Small molecule TrkB agonist deoxygedunin protects nigrostriatal dopaminergic neurons from 6-OHDA and MPTP induced neurotoxicity in rodents

Neuropharmacology 99 (2015) 448e458

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Small molecule TrkB agonist deoxygedunin protects nigrostriatal dopaminergic neurons from 6-OHDA and MPTP induced neurotoxicity in rodents Shuke Nie a, 1, Yan Xu a, 1, Guiqin Chen a, Kai Ma a, Chao Han a, Zhenli Guo c, **, Zhentao Zhang d, Keqiang Ye b, Xuebing Cao a, * a

Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA c Department of Neurology, Xinhua Hospital of Hubei Province, Wuhan 430015, China d Department of Neurology, Renmin Hospital of Wuhan University, Wuhan 430060, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 October 2014 Received in revised form 10 August 2015 Accepted 11 August 2015 Available online 14 August 2015

Dopaminergic neurons loss in the substantia nigra (SN) and dopamine (DA) content loss in the striatum correlate well with disease severity in Parkinson's disease (PD). Brain-derived neurotrophic factor (BDNF) is a member of neurotrophin family and is necessary for the survival and development of DA neurons in the SN. Deficits in BDNF/TrkB receptors signaling contribute to the dysfunction of PD. Deoxygedunin, a derivative of gedunin produced from Indian neem tree, binds TrkB receptor and activates TrkB and its downstream signaling cascades in a BDNF-independent manner, and possesses neuroprotective effects in vitro and in vivo. In this study, we tested the neuroprotective effects of deoxygedunin in 6hydroxydopamine (6-OHDA)-lesioned rat model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of Parkinson's disease. Rats were treated with deoxygedunin 5 mg/kg (i.p.) for one month started two weeks before 6-OHDA lesion (pre-treatment), or for two weeks right after lesion (post-treatment), with isovolumetric vehicle as control and normal. Mice were given deoxygedunin 5 mg/kg (i.p.) for 2 weeks and administrated with MPTP twice at the dose of 20 mg/kg (i.p.) on day 7. The results revealed that pretreatment with deoxygedunin improved PD models' behavioral performance and reduced dopaminergic neurons loss in SN, associated with the activation of TrkB receptors and its two major signaling cascades involving mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Thus, our current study indicates that deoxygedunin, as a small molecule TrkB agonist, displays prominent neuroprotective properties, providing a novel therapeutic strategy for treating Parkinson's disease. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Parkinson's disease Deoxygedunin Neuroprotection Brain derived neurotrophic factor 6-Hydroxydopamine MPTP

1. Introduction Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and exerts its biological functions through two receptors: the p75 neurotrophin receptor (p75NTR) and the TrkB

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S. Nie), [email protected] (Y. Xu), [email protected] (G. Chen), [email protected] (K. Ma), [email protected] (C. Han), [email protected] (Z. Guo), zhentao.zhang@emory. edu (Z. Zhang), [email protected] (K. Ye), [email protected] (X. Cao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neuropharm.2015.08.016 0028-3908/© 2015 Elsevier Ltd. All rights reserved.

receptor tyrosine kinase (Huang and Reichardt, 2003; Kaplan and Miller, 2000). BDNF binding to TrkB triggers its dimerization and autophosphorylation of tyrosine residues in its intracellular domain, resulting in activation of the three major signaling pathways involving Ras/Raf/MAPK, PI3K/Akt and PLC-g. BDNF protects neurons from glutamate toxicity (Lindholm et al., 1993) and rescues neurons from programmed cell death (Leeds et al., 2005), reduces ischemic injury (Kurozumi et al., 2004; Schabitz et al., 2000) and improves functional recovery and post-injury regeneration (Koda et al., 2004). Moreover, BDNF is of particular therapeutic interest because of its neurotrophic actions on neuronal populations involved in neurodegenerative diseases (Askanas, 1995; Lindsay, 1996; Siegel and Chauhan, 2000). In addition to promoting

