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7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents
Accepted Manuscript Title: 7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents Author: Dandan Luo Ying Shi Jun Wang Qing Lin Yi Sun Keqiang Ye Qiao Yan Hai Zhang PII: DOI: Reference:
S0304-3940(16)30180-X http://dx.doi.org/doi:10.1016/j.neulet.2016.03.042 NSL 31935
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Neuroscience Letters
Received date: Revised date: Accepted date:
6-2-2016 21-3-2016 23-3-2016
Please cite this article as: Dandan Luo, Ying Shi, Jun Wang, Qing Lin, Yi Sun, Keqiang Ye, Qiao Yan, Hai Zhang, 7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents Dandan Luoa, Ying Shib, Jun Wangb, Qing Linb, Yi Suna,c, Keqiang Yed, Qiao Yana,*, Hai Zhangb,1,** a
Stem Cell Translational Research Center, TongJi Hospital, TongJi University School of Medicine, ShangHai
200065, China b
Department of Biology, Sundia MediTech Company, Ltd. 388 Jialilve Road, Zhangjiang Hightech Park,
Shanghai 201203, China c
Neuropsychiatric Institute, Medical Retardation Research Center, University of California, Los Angeles, Los
Angeles, CA 90095, USA d
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA
30322, USA Corresponding authors with postal and email addresses: * Qiao Yan: Stem Cell Translational Research Center, TongJi Hospital, TongJi University School of Medicine, ShangHai 200065, China. Email: [email protected] 1,** Hai Zhang: 1 Present address: Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Email: [email protected] Highlights · 7,8-DHF alleviated akinesia of 6-OHDA rat model of parkinsonism by protecting dopaminergic neurons · 7,8-DHF elevated TrkB phosphorylation in the SN in 6-OHDA rat model · 7,8-DHF protected acute MPTP neurotoxicity in mice
Abstract Brain-derived neurotrophic factor (BDNF) is a notably important neurotrophin which regulates neuronal survival and differentiation in the nervous system. However, its clinical usage is particularly limited. 7,8-dihydroxyflavone (7,8-DHF), which acts as a selective agonist of BDNF receptor TrkB, is reported to possess neuroprotective effects both in vitro and in vivo. Here we explored the potent neuroprotective effects of 7,8-DHF in 6-OHDA induced rat and MPTP induced mouse model of Parkinsonism. The results demonstrated that treatment with 7,8-DHF in drinking water for four weeks (two weeks before 6-OHDA + two weeks after 6-OHDA lesion) significantly improved dopamine-mediated behaviors in 6-OHDA rat model, and prevented the loss of dopaminergic neurons in the substantia nigra (SN). Phospho-Y816-TrkB immunostaining showed that TrkB phosphorylation was significantly elevated in the SN in 7,8-DHF pretreated group, indicating 7,8-DHF activated TrkB and likely contributed to its neuroprotective effects. 7,8-DHF also protected acute MPTP neurotoxicity in mice but did not affect the climbing behavior in pole test. Thus our study indicates the neuroprotective properties of 7,8-DHF through the activation of TrkB, which provides a novel therapeutic treatment for Parkinson’s disease. Key Words: 7,8-dihydroxyflavone; Neuroprotection; Brain derived neurotrophic factor; 6-hydroxydopamine; MPTP; Parkinson’s disease.
