study, 3-NP caused a sig544 nificant elevation in levels of IL-1b and TNF-a in the stri545 atum. Treatment with hemin declined levels of cytokines 546 significantly, although, the decline was most prominent 547Q10 in a combination group of lithium and hemin. In addition, 548 IL-1b promotes excitotoxicity (Pearson et al., 1999) and 549 TNF-a activates microglia in vitro (Drew and Chavis 550 2000); Administration of hemin inhibits IL-1b (reduced 551 excitotoxicity) and inhibits microglia activity, this results 552 in a decrease in the expression of TNF-a, iNOS (protein 553 and mRNA) and NO release, leading to less neuronal 554 degeneration and apoptosis (Drew and Chavis, 2000). 555 Data obtained suggest that a low-dose combination of 556 prophylactic administration of hemin and LiCl provided a 557 more pronounced effect in preventing the development 558 of 3-NP-induced neurotoxicity. This suggests that 559 concurrent pharmacological modulation of HO-1 and 560 GSK-3b may represent a better therapeutic strategy to 561 provide optimal neuroprotection. 507 508

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Acknowledgments—Authors are thankful to the All India Council of Technical Education (AICTE), New Delhi for providing financial assistance under the Research Promotion Scheme (8023/RID/ RPS-46/2011-12) to Dr. Puneet Kumar Bansal.

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(Accepted 11 December 2014) (Available online xxxx)

Please cite this article in press as: Khan A et al. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3b modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.12.018

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