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neuronal survival, BDNF/TrkB also plays a critical role in mediating synaptogenesis and activity-induced modification of synaptic transmission, regulating LTP and long-term memory (Lu et al., 2008; Minichiello, 2009). Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which affects 1.5% of the population older than 65 years (Connolly and Lang, 2014). The pathological feature of PD is characterized by the loss of dopaminergic neurons in the SN and the loss of dopamine content in the striatum. BDNF is a member of neurotrophin family, which are protein molecules regulating neuronal survival, growth, differentiation and function in the nervous system. BDNF binding to TrkB receptor triggers its dimerization and activation of MAPK, PI3K/Akt and PLC-g signaling cascades. Because of its survival-promoting activity and essential for the development of DA neurons in the SN, BDNF has been investigated extensively in PD related setting. BDNF (Klein et al.,1999; Razgado-Hernandez et al., 2015) attenuates the lesion-induced loss of nigrostriatal dopaminergic neurons in animal models of PD. Expressing BDNF into either the striatum or the midbrain attenuates 6-OHDA-induced losses of nigrostriatal neurons (Benraiss et al., 2012; Yoshimoto et al., 1995), and inhibiting BDNF expression cause the death of SNpc dopaminergic neurons and behavioral deficits characteristic of neurotoxic models of PD in rodents (Porritt et al., 2005). Moreover, BDNF elicits behavioral improvement and motor dysfunction in a rat model of PD (Yoshimoto et al.,1995), and physical training exert its antidepressant effect and neuroprotection in the striatum and the hippocampus via increasing the BDNF levels in 6-OHDA-treated models of PD (Tuon et al., 2014). The serum and cerebrospinal fluid levels of BDNF were significantly reduced in PD patients when compared to healthy subjects (Nagatsu and Sawada, 2005; Scalzo et al., 2010). Serum BDNF level is also a biomarker for early recognition in the PD patients with cognitive impairment (Angelucci et al., 2015), but BDNF Val66Met polymorphism is not associated with the risk of developing PD in a meta-analysis study (Mariani et al., 2015). However, the clinical trials with recombinant BDNF were disappointing. Presumably, it is due to its short half-life, and it does not pass through blood brain barrier (BBB) (Thoenen and Sendtner, 2002). To circumvent the pharmacokinetic curbs of BDNF, we developed a cell-based assay to identify small molecules that mimic the neurotrophic activities of BDNF. We reported that deoxygedunin selectively activates TrkB, but not TrkA or TrkC (Jang et al., 2010a). It provokes TrkB activation in mouse brain upon intraperitoneal or oral administration. It passes through the brain blood barrier and displays no chronic toxicity. Deoxygedunin exerts the neurotrophic effects in a TrkB-dependent manner. It demonstrates promising therapeutic efficacy in various cellular and animal models. Deoxygedunin is a derivative of gedunin produced from Indian neem tree. Recently, we have shown that it directly binds to the extracellular domain of TrkB receptors and promotes their activation, acting as a small molecular agonist. Moreover, administration of deoxygedunin into mice exhibits potent neuroprotective, learning and memory enhancement and anti-depressant effects, which were mediated by TrkB receptors (Jang et al., 2010a). In the present study, we investigated its potential neuroprotective effects in 6-OHDA-lesioned rat model and MPTP induced mice model of PD by evaluating the behavioral performance and histological analysis of SN. We found that deoxygedunin robustly activates TrkB and its downstream signals and reveals promising therapeutic efficacy toward PD. 2. Materials and methods 2.1. Animals Male SpragueeDawley rats, weighing 220e250 g, and male C57/BL6 mice, 8e9 weeks old, were purchased from HFK

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Bioscience Company (Beijing, China) and Huazhong University of Science and Technology Laboratory Animal Center (Wuhan, China). They were housed under 21e23
C with 12 h light:dark cycle with food and water available ad libitum. All of the experimental protocols were carried out in accordance with the Rules of Animal Care and Use Committees of Huazhong University of Science and Technology (HUST), which is fully compatible with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No.8023, revised 1978). Maximum efforts were made to reduce the number of animals used and to minimize discomfort. 2.2. Materials 6-Hydroxydopamine hydrobromide, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (M0896) and apomorphine hydrochloride were purchased from SigmaeAldrich (USA). Deoxygedunin was purchased from Gaia Chemical Company (catalog no.L4250), and was dissolved in solvents contained 5% DMSO, 15% Tween 20 and 80% physiological saline (0.9% sodium chloride). 1% sodium pentobarbital was purchased from Wuhan Union hospital (China). DAB kit for IHC (K5007, DAKO), Rabbit polyclonal tyrosine hydroxylase antibody (ab112, Abcam, USA), Goat polyclonal Tyrosine Hydroxylase antibody (ab101853, Abcam), rabbit monoclonal BDNF antibody [EP1293] (ab108383, Abcam), rabbit anti-TrkB (ab33655, Abcam) and rabbit antiphospho-TrkB (Y816) (ab75173, Abcam) were purchased from Abcam company; p44/42 MAPK (Erk1/2) Rabbit mAb (#4695), phospho-p44/42 MAPK (Erk1/2) Rabbit mAb (#4370), Akt (pan) (C67E7) Rabbit mAb (#4691), phospho-Akt (Ser473) (D9E) XP Rabbit mAb (#4060), rabbit caspase-3 Antibody (#9662), rabbit Bcl-xl (54H6) Antibody (#2764), mouse anti-b actin (8H10D10) antibody (#3700) and HRP-conjugated secondary anti-rabbit or anti-mouse antibody were purchased from Cell Signaling Technology. All chemicals not included above were purchased from SigmaeAldrich. 2.3. 6-OHDA-lesioned rat model Each group contained 10 rats. The first group was the pretreatment group. Rats were treated with deoxygedunin given at the dose of 5 mg/kg daily by intraperitoneal injection (i.p.) for one month started two weeks before 6-OHDA lesions (pretreatment group, n ¼ 10). The rats in other three groups were given intraperitoneally with isovolumetric vehicle daily for two weeks before the operation. All the rats except the normal groups were then subject to unilateral intrastriatal injection of 6-OHDA. Rats were anesthetized with 40 mg/kg 1% sodium pentobarbital. They were stereotaxic injected with total dose of 20 mg of fresh prepared 6-OHDA (calculated as free base) into two sites of the left striatum, using the following coordinates (in mm relative to Bregma): AP þ0.5, L 3.0, DV 4.0; AP 0.5, L 3.5, DV 5.0 (Fig. 1A). The injection rate was 1 ml/min and a total of 2 ml was injected at each site. The needle was left in place for 2 min before retracting. After the lesion, deoxygedunin treatment was continued for two more weeks. The second group was given deoxygedunin 5 mg/kg (i.p.) one hour after 6-OHDA lesion and last for 2 weeks (post-treatment group, n ¼ 10). The third group was treated with 6-OHDA lesion without deoxygedunin intervention (control group, n ¼ 10). The fourth group was normal group (n ¼ 10). Control group with 6-OHDA lesion and normal group without 6-OHDA lesion were injected intraperitoneally with isovolumetric vehicle. A total of three rats (one in pre-treatment group, one in post-treatment group and one in control group) were excluded from the 6-OHDA-lesioned