1
1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disease in aged populations and is characterized by gradual degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) [1]. Currently the main drug therapy for PD is levodopa. However, levodopa cannot prevent the progress of neurodegeneration, thus neuroprotective therapies have been focused in recent decades. Brain derived neurotrophic factor (BDNF) is a notably important neurotrophin which activates its receptor tyrosine kinase receptor B (TrkB), leading to the autophosphorylation of TrkB and activation of downstream signalings such as MAPK, PI3K and PLC [2]. BDNF promotes the survival of dopaminergic neurons [3], and is necessary for the development of DA neurons in the SN [4]. In animal models of PD, BDNF prevents the degeneration of dopaminergic neurons of the SN and improves motor function [5]. In PD patients, the expression of BDNF is reduced in the nigrostriatum and the serum [6, 7]. Moreover, the serum level of BNDF is strongly correlated with cognitive performance in PD patients [8], indicating possible association of BDNF with the survival of dopaminergic neurons and the pathology of PD. Because of the remarkable neurotrophic actions of BDNF on neurons, it has been involved in particular therapeutic interest in neurodegenerative diseases. However, the clinical trials using recombinant BDNF have been disappointingly negative [9], presumably because of poor delivery and short half-life. Small molecules have been developed to stimulate BDNF expression or activate its receptor, which were expected to display neuroprotective potencies [10, 11]. Recently, it was reported that 7,8-dihydroxyflavone (7,8-DHF) acted as a potent TrkB agonist, which could penetrate the blood brain barrier [12], therefore trigged TrkB phosphorylation [13, 14] and exerted protective effects both in cytotoxic models [15, 16] and several neurodegenerative animal models [17, 18]. In the current study, we explored the potential neuroprotective effects of 7,8-DHF in rat 6-hydroxydopamine (6-OHDA) model and mouse 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) model using behavioral and histological analyses. And the possible mechanism underlying the effects of 7,8-DHF via TrkB was further studied. 2. Materials and Methods 2.1. Animals Male Sprague-Dawley rats (200-250g) and male C57/BL6 mice (8-9 weeks old) were purchased from SLAC laboratory animal center (Shanghai, China) and housed at 22-24℃ facility with 12h light/dark cycle. Food and water were provided ad libitum. All the experimental protocols were carried in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Institutional Animal Care and Use Committee. 2.2. Materials Apomorphine hydrochloride, 6-OHDA hydrobromide and MPTP were ordered from Sigma (St. Louis, MO 63130); 7,8-DHF was purchased from TCI (Tokyo, Japan); Rabbit anti-tyrosine hydroxylase (TH) antibody was ordered from Millipore (Billerica, MA 01821). Rabbit anti-phospho (Y816)-TrkB was ordered from Abcam (Cambridge, MA 02139); Biotinylated secondary antibody, HRP-conjugated streptavidin and 3,3′-Diaminobenzidine (DAB) were products of Jackson ImmunoResearch (West Grove, PA 19390). 2.3. 6-OHDA rat model and DHF treatment Rats were anesthetized with 60 mg/kg sodium pentobarbital for stereotaxic surgery. 6-OHDA was dissolved in artificial cerebral spinal fluid and was injected into two sites of the left striatum: AP +0.5, L -2.5, DV -5.0; AP -0.5, L -4.0, DV -5.0 (mm from Bregma). Each site received 10μg/2μl 6-OHDA. 7,8-DHF was dissolved in drinking water at the concentration of 160mg/L by gradually dropping 1M NaOH into the water 2
and the final pH was 7.4-7.6. The volume of water that rats drank slightly varied each day, so the dose they received was 12~16mg/kg per day. The study involved two different schedules of DHF treatment (Fig. 1A): a. Pretreatment (Pre) - 7,8-DHF treatment for 14 days before 6-OHDA lesion and for another 14 days after lesion; b. Post-treatment (Post) - 7,8-DHF treatment for 14 days only after lesion. Control group was 6-OHDA lesioned and drank regular water. 2.4. Behavioral tests in rat model Two weeks after 6-OHDA lesion, the akinesia behaviors were assessed with the following tests by experimenters blinded to the treatment design. 2.4.1. Cylinder test Rats were placed in a transparent cylinder (22cm in diameter, 30cm in height), and the number of wall contact for left, right or both forelimbs was counted respectively until the total number reached 20. The motor performance for each limb was expressed as the percent use of the limb relative to the total number. 2.4.2. Apomorphine induced rotation Rats were placed in a round enclosure with 40cm in diameter. They were injected subcutaneously with 0.25mg/kg apomorphine and waited for 5min. Then the number of contralateral turning was counted manually for another 5min. 2.4.3. Adjusting step test Rats were held by the experimenter with the hindlimbs gently fixed and slightly elevated. It was moved slowly sideways (90cm of distance in 5s) with one of its forepaws touching the table and the other fixed, first in the forehand direction, then in the backhand direction. The number of adjusting steps for each forelimb on both directions was recorded respectively. 