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Fig. 1. Schematic illustration of experimental designs and effects of deoxygedunin on the behavioral performance of 6-OHDA lesioned rat model. 30 rats were stereotaxic injected with total dose of 20 mg of fresh prepared 6-OHDA (calculated as free base) into two sites of the left striatum (in mm relative to Bregma): AP þ0.5, L 3.0, DV 4.0; AP 0.5, L 3.5, DV 5.0 (A). Schematic representation of the 6-OHDA lesioned rat mode experimental design (B). Effects of deoxygedunin on the Cylinder test in the four groups (C). Deoxygedunin improves locomotion function in 6-OHDA-treated rats. Compared with pre-treatment and normal groups, the numbers of right hand contact in the control group was significantly reduced (*p < 0.05). The number of both paw use in normal group was significantly more than other three groups (##p < 0.001). Effects of deoxygedunin on adjusting step test (D). *p < 0.05, **p < 0.01, significant difference between the two indicated groups. Compared with normal group, the rats in the control group exhibited damaged right forelimb locomotion function. Pretreatment with deoxygedunin for two weeks before 6-OHDA-lesioned surgery prevented this decrease, indicating reversion of right forelimb akinesia (p < 0.05). Effects of deoxygedunin on rotational behavior in 6-OHDA lesion rat model (E). Pre-treatment with deoxygedunin significantly reduced the number of contralateral rotation. *p < 0.05, **p < 0.01, significant difference between the two indicated groups. Data were presented as mean ± SEM. n ¼ 9.

rat model study because of death and operation failure during the experimental period. 2.4. MPTP neurotoxicity mouse model Thirty mice were randomly divided into three groups (n ¼ 10 in each group). Mice were given deoxygedunin 5 mg/kg (i.p) for 2 weeks before and after MPTP injection (deoxygedunin group, n ¼ 10). On day 7, animals in deoxygedunin group and MPTP group (n ¼ 10) were administrated with MPTP (i.p) twice at the dose of 20 mg/kg each (2 h apart) for one week. The third group was injected intraperitoneally with isovolumetric vehicle (Normal group, n ¼ 10). Behavior test of the animals were performed on day 14. No animals were excluded from the MPTP-induced neurotoxicity mouse model study. The weigh of the mice in each group were measured every two days.

2.5. Behavioral tests Two weeks after the 6-OHDA-lesioned operation, the rats in four groups (9 in pre-treatment group, 9 in post-treatment, 9 in control group and 10 in normal group) were assessed for akinesia with cylinder test, adjusting step test and apomorphine induced rotation test. MPTP-treated mice (10 in each group) were evaluated with pole test according to a well established protocol to assess the locomotion activity (Fig. 1B). All behavioral tests were done in a blinded fashion. 2.5.1. Cylinder test Rats in each group were placed in a transparent cylinder (22 cm in diameter and 30 cm height). They were acclimatized in the transparent cylinders for 5 min prior to test. Animal in the cylinder would support its body with one or both of its forelimbs. Then, the

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2.5.4. Pole test Mice in each group were placed head-up on the top of a vertical pole (50 cm long and 1 cm in diameter). The base of the pole was placed in the home cage. The mice would turn its head downward and climb along the pole to the home cage. Time of the mouse head orient downward (named as Tturn) and total time from the top of the pole to home cage as an indication of the locomotion activity (TLA) were recorded. Before the final behavioral assessment, mice in each group received training for three days. Each mouse received five successive trials for average.

incubated with EDTA (PH 9.0) antigen retrieval buffer microwavetreated to retrieve antigens, processed slices were blocked with 3% BSA for 30 min at room temperature, and then incubated in a humidified chamber with diluted primary antibody in blocking buffer overnight at 4
C overnight. The following primary antibodies were used: rabbit polyclonal tyrosine hydroxylase antibody (1:500, Abcam) for SNpc in the 6-OHDA lesioned rats, rabbit antiphosphorylated-TrkB (Y816) (1:100, Abcam) and goat polyclonal Tyrosine Hydroxylase antibody (1:250, Abcam) for the striatum and SNpc in the MPTP-treated mouse. For double staining, various combinations of antibodies were selected. After being washed three times at 5 min with 1  PBS, sections were incubated in dark with an appropriately diluted Alexa Fluor 488-coupled secondary antibodies for 50 min. After 10 min incubation in 100 ml 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) solution (1:1000) and a PBS wash, fluorescent images of SNpc TH staining in 6-OHDA lesioned rat and striatum TH staining in MPTP-treated mouse were examined on an OLYMPUS IX71 fluorescent microscope. Co-expression of TH and phospho-TrkB (Y816) in the substantia nigra compact part of the MPTP neurotoxicity mouse were evaluated by a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Tokyo, Japan). Then the optical density was compared and positive cells were counted by the image J software. Briefly, immunofluorescent images were changed into 8-bit type (gray) and then were inverted (light background), and later processed into binary image. Remove despeckle and outliers (10 in the threshold) in the binary images. Threshold the image using the automated routine. Number of positive neurons was calculated using the “analyze particles” plugin of Image J with the following steps: Analyze / Analyze Particles: Enter 20 as the minimum particle size, toggle “Show Outlines”, check “Display Results”, “Summarize” and “Record Stats” and click “OK”. The optical densities were calculated using the ratio of integrated density (IOD) to area with the “Measure” plugin of Image J.

2.6. Tissue preparation

2.8. Immunohistochemistry staining

After the completion of behavioral tests, all the animals were deeply anesthetized. Four animals form each group were transcardially perfused with 30 ml saline plus 4% paraformaldehyde solution in 0.1M phosphate buffer (pH7.4) for 8 min. Brains were removed quickly from the skull and immerse overnight in 4% PFA at 4
C overnight. Afterward, the area of striatum (CPU) and substantial nigra (SN) were cut coronally using a rodent brain matrice (RWD Lifescience, China), subjected to dehydration, cleared with dimethyl benzene, and eventually embedded in paraffin blocks for the next immunofluorescent and immunohistochemistry staining. Other four animals form each group were transcardially perfused with 15 ml phosphate-buffered saline and were immediately decapitated. Ventral midbrain containing the substantia nigra of the lesioned hemisphere of each rat were obtained and frozen in liquid nitrogen and stored at 80
C for biochemical analysis.

After paraffin embedding, the tissues were routinely sliced into 5 mm sections. Slides with mounted sections are placed in 37
C heater overnight at room temperature. For 6-OHDA-lesioned rat models, three SNpc sections from Bregma 4.5, 5.0 and 5.5 mm were used for phospho-TrkB positive cells counting. Meanwhile, one section from Bregma þ2.5 was used for the TH Immunohistochemistry staining of the lesioned striatum, assessing the neuroprotection effect of deoxygedunin on the surrounding tissue at the injection site (n ¼ 4). For MPTP-treated mice, three SNpc sections from Bregma 3.0, 3.3 and 3.6 mm were used for TH positive cell counting (n ¼ 4). Then all slides were de-waxed by xylene twice for 15 min each, and were hydrated with 100% ethanol twice for 5 min each, 95% ethanol twice for 5 min each, and 70% ethanol twice for 5 min each and then rinsed in distilled water. The sections were immersed in EDTA (PH 9.0) antigen retrieval buffer, followed by washing three times at 5 min with PBS (PH 7.4), and then were immersed in 3% basic hydrogen peroxide for 25 min in dark to block the activity of endogenous peroxidase, followed by the above washing steps. Processed slices were blocked with 3% BSA for 30 min at room temperature, and then incubated in a humidified chamber with diluted primary antibody in blocking buffer overnight at 4
C overnight. Tyrosine hydroxyls (TH) antibody (1:800, Abcam) was used to determine dopaminergic neuron counts in the substantia nigra and nerve terminal density in the striatum, and anti-phosphorylated-TrkB (Y816) (1:1000, Abcam) was used to test TrkB activation. After washing three times at 5 min with PBS, all sections were subsequently incubated with biotinylated goat antirabbit IgG at 37
C for 30 min and incubated with Horseradish

numbers of left, right or both forelimb(s) wall contacts were countered until total number of wall contact reached 20 times. Each behavior was expressed as percent use of left, right or both limb(s) relative to the total number. 2.5.2. Adjusting step test The hind limbers of rats were held by the experimenters and slightly raising the hind part of the body. The forelimb of the rat not to be tested was also fixed, with only the other forepaw touching the table. The rat was moved slowly along the table (90 cm in 5 s), first use the right forehand (from left to right direction) then the left forehand (from right to left direction). The number of adjusting steps of each left and right forelimbs on both directions was recorded individually. 2.5.3. Apomorphine induced rotation test After completing the above two behavioral tests, rats in the four groups were injected with apomorphine (0.05 mg/kg saline, subcutaneous injection at the neck area of the rats) which induced spontaneous contralateral rotations. They were acclimatized in the transparent cylinders for 5 min before the test. Then the number of contralateral rotation was countered for the first 5 min after injection.

2.7. Immunofluorescent staining After paraffin embedding, the tissues were routinely sliced into 5 mm sections. Slides with mounted sections are placed in 37
C heater overnight at room temperature. For 6-OHDA-lesioned rat models, three SNpc sections from Bregma 4.5, 5.0 and 5.5 mm were used for TH positive cells counting. For MPTP-treated mice, three SNpc sections from Bregma 3.0, 3.3 and 3.6 mm and one striatal section from Bregma þ1.0 mm were used for TH and p-TrkB immunofluorescent staining (n ¼ 4). Then all slides underwent deparaffinization with xylene and dehydration with ethanol at different concentration, and then rinsed in distilled water. After