2.5. Acute MPTP mouse model 7,8-DHF water was prepared as described above. Mice were given 7,8-DHF or regular water continuously for 14 days. On the 7th day, they were injected intraperitoneally with 20mg/kg MPTP twice at 2h apart. Pole test was performed on day 15. Mice were placed with the head upward on the top of a vertical pole with 1.2cm in diameter and 50cm long, and the base of the pole was fixed in the animal’s home cage. The time spent to orient downward (Tturn) and the total time spent to descend (TLA) was recorded. Each mouse was tested for five successive trials to get the average. 2.6. Immunohistochemistry staining After the completion of behavioral tests, the animals were perfused with 4% paraformaldehyde (in 0.01M PBS, pH 7.4). The brains were removed and further fixed overnight and then transferred to 30% sucrose solution till sunk. The brains were cut into 30μm coronal sections on a cryostat microtome. The sections were incubated with TH antibody (1:1000) overnight at 4℃ for the detection of dopaminergic neurons in the SN and DAergic terminals in the striatum; or with anti-phospho(Y816)-TrkB (1:1000) antibody for the detection of TrkB activation in the SN, followed by biotinylated secondary antibody and HRP-conjugated streptavidin. The sections were developed with DAB and photographed with an Olympus DP72 camera connected to BX51 microscope. 2.7. SN cell counting For each rat, three SN sections at Bregma -4.8, -5.3 and -5.8 mm were used for TH+ or p-TrkB+ cell counting. For mice, we stained three SN sections at Bregma -3.0, -3.3 and -3.6 mm for each animal. Cell counting was conducted by an experimenter who did not know the treatment condition, and the result for each animal was the summation of the number from its three sections. 2.8. Statistic analysis All data were expressed as mean ± SEM and analyzed by the software GraphPad Prism. Two-way ANOVA followed by post-hoc t-test was performed for cylinder test and adjusting step test; other data were 3
analyzed with one-way ANOVA followed by Tukey test. Significant level was set at p<0.05. 3. Results 3.1. 7.8-DHF improved the behavioral performance in rat 6-OHDA model Rats were treated as described in Fig. 1A and the akinesia symptoms were assessed with a series of behavioral tests. As shown in Fig. 1B, a two-way ANOVA on forepaw use in cylinder test revealed a main effect of the side of forepaw (F(2,78)=36.02, p<0.0001) and treatment (F(2,78)=3.989, p=0.0258), and a significant interaction (F(4,78)=3.91, p=0.0093). The right forepaw use was significantly decreased for both control (p=0.0032) and Post (p=0.0455) groups compared to their left side. And pretreatment with 7,8-DHF significantly improved the right forepaw use compared with control group (p=0.0243), indicating pretreatment with 7,8-DHF protected 6-OHDA-impaired forepaw use. To assess the extent of dopamine depletion, apomorphine induced contralateral rotation was conducted. As shown in Fig. 1C, there was a significant effect of treatment on the number of rotation (F(2,63)=3.617, p=0.0344). It was significantly declined for Pre (p=0.0115) but not Post group, suggested protection of DA neurons by 7,8-DHF pretreatment. For adjusting step test, a two-way ANOVA on forelimb step revealed a main effect of the side of forepaw (F(2,78)=16.02, p<0.001) and treatment (F(2,78)=9.17, p=0.0138), but not interaction (F(4,78)=3.72, ns). The right forepaw stepping was significantly damaged for both control (p=0.0048) and Post (p=0.0086) groups compared to their left side. Post hoc analysis revealed that the dysfunction was significantly rescued by 7,8-DHF pretreatment (Fig. 1D, p=0.0084). None of the rats showed notable step deficit in the backhand direction test (Fig. 1E). 3.2. 7,8-DHF protected 6-OHDA induced DA neurons loss in rats After the completion of behavioral tests, brain sections of the SN and the striatum were immunohistochemically stained with TH antibody. As demonstrated in Fig. 2, 7,8-DHF pretreatment while not post-treatment significantly preserved DA neurons in the SN compared with control and was confirmed by further quantification data (Fig. 2D, F(2,26)=4.851, p=0.0162). Meanwhile, TH staining of the striatum also revealed that 6-OHDA-induced DA terminals depletion in the striatum was rescued by pretreatment but not post-treatment with 7,8-DHF (Fig. 2E-G). 3.3. TrkB phosphorylation was elevated by 7,8-DHF pretreatment Since 7,8-DHF acts as a TrkB agonist, we immunostained the SN sections with p-Y816-TrkB antibody to determine if the treatment could activate TrkB in rat 6-OHDA model (Fig. 3). TrkB phosphorylation was significantly increased by 7,8-DHF pretreatment compared with control (Fig. 3D, F(2,26)=4.625, p=0.0191), but not in Post group, indicating activating of TrkB by the pretreatment of 7,8-DHF. 3.4. Neuroprotective effects of 7,8-DHF in acute MPTP mouse model Previous studies have reported that 7,8-DHF protected DA neurons in acute MPTP mouse model [13], and prevented DA neurons loss and restored motor deficit in a progressive MPTP model [19], here we also wanted to know if the oral treatment with 7,8-DHF in the present study could improve the behavioral performance in acute MPTP model. Two doses of MPTP injection induced a 55% and significant decrease of the number of TH+ neurons in the SN compared with normal group (Fig. 4). Similar with the reported results, 7,8-DHF significantly preserved the TH+ cells (Fig. 4D, F(2,14)=4.77, p=0.0265), confirmed a neuroprotective effect on MPTP induced neurotoxicity. However, we did not observe any significant behavior impairment in 4
pole test (Fig. 4E, p=0.21 for Tturn and p=0.37 for TLA). This was probably due to limited dopaminergic degeneration by acute MPTP, which was insufficient to induce motor damage. 4. Discussion In the present study, we found that the TrkB agonist 7,8-DHF protected 6-OHDA and MPTP induced dopamine neurons degeneration in rodents. Pretreatment with 7,8-DHF improved the behavior performance significantly in 6-OHDA rat model. Furthermore, chronic treatment with 7,8-DHF induced TrkB phosphorylation in the SN of rats, indicating the activation of TrkB, which probably contributed to its neuroprotective actions. Flavonoids are a class of polyphenol compounds that are widely distributed in the plant. 7,8-DHF is a bioactive high-affinity TrkB agonist which provokes TrkB dimerization, autophosphorylation and activation of down-stream MAPK signaling [13]. 7,8-DHF has been demonstrated to have various biological effects, including anti-oxidative effects [15] and neuro-regenerating effects in traumatic neuro-injury models [20, 21]. Furthermore, 7,8-DHF displayed antidepressant effects [22] and potentiated spatial memory through TrkB pathways [17]. These findings provide the possibility that 7,8-DHF would play multiple functions in the central nervous system. Many studies have demonstrated the potential link of BDNF with the pathophysiology of PD. In PD patients, the serum level of BDNF was significantly decreased [7], which has been reported to strongly correlate with the cognitive impairment of PD patients [8]. And BDNF Val66Met, a functional polymorphism, was also reported to be associated with cognitive impairment [23]. Moreover, the disturbance of TrkB signaling induced the deposit of α-synuclein, the hallmark of PD, and led to the loss of TH positive cells in the SN [24]. Thus the dysfunction of BDNF/TrkB signaling plays an important role in the development of PD. Due to the neuroprotective activity, BDNF was heavily studied in the nigrostriatal dopaminergic system. Transplantation of BDNF-secreting cells has been published to rescue the neuron injury and promote the motor function in rodent PD models [25, 26]. In addition, some potent neuroprotective strategies, such as monoamine oxidase inhibitors, also increased the expression of BNDF in cultured cells, PD models [27], as well as in PD patients [28]. Exercise likewise has been demonstrated to be an effective way to improve the expression of BDNF, which provided benefits to PD models and patients [29, 30]. Nevertheless, the clinical trials for neurotrophins have been disappointing due to the poor permeability through the blood brain barrier and a short serum half-life. Thus as a small molecule TrkB agonist, 7,8-DHF has a better chance to mimic the profound neuroprotective effects of BDNF in a translational point of view. In our current study, pretreatment with 7,8-DHF preserved DA-mediated behaviors in rat 6-OHDA model and improved the expression of phospho-TrkB in the SN. These results indicated a correlation between TrkB activation and the neuroprotective effects of 7,8-DHF. However, since there were greater neurons survived in DHF pretreatment group, the elevated expression of phospho-TrkB may also be due to the preserved cell quantity. Future studies, such as the detecting of the TrkB downstream signalings and using of transgenic tools, could be performed to further confirm the molecular mechanisms of the neuroprotective effects of 7,8-DHF. 5. Conclusion The TrkB agonist 7,8-DHF protects the loss of DA neurons induced by 6-OHDA and MPTP in rodents. Pretreatment with 7,8-DHF improves the behavioral performances in rat 6-OHDA model, which is probably 5
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Figure legends Figure 1. Effects of 7,8-DHF on locomotor function in 6-OHDA model. A. Experimental design and 7,8-DHF treatment schedule. B. Cylinder test. 6-OHDA significantly reduced the right forepaw use in rats, while it was improved by 7,8-DHF pretreatment (* p=0.0243 compared with the right side of control group). n=9-10. C. Apomorphine induced rotation. The number of contralateral rotation for pretreatment group was significantly decreased compared with control. n=22. D & E. Adjusting step test. 6-OHDA damaged the right forelimb stepping compared with its left side in the forehand direction. 7,8-DHF pretreatment significantly reversed the right forelimb dysfunction (* p=0.0084 compared with the right side of control group). n=9-10. None of the rats showed deficit in the backhand direction. Data were expressed as mean ± SEM.