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peroxidase labeled streptavidin fluid at 37
C for 30 min, followed by DAB solutions for 5 min, counterstained with Harris hematoxylin for 3 min, dehydrated, and eventually cover slipped. Sections were digitally captured through an Olympus camera connected to the microscope under the same light intensity, and analyzed using the software Imagine J by an independent experimenter blind to the sections. Then the optical density of TH immunohistochemistry of the lesioned striatum was compared and positive p-TrkB immunoreactive cells were counted by the image J software. Firstly, Immunohistochemistry images were also changed into 8-bit type (gray) but without invertion, which is different from the analysis of immunofluorescent image. Then the next step is the same as the above. Briefly, the images were converted to a binary. Threshold the image using the automated routine. Number of positive neurons and the optical density was calculated using the “analyze particles” or “measure” plugin of Image J. 2.9. Brain protein extraction and western blot analysis After the evaluation of animal behavioral tests, the rats (n ¼ 4) and mouse (n ¼ 4) were decapitated for the ventral midbrain containing the substantia nigra followed the methods of Serge Przedborski (Jackson-Lewis and Przedborski, 2007). Store the brain tissues at 80
C until analysis. Brain protein was extracted using a commercial protein extraction kit (P0028, Beyotime Company, China). Briefly, SN tissues were homogenized in RIPA lysis buffer containing protease inhibitor PMSF (PMSF:RIPA ¼ 1:99), cocktail (Roche, Canada) and phosphatase inhibitor PhosStop (Roche, Canada) on ice, and centrifuged for 15 min at 12,000 g, 4
C. The supernatant was collected and measured for the protein concentration using a BCA Protein Assay Kit (P0012, Beyotime Company, China). Then the supernatant was boiled in 10X SDS loading buffer with the proportion of 4:1. A total of 40 mg protein of each sample were loaded onto SDS-PAGE. After SDS-PAGE, the protein samples were transferred to a PVDF membrane. The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and incubated with a variety of primary antibodies overnight at 4
C: Rabbit polyclonal tyrosine hydroxylase antibody (1:800, Gene Tex), rabbit monoclonal anti-BDNF (1:1000, Abcam), rabbit polyclonal anti-TrkB (1:2000, Abcam) and rabbit polyclonal anti-phospho-TrkB (Y816) (1:1000, Abcam), rabbit monoclonal anti-p44/42 MAPK (Erk1/2) (1:2000, Cell Signaling), rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (1:2000, Cell Signaling), rabbit monoclonal anti-Akt (pan) (1:1000, Cell Signaling), rabbit monoclonal anti-phospho-Akt (Ser473) (D9E)XP (1:2000, Cell Signaling), rabbit polyclonal caspase 3 Antibody (1:1000, Cell Signaling), rabbit monoclonal Bcl-xl (54H6) Antibody (1:1000, Cell Signaling), and mouse monoclonal anti-b actin (8H10D10) (1:1000, Cell Signaling). Then, the membranes were washed with TBST for three times (8 min/times) and incubated with HRP-conjugated secondary anti-rabbit (1:5000, Cell Signaling) or anti-mouse antibody (1:4000, Cell Signaling) for 2 h at room temperature. After washing, ECL was loaded onto the membrane to detect the immunoreaction using the Bio-Rad imaging system. The branes were analyzed with Imagine J software. Relative band intensities were calculated as a ratio of the phosphorylated protein to total protein for TrkB, Erk1/2 and Akt. For TH, BDNF, caspase-3 and Bcl-xl, relative band intensities were measured with b-actin serving as internal control.

analyzed with one-way ANOVAs followed by Tukey's multiple comparison test (more than two groups). Significance level was set when P < 0.05. 3. Results 3.1. Deoxygedunin improves behavior performance in 6-OHDAlesioned rats The preclinical evidence supports that BDNF is useful as a therapeutic agent for various neurological disorders. To explore whether the small TrkB agonist deoxygedunin exhibits any therapeutic effect in PD, we employed 6-OHDA-lesioned PD rat model, and pretreated or post-treated the animals with 5 mg/kg of deoxygedunin. Two weeks after 6-OHDA-lesioned surgery, cylinder test was used to evaluate the use frequency of each limb in rats. Compared with pre-treatment and normal groups, the numbers of right hand contact in the control group was significantly reduced (20.63 ± 2.203 vs 10.0 ± 1.336, p ¼ 0.036; 22.50 ± 3.27 vs 10.0 ± 1.336, p ¼ 0.007), indicating impaired right (contralateral side) paw use (Fig. 1C). The number of both paw use in normal group was significantly more than other three groups (F ¼ 31.936, p ¼ 0.000). In addition to the normal group, number of right paw use in other three group was less than the number of left paw use (F ¼ 5.152, p ¼ 0.005). There was no statistical significance in the number of right paw use between the post-treatment and the control group (14.38 ± 2.397 vs 10.0 ± 1.336, p ¼ 0.645), but the number of right paw use in the pre-treatment group was more than post-treatment group and there was statistical significance in the number of right paw use between the pre-treatment and the control group, suggesting the protection of locomotion function with deoxygedunin pretreatment. Adjusting step test is one of the parameter that deeply affected by the extent of 6-OHDA-induced lesion in rats. As shown in Fig. 1D, in the forehand test, the rats in the control group exhibited damaged right forelimb (lesion contralateral side) motor function, which was significantly decreased compared to the normal group (5.25 ± 0.366 vs 9.10 ± 0.314, p ¼ 0.001) and the pre-treatment group (5.25 ± 0.366 vs 8.13 ± 0.350, p ¼ 0.030). Pretreatment with deoxygedunin for two weeks before 6-OHDA-lesioned surgery prevented this decrease, indicating reversion of right forelimb akinesia (p < 0.05). However, deoxygedunin did not exert the protective effect if administrated immediately after 6-OHDA lesion, which implies lacking of neuroprotective effects with posttreatment (6.75 ± 1.278 vs 5.25 ± 0.366, p ¼ 0.431). To further assess the neuroprotective effects of deoxygedunin on dopamine depletion in PD rat model, apomorphine-induced rotation was examined and the number of turning was showed in Fig. 1E. We found that striatum injection of 6-OHDA resulted in moderatespeed rotation to the contralateral direction in the control group. Notably, compared to the control group, treatment with deoxygedunin significantly reduced the number of contralateral rotation in the pre-treatment group and in the post-treatment group (45.38 ± 3.246 vs 27.88 ± 1.125, p ¼ 0.000; 45.38 ± 3.246 vs 36.13.0 ± 0.895, p ¼ 0.003), suggesting that this compound could protect the denervation of dopaminergic pathways. What's more, there was statistical significance in the number of contralateral rotation between the pre-treatment and the post-treatment group (27.88 ± 1.125 vs 36.13.0 ± 0.895, p ¼ 0.010), suggesting the neuroprotection of locomotion function with deoxygedunin pretreatment.