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Figure 2. TH staining of the SN and striatum of 6-OHDA model. Rats were treated as described in Fig. 1A. Brain sections containing the SN (A-C) and striatum (E-G) were immunostained with TH antibody. The cell counting data of the SN revealed that 7,8-DHF pretreatment prevented 6-OHDA-induced DA neurons loss in the left SN (D). n=9-10. Scale bar in 2C = 200μm and in 2G = 1mm. Arrows in E-F directed the lesioned area of the striatum.
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Figure 3. Phosphorylated-TrkB immunostaining of the SN. Rats were treated as described in Fig. 1A. SN sections were immunostained with phosphor-Y816-TrkB antibody. Representative sections of the left SN were shown for each group. A: control; B: post-treatment; C: pretreatment. Scale bar = 100μm. Data were expressed as mean ± SEM. n=9-10. The result revealed that TrkB phosphorylation was significantly increased in 7,8-DHF pretreated animals.
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Figure 4. Neuroprotective effects of 7,8-DHF in acute MPTP mouse model. Mice were treated as described in the Methods section 2.5. Pole test was performed and the SN sections were stained with TH antibody. A: normal; B: MPTP + regular water; C: MPTP + 7,8-DHF. D: MPTP significantly degenerated the DA neurons of the SN, which was rescued by 7,8-DHF pretreatment. Data were expressed as mean ± SEM. n=5-6. Scale bar = 100 μm. E. The time to orient downward (Tturn) and total time to descend (TLA) in pole test. There was no significant difference between groups.
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S0304-3940(16)30180-X http://dx.doi.org/doi:10.1016/j.neulet.2016.03.042 NSL 31935
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
6-2-2016 21-3-2016 23-3-2016
Please cite this article as: Dandan Luo, Ying Shi, Jun Wang, Qing Lin, Yi Sun, Keqiang Ye, Qiao Yan, Hai Zhang, 7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
7,8-dihydroxyflavone protects 6-OHDA and MPTP induced dopaminergic neurons degeneration through activation of TrkB in rodents Dandan Luoa, Ying Shib, Jun Wangb, Qing Linb, Yi Suna,c, Keqiang Yed, Qiao Yana,*, Hai Zhangb,1,** a
Stem Cell Translational Research Center, TongJi Hospital, TongJi University School of Medicine, ShangHai
200065, China b
Department of Biology, Sundia MediTech Company, Ltd. 388 Jialilve Road, Zhangjiang Hightech Park,
Shanghai 201203, China c
Neuropsychiatric Institute, Medical Retardation Research Center, University of California, Los Angeles, Los
Angeles, CA 90095, USA d
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA
30322, USA Corresponding authors with postal and email addresses: * Qiao Yan: Stem Cell Translational Research Center, TongJi Hospital, TongJi University School of Medicine, ShangHai 200065, China. Email: [email protected] 1,** Hai Zhang: 1 Present address: Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Email: [email protected] Highlights · 7,8-DHF alleviated akinesia of 6-OHDA rat model of parkinsonism by protecting dopaminergic neurons · 7,8-DHF elevated TrkB phosphorylation in the SN in 6-OHDA rat model · 7,8-DHF protected acute MPTP neurotoxicity in mice
Abstract Brain-derived neurotrophic factor (BDNF) is a notably important neurotrophin which regulates neuronal survival and differentiation in the nervous system. However, its clinical usage is particularly limited. 7,8-dihydroxyflavone (7,8-DHF), which acts as a selective agonist of BDNF receptor TrkB, is reported to possess neuroprotective effects both in vitro and in vivo. Here we explored the potent neuroprotective effects of 7,8-DHF in 6-OHDA induced rat and MPTP induced mouse model of Parkinsonism. The results demonstrated that treatment with 7,8-DHF in drinking water for four weeks (two weeks before 6-OHDA + two weeks after 6-OHDA lesion) significantly improved dopamine-mediated behaviors in 6-OHDA rat model, and prevented the loss of dopaminergic neurons in the substantia nigra (SN). Phospho-Y816-TrkB immunostaining showed that TrkB phosphorylation was significantly elevated in the SN in 7,8-DHF pretreated group, indicating 7,8-DHF activated TrkB and likely contributed to its neuroprotective effects. 7,8-DHF also protected acute MPTP neurotoxicity in mice but did not affect the climbing behavior in pole test. Thus our study indicates the neuroprotective properties of 7,8-DHF through the activation of TrkB, which provides a novel therapeutic treatment for Parkinson’s disease. Key Words: 7,8-dihydroxyflavone; Neuroprotection; Brain derived neurotrophic factor; 6-hydroxydopamine; MPTP; Parkinson’s disease.
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1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disease in aged populations and is characterized by gradual degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) [1]. Currently the main drug therapy for PD is levodopa. However, levodopa cannot prevent the progress of neurodegeneration, thus neuroprotective therapies have been focused in recent decades. Brain derived neurotrophic factor (BDNF) is a notably important neurotrophin which activates its receptor tyrosine kinase receptor B (TrkB), leading to the autophosphorylation of TrkB and activation of downstream signalings such as MAPK, PI3K and PLC [2]. BDNF promotes the survival of dopaminergic neurons [3], and is necessary for the development of DA neurons in the SN [4]. In animal models of PD, BDNF prevents the degeneration of dopaminergic neurons of the SN and improves motor function [5]. In PD patients, the expression of BDNF is reduced in the nigrostriatum and the serum [6, 7]. Moreover, the serum level of BNDF is strongly correlated with cognitive performance in PD patients [8], indicating possible association of BDNF with the survival of dopaminergic neurons and the pathology of PD. Because of the remarkable neurotrophic actions of BDNF on neurons, it has been involved in particular therapeutic interest in neurodegenerative diseases. However, the clinical trials using recombinant BDNF have been disappointingly negative [9], presumably because of poor delivery and short half-life. Small molecules have been developed to stimulate BDNF expression or activate its receptor, which were expected to display neuroprotective potencies [10, 11]. Recently, it was reported that 7,8-dihydroxyflavone (7,8-DHF) acted as a potent TrkB agonist, which could penetrate the blood brain barrier [12], therefore trigged TrkB phosphorylation [13, 14] and exerted protective effects both in cytotoxic models [15, 16] and several neurodegenerative animal models [17, 18]. In the current study, we explored the potential neuroprotective effects of 7,8-DHF in rat 6-hydroxydopamine (6-OHDA) model and mouse 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) model using behavioral and histological analyses. And the possible mechanism underlying the effects of 7,8-DHF via TrkB was further studied. 2. Materials and Methods 2.1. Animals Male Sprague-Dawley rats (200-250g) and male C57/BL6 mice (8-9 weeks old) were purchased from SLAC laboratory animal center (Shanghai, China) and housed at 22-24℃ facility with 12h light/dark cycle. Food and water were provided ad libitum. All the experimental protocols were carried in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Institutional Animal Care and Use Committee. 2.2. Materials Apomorphine hydrochloride, 6-OHDA hydrobromide and MPTP were ordered from Sigma (St. Louis, MO 63130); 7,8-DHF was purchased from TCI (Tokyo, Japan); Rabbit anti-tyrosine hydroxylase (TH) antibody was ordered from Millipore (Billerica, MA 01821). Rabbit anti-phospho (Y816)-TrkB was ordered from Abcam (Cambridge, MA 02139); Biotinylated secondary antibody, HRP-conjugated streptavidin and 3,3′-Diaminobenzidine (DAB) were products of Jackson ImmunoResearch (West Grove, PA 19390). 2.3. 6-OHDA rat model and DHF treatment Rats were anesthetized with 60 mg/kg sodium pentobarbital for stereotaxic surgery. 6-OHDA was dissolved in artificial cerebral spinal fluid and was injected into two sites of the left striatum: AP +0.5, L -2.5, DV -5.0; AP -0.5, L -4.0, DV -5.0 (mm from Bregma). Each site received 10μg/2μl 6-OHDA. 7,8-DHF was dissolved in drinking water at the concentration of 160mg/L by gradually dropping 1M NaOH into the water 2
and the final pH was 7.4-7.6. The volume of water that rats drank slightly varied each day, so the dose they received was 12~16mg/kg per day. The study involved two different schedules of DHF treatment (Fig. 1A): a. Pretreatment (Pre) - 7,8-DHF treatment for 14 days before 6-OHDA lesion and for another 14 days after lesion; b. Post-treatment (Post) - 7,8-DHF treatment for 14 days only after lesion. Control group was 6-OHDA lesioned and drank regular water. 2.4. Behavioral tests in rat model Two weeks after 6-OHDA lesion, the akinesia behaviors were assessed with the following tests by experimenters blinded to the treatment design. 