2.10. Statistic analysis Data are presented as mean ± SEM. Statistical analysis was carried out by SPSS20 software. Student's t-tests were used when only two groups were compared. Statistical evaluation was

3.2. Deoxygedunin protects dopaminergic neurons in 6-OHDAlesioned rat model After the behavioral test battery, the integrity of dopaminergic

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neurons in the SNpc and their terminal fibers in striatum were evaluated by immunofluorescent and immunohistochemistry with dopaminergic neuron specific marker, anti-TH antibody. We found that there were fewer TH positive neurons in the similar anatomic level sections of SN in the control rats than deoxygedunin-treated rats and normal rats (Fig. 2A and B). This observation was further quantified with cell counting in a double blind way and analyzed by western blotting of the SN lysates (Fig. 2C and D). The cell counting results indicated a statistically significant preservation of TH positive dopaminergic SN neurons in the deoxygedunin pretreatment and post-treatment groups compared with control group (p < 0.01) (Fig. 2A). The western blotting of TH in SN further confirmed the findings. The ratio of TH/b-action in pretreatment group was significantly higher than control and post-treatment groups (p < 0.05). Meanwhile, there was statistical significance in the expression levels of tyrosine hydroxylase between the pretreatment and the post-treatment group, indicating the neuroprotection of pretreatment with deoxygedunin (p < 0.05) (Fig. 2C

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and D). The striatum injection of 6-OHDA also resulted in decrease of the TH positive DA terminals in the striatal region (Section on bregma þ2.5) of the control and post-lesioned treatment rats (Fig. 2E and F). Hence, the pretreatment with deoxygedunin resulted in noticeable protective effects on the striatal TH staining. 3.3. Deoxygedunin activates TrkB signalings The activation of TrkB through its autophosphorylation likely contributed to its neuroprotective effects. SN dopaminergic neurons express BDNF specific receptor TrkB and the neuroprotective effects of deoxygedunin may result from its TrkB agonistic activity on these neurons, it is important to demonstrate whether treatment with deoxygedunin could activate TrkB. To explore whether pretreatment with deoxygedunin could indeed activate TrkB, we intraperitoneally injected the rats with deoxygedunin or isovolumetric vehicle beginning at two weeks before 6-OHDAlesioned surgery. Then we performed immunohistochemistry to

Fig. 2. Tyrosine hydroxylase immunostaining of substantia nigra (SN) and striatum in 6-OHDA lesioned rat model reveal that deoxygedunin protected dopaminergic neurons. (A) Tyrosine hydroxylase immunofluorescent of substantia nigra pars compacta (SNpc). Scale bar ¼ 100 mm. We found that there were fewer TH positive neurons in the similar anatomic level sections of SN in the control rats than deoxygedunin-treated rats and normal rats. Compared to the post-treatment group, pre-treatment with deoxygedunin significantly attenuated the loss of dopaminergic neurons in SNpc (A and B). The western blotting of TH in SN further reconfirmed the findings (C and D). *p < 0.05, **p < 0.01, significant difference between the two indicated groups. The striatum injection of 6-OHDA also resulted in decrease of the TH positive DA terminals in the striatal region (Section on bregma þ2.5) of the control and post-lesioned treatment rats (E and F). Scale bar ¼ 1000 mm (up)/100 mm (down). Data were presented as mean ± SEM. n ¼ 4.

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examine TrkB activation. Rat brain sections contained the SNpc regions were stained with p-TrkB (Y816) to assess TrkB activation. As shown in Fig. 3A, TrkB phosphorylation in SNpc regions was significantly elevated in the pretreatment group compared with control group (p < 0.01), and it was higher in the pretreatment group than in the post-treatment group (p < 0.05), indicating pretreatment with deoxygedunin activates TrkB markedly. But there was no statistical significance between the control group and post-treatment group (Fig. 3B). As expected in the analysis of western blotting, the TrkB receptors were more prominently phosphorylated in pre-treatment group than other three groups, as

were the downstream pathways involving MAPK and PI3K/Akt pathways, and there was no significant difference in the expression of BDNF in the SN between each group (Fig. 3C and E). The quantitative ratios of p-TrkB/TrkB, p-MAP/MAPK and p-Akt/Akt were summarized in Fig. 3H. Noticeably, this effect by deoxygedunin was independent of altering BDNF expression in the animals (Fig. 3G). As expected, the expression of Bcl-XL, a marker for cell survival, was evidently higher in deoxygedunin-treated groups than control group (Fig. 3D and F). Combining with the behavioral tests and Immunohistochemistry staining results, we concluded that pretreatment with deoxygedunin activates TrkB receptor and

Fig. 3. Deoxygedunin activates TrkB signaling pathways. TrkB phosphorylation in SNpc regions was significantly elevated in the pretreatment group compared with control group (p < 0.01), and it was higher in the pretreatment group than in the post-treatment group (p < 0.05), indicating pretreatment with deoxygedunin activated TrkB markedly (A and B). Scale bar ¼ 100 mm, *p < 0.05, **p < 0.01, compared with the two indicated groups, n ¼ 4. TrkB receptors were more phosphorylated in pre-treatment group than other three groups, as were the downstream pathways involving MAPK and PI3K/Akt pathways, and there was no significant difference in the expression of BDNF in the SN between each group (C and E). The expression of Bcl-XL was evidently higher in deoxygedunin-pretreated groups than control group (D and F). *p < 0.05, **p < 0.01, n ¼ 4, compared with the two indicated groups. Data were presented as mean ± SEM.

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downstream signaling pathways in a BDNF-independent manner. 3.4. Deoxygedunin is neuroprotective in MPTP-treated mice To further assess the neuroprotective effects of deoxygedunin in MPTP-induced neurotoxicity model in mice, pole test was used to assess the locomotion performance. Since pre-treatment with deoxygedunin revealed more prominent neuroprotective actions,

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we employed the pretreatment paradigm in the MPTP-induced PD model. We found that the weigh in MPTP-treated mice were much lower than normal group for the neurotoxicity of MPTP (p < 0.05), but pre-treatment with deoxygedunin ameliorated the loss of weight (Fig. 4A). MPTP treated mice without deoxygedunin treatment had longer time to orient downward (Tturn) and to descend (TLA) than normal animals, indicating impaired motor function (p < 0.05). Pretreatment with deoxygedunin (Fig. 4B) decreased

Fig. 4. The weigh of MPTP-treated mice were much lower than normal group due to the neurotoxicity of MPTP (p < 0.05), but pre-treatment with deoxygedunin ameliorated the loss of weight (*p < 0.05, **p < 0.01), n ¼ 10. Effects of deoxygedunin on pole test in MPTP-treated and normal mice. Pretreatment with deoxygedunin decreased Tturn and TLA compared to the MPTP group (B). *p < 0.05, significant difference between the two indicated groups (n ¼ 10). The numbers of TH positive neurons were much fewer in MPTP treated mice than in normal and deoxygedunin group, scale bar ¼ 100 mm (C). Positive cell counting revealed that the number of TH positive neurons in MPTP group was significantly reduced compared with normal control and deoxygedunin group (p < 0.05) (D). Immunoblotting also validated this observation (E and F). The MPTP neurotoxicity also resulted in decrease of the TH positive DA terminals in the striatal region of the control group compared with normal group (p < 0.05). Scale bar ¼ 500 mm (up)/100 mm (down) (G and H). *p < 0.05, **p < 0.01, n ¼ 4, compared with the two indicated groups. Data were presented as mean ± SEM.

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Tturn and TLA compared to the MPTP group (p < 0.05). After the pole test, TH immunostaining was performed to evaluate MPTP induced neurotoxicity on SN dopaminergic neurons. As shown in Fig. 4C, the numbers of TH positive neurons were much fewer in MPTP treated mice than in normal and deoxygedunin group. Positive cell counting revealed that the number of TH positive neurons in MPTP group was significantly reduced compared with normal control and deoxygedunin group (p < 0.05) (Fig. 4D). Immunoblotting also validated this observation (Fig. 4E and F). The MPTP neurotoxicity also resulted in decrease of the TH positive DA terminals in the striatal region of the control group compared with normal group (p < 0.05). The mean optical density of striatal TH immunofluorescent staining in the deoxygedunin group was higher than MPTP group, but there was no statistical difference (Fig. 4G and H). The probable underlying mechanism is due to the duration of MPTP injection is merely 7 days. Combining with the above behavioral tests and results of Immunohistochemistry staining, pretreatment with deoxygedunin could protect MPTP-induced neurotoxicity on DA neurons in PD mouse model.

3.5. Deoxygedunin activates TrkB receptors and signal cascades in MPTP-treated mice To explore the mechanism of the neuroprotective effects of deoxygedunin on MPTP-induced neurotoxicity model in mice, we conducted immunofluorescent staining and western blotting analysis to examine the activation of TrkB receptor and its downstream signaling pathways. As expected, we found the coexpression of TH and phospho-TrkB (Y816) in the substantia nigra of the MPTP neurotoxicity mouse (Fig. 5A). The mean optical density of co-expression cells is higher in the deoxygedunin group than MPTP group (p < 0.05) (Fig. 5B). We also discovered that the TrkB receptor was more prominently phosphorylated in deoxygedunin group than MPTP and normal groups, so were the downstream pathways involving MAPK and PI3K/Akt pathways (Fig. 5C and D), and there was no significant difference in the expression of BDNF in the SN between each group (Fig. 5E and F). As expected, we found that deoxygedunin protected neurons from apoptosis by upregulating the Bcl-xl expression and suppressing the activation of

Fig. 5. Co-expression of TH and phospho-TrkB (Y816) in the substantia nigra of the MPTP neurotoxicity mouse. Scale bar ¼ 50 mm (A). The mean optical density of co-expression cells is higher in the deoxygedunin group than MPTP group (p < 0.05) (B). TrkB receptor was more prominently phosphorylated in deoxygedunin group than MPTP and normal groups, so were the downstream pathways involving MAPK and PI3K/Akt pathways (C and D). There was no significant difference in the expression of BDNF in the SN between each group (E and F). Deoxygedunin protected neurons from apoptosis by up-regulating the Bcl-xl expression and suppressing the activation of caspase-3 (C, G and H). *p < 0.05, **p < 0.01, significant difference between the two indicated groups. Data were presented as mean ± SEM. n ¼ 4.