2.4.1. Cylinder test Rats were placed in a transparent cylinder (22cm in diameter, 30cm in height), and the number of wall contact for left, right or both forelimbs was counted respectively until the total number reached 20. The motor performance for each limb was expressed as the percent use of the limb relative to the total number. 2.4.2. Apomorphine induced rotation Rats were placed in a round enclosure with 40cm in diameter. They were injected subcutaneously with 0.25mg/kg apomorphine and waited for 5min. Then the number of contralateral turning was counted manually for another 5min. 2.4.3. Adjusting step test Rats were held by the experimenter with the hindlimbs gently fixed and slightly elevated. It was moved slowly sideways (90cm of distance in 5s) with one of its forepaws touching the table and the other fixed, first in the forehand direction, then in the backhand direction. The number of adjusting steps for each forelimb on both directions was recorded respectively. 2.5. Acute MPTP mouse model 7,8-DHF water was prepared as described above. Mice were given 7,8-DHF or regular water continuously for 14 days. On the 7th day, they were injected intraperitoneally with 20mg/kg MPTP twice at 2h apart. Pole test was performed on day 15. Mice were placed with the head upward on the top of a vertical pole with 1.2cm in diameter and 50cm long, and the base of the pole was fixed in the animal’s home cage. The time spent to orient downward (Tturn) and the total time spent to descend (TLA) was recorded. Each mouse was tested for five successive trials to get the average. 2.6. Immunohistochemistry staining After the completion of behavioral tests, the animals were perfused with 4% paraformaldehyde (in 0.01M PBS, pH 7.4). The brains were removed and further fixed overnight and then transferred to 30% sucrose solution till sunk. The brains were cut into 30μm coronal sections on a cryostat microtome. The sections were incubated with TH antibody (1:1000) overnight at 4℃ for the detection of dopaminergic neurons in the SN and DAergic terminals in the striatum; or with anti-phospho(Y816)-TrkB (1:1000) antibody for the detection of TrkB activation in the SN, followed by biotinylated secondary antibody and HRP-conjugated streptavidin. The sections were developed with DAB and photographed with an Olympus DP72 camera connected to BX51 microscope. 2.7. SN cell counting For each rat, three SN sections at Bregma -4.8, -5.3 and -5.8 mm were used for TH+ or p-TrkB+ cell counting. For mice, we stained three SN sections at Bregma -3.0, -3.3 and -3.6 mm for each animal. Cell counting was conducted by an experimenter who did not know the treatment condition, and the result for each animal was the summation of the number from its three sections. 2.8. Statistic analysis All data were expressed as mean ± SEM and analyzed by the software GraphPad Prism. Two-way ANOVA followed by post-hoc t-test was performed for cylinder test and adjusting step test; other data were 3
analyzed with one-way ANOVA followed by Tukey test. Significant level was set at p<0.05. 3. Results 3.1. 7.8-DHF improved the behavioral performance in rat 6-OHDA model Rats were treated as described in Fig. 1A and the akinesia symptoms were assessed with a series of behavioral tests. As shown in Fig. 1B, a two-way ANOVA on forepaw use in cylinder test revealed a main effect of the side of forepaw (F(2,78)=36.02, p<0.0001) and treatment (F(2,78)=3.989, p=0.0258), and a significant interaction (F(4,78)=3.91, p=0.0093). The right forepaw use was significantly decreased for both control (p=0.0032) and Post (p=0.0455) groups compared to their left side. And pretreatment with 7,8-DHF significantly improved the right forepaw use compared with control group (p=0.0243), indicating pretreatment with 7,8-DHF protected 6-OHDA-impaired forepaw use. To assess the extent of dopamine depletion, apomorphine induced contralateral rotation was conducted. As shown in Fig. 1C, there was a significant effect of treatment on the number of rotation (F(2,63)=3.617, p=0.0344). It was significantly declined for Pre (p=0.0115) but not Post group, suggested protection of DA neurons by 7,8-DHF pretreatment. For adjusting step test, a two-way ANOVA on forelimb step revealed a main effect of the side of forepaw (F(2,78)=16.02, p<0.001) and treatment (F(2,78)=9.17, p=0.0138), but not interaction (F(4,78)=3.72, ns). The right forepaw stepping was significantly damaged for both control (p=0.0048) and Post (p=0.0086) groups compared to their left side. Post hoc analysis revealed that the dysfunction was significantly rescued by 7,8-DHF pretreatment (Fig. 1D, p=0.0084). None of the rats showed notable step deficit in the backhand direction test (Fig. 1E). 