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caspase 3 (Fig. 5C, G and H). Together, our findings support that pretreatment with deoxygedunin activates TrkB receptor and downstream signaling pathways and demonstrates robust neuroprotective activity in MPTP-treated mice. 4. Discussion In the current study, we demonstrated that deoxygedunin, as a potent TrkB agonist, displays remarkable neuroprotective effects in 6-OHDA-lesioned rat model and MPTP-lesioned mouse model of PD. Long-term treatment with deoxygedunin attenuated dopaminergic neurons loss and activated TrkB signaling pathways in SN, which was probably associated with its neuroprotective property. Employing TrkB knockout mice and TrkB knockin mutant mice, we demonstrated that deoxygedunin exerts its neuroprotective activity is TrkB-dependent upon oral administration. Moreover, it triggers TrkB receptor activation in a BDNF-independent way (Jang et al., 2010b). In this study, we show that deoxygedunin displays remarkably neuroprotective activity in two well-established PD models presumably via activating TrkB receptors. Noticeably, deoxygedunin pretreatment exhibits more stable and constant neuroprotective activity than post-treatment paradigm (Figs. 2 and 3), indicating that deoxygedunin might possess more prominent protective than therapeutic actions. On the other hand, it remains unclear of the brain exposure of deoxygedunin. To translate this promising small molecule into a clinical useful pharmacological agent, certainly, more work is necessary in determining its in vivo pharmacokinetic profiles and oral bioavailability. BDNF is one of the key molecules to regulate neuronal survival, growth, differentiation and function in the nervous system (Hyman et al., 1991). BDNF exerts its neuroprotective effects through activating its specific receptor TrkB, stimulating cell proliferation pathways, such as PI-3-kinase and MAPK, and inhibiting apoptosis signaling pathways. Deficiency of the BDNF/TrkB signaling pathway has been believed to play an important role in the occurrence and development of PD (Murer et al., 2001). However, recombinant BDNF failed to show demonstrable benefit to patients with amyotrophic lateral sclerosis in a large scale phase III trial, for its in vivo instability and poor pharmacokinetics (Fletcher et al., 2008). As a potent small molecule TrkB agonist, deoxygedunin overcomes the insurmountable intrinsic problem associated with proteins like BDNF, and demonstrates the profound neuroprotective effects (Jang et al., 2010b). Deoxygedunin directly binds TrkB receptors and provokes its dimerization. In contrast, it doesn't bind to TrkA at all, indicating the selectivity and affinity between the TrkB receptor and deoxygedunin. In our previous study, the protective effects of deoxygedunin on cortical neurons are completely reversed by the pretreatment of TrkB receptor antagonist, K252a. Meanwhile, deoxygedunin-provoked downstream Akt signaling also reduced by K252a antagonist. 1NMPP1, a TrkB F616A inhibitor, was also discovered to greatly diminish dexoygedunin's protective effect in TrkB F616A knock-in mice (Jang et al., 2010a). Thus, deoxygedunin selectively activates TrkB but not TrkA or TrkC in a BDNFindependent manner, and prevents neuronal apoptosis in a TrkBdependent manner. Gedunin, a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), has demonstrated the ability to exhibit antimalarial, insecticidal, and most recently anticancer activity (Khalid et al., 1989; Uddin et al., 2007). The antitumor activity of gedunin was explored through the use of the connectivity map (Lamb et al., 2006). Lamb et al. found, via high connectivity scores with GDA, 17-AAG, and 17-DMAG, that gedunin exhibited its antiproliferative activity through Hsp90 modulation. Gedunin was recently shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding machinery and to induce

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the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors. Currently, there are no reports about neuronal protective role by gedunins. Glutamate and kainic acid (KA) einduced excitotoxicity is the common cause of the neuronal cell death in neurodegenerative disease (Chase and Oh, 2000; Sun et al., 1992). In our previous study, gedunin derivatives suppress glutamate-induced or OGD-triggered neuronal apoptosis in a doesdependent manner with deoxygedunin revealing a more potent protective effect. Meanwhile, neurotoxicity initiated by KA can also be blocked by deoxygedunin in vitro and in vivo (Jang et al., 2010a). Deoxygedunin also reveals potent therapeutic effect in an animal model of depression as a potent TrkB agonist. Moreover, it protects vestibular ganglion from degeneration in BDNF / mice. Thus, deoxygedunin mimics BDNF and possesses robust neurotrophic activities, and it is a promising lead compound for antidepressant drug development. In the current study, we show that deoxygedunin also exhibits profound neuroprotective actions in two different PD models. Deoxygedunin protects the loss of DA neurons induced by 6-OHDA and MPTP in rodents. It also improved the behavior performances on PD models. Long-term use of deoxygedunin activates the TrkB, which is a potential mechanism underlying its neuroprotective effects. Other TrkB agonists or drugs including BDNF, N-acetylserotonin (NAS) derivatives such as HIOC, gedunin derivatives except deoxygedunin, 7,8-dihydroxyflavone (7,8-DHF) and so on, have been widely investigated in the neurodegenerative disease (Alzheimer's disease and amyotrophic lateral sclerosis), stroke, depression, and so on (Jang et al., 2010a, 2010b; Liu et al., 2014; Wang et al., 2014; Zhang et al., 2014). HIOC derived from NAS is much more stable than NAS and it exhibits a much stronger effect in provoking TrkB activation in mouse brain and tetinas. Deoxygedunin and 7,8-DHF both display comparable agonistic activity on TrkB receptor and mimic BDNF's neuroprotective effect in animal models. For example, in a forced swim test, both 7,8-DHF and dexoygedunin (5 mg/kg) remarkably reduced the immobility with deoxygedunin revealing a more potent antidepressant effect, and this effect can be blocked by 1NMPP1, a TrkB F616A inhibitor (Jang et al., 2010a). 7,8DHF and NAS share the same binding pocket on TrkB ECD, and it is different from the motif on TrkB ECD of dexoygedunin. Presumably, that's the underlying mechanism that these TrkB agonists exert different neuroprotective and antidepressant actions. Collectively, our study provides not only a novel tool to explore the molecular mechanism of BDNF/TrkB system in PD but also an innovative therapeutic agent for treating the neurodegenerative diseases including PD. Neuroprotective effects of deoxygedunin on the two classical acute or subacute damage models of PD have been investigated in the present study, however the neurotrophic activities of dexoygedunin on other animal models such as rotenone models for Parkinson's disease deserves further study. 5. Conclusion Our study indicated that deoxygedunin as a potent TrkB agonist, displays remarkable neuroprotective effects in 6-OHDA-lesioned rat model and MPTP-lesioned mouse model of PD in a BDNFindependent way, providing a novel therapeutic strategy for treating Parkinson's disease. Conflict of interest All of the authors declare no conflict of interest. Acknowledgments This research was supported by the National Natural Science

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