3.2. 7,8-DHF protected 6-OHDA induced DA neurons loss in rats After the completion of behavioral tests, brain sections of the SN and the striatum were immunohistochemically stained with TH antibody. As demonstrated in Fig. 2, 7,8-DHF pretreatment while not post-treatment significantly preserved DA neurons in the SN compared with control and was confirmed by further quantification data (Fig. 2D, F(2,26)=4.851, p=0.0162). Meanwhile, TH staining of the striatum also revealed that 6-OHDA-induced DA terminals depletion in the striatum was rescued by pretreatment but not post-treatment with 7,8-DHF (Fig. 2E-G). 3.3. TrkB phosphorylation was elevated by 7,8-DHF pretreatment Since 7,8-DHF acts as a TrkB agonist, we immunostained the SN sections with p-Y816-TrkB antibody to determine if the treatment could activate TrkB in rat 6-OHDA model (Fig. 3). TrkB phosphorylation was significantly increased by 7,8-DHF pretreatment compared with control (Fig. 3D, F(2,26)=4.625, p=0.0191), but not in Post group, indicating activating of TrkB by the pretreatment of 7,8-DHF. 3.4. Neuroprotective effects of 7,8-DHF in acute MPTP mouse model Previous studies have reported that 7,8-DHF protected DA neurons in acute MPTP mouse model [13], and prevented DA neurons loss and restored motor deficit in a progressive MPTP model [19], here we also wanted to know if the oral treatment with 7,8-DHF in the present study could improve the behavioral performance in acute MPTP model. Two doses of MPTP injection induced a 55% and significant decrease of the number of TH+ neurons in the SN compared with normal group (Fig. 4). Similar with the reported results, 7,8-DHF significantly preserved the TH+ cells (Fig. 4D, F(2,14)=4.77, p=0.0265), confirmed a neuroprotective effect on MPTP induced neurotoxicity. However, we did not observe any significant behavior impairment in 4
pole test (Fig. 4E, p=0.21 for Tturn and p=0.37 for TLA). This was probably due to limited dopaminergic degeneration by acute MPTP, which was insufficient to induce motor damage. 4. Discussion In the present study, we found that the TrkB agonist 7,8-DHF protected 6-OHDA and MPTP induced dopamine neurons degeneration in rodents. Pretreatment with 7,8-DHF improved the behavior performance significantly in 6-OHDA rat model. Furthermore, chronic treatment with 7,8-DHF induced TrkB phosphorylation in the SN of rats, indicating the activation of TrkB, which probably contributed to its neuroprotective actions. Flavonoids are a class of polyphenol compounds that are widely distributed in the plant. 7,8-DHF is a bioactive high-affinity TrkB agonist which provokes TrkB dimerization, autophosphorylation and activation of down-stream MAPK signaling [13]. 7,8-DHF has been demonstrated to have various biological effects, including anti-oxidative effects [15] and neuro-regenerating effects in traumatic neuro-injury models [20, 21]. Furthermore, 7,8-DHF displayed antidepressant effects [22] and potentiated spatial memory through TrkB pathways [17]. These findings provide the possibility that 7,8-DHF would play multiple functions in the central nervous system. Many studies have demonstrated the potential link of BDNF with the pathophysiology of PD. In PD patients, the serum level of BDNF was significantly decreased [7], which has been reported to strongly correlate with the cognitive impairment of PD patients [8]. And BDNF Val66Met, a functional polymorphism, was also reported to be associated with cognitive impairment [23]. Moreover, the disturbance of TrkB signaling induced the deposit of α-synuclein, the hallmark of PD, and led to the loss of TH positive cells in the SN [24]. Thus the dysfunction of BDNF/TrkB signaling plays an important role in the development of PD. Due to the neuroprotective activity, BDNF was heavily studied in the nigrostriatal dopaminergic system. Transplantation of BDNF-secreting cells has been published to rescue the neuron injury and promote the motor function in rodent PD models [25, 26]. In addition, some potent neuroprotective strategies, such as monoamine oxidase inhibitors, also increased the expression of BNDF in cultured cells, PD models [27], as well as in PD patients [28]. Exercise likewise has been demonstrated to be an effective way to improve the expression of BDNF, which provided benefits to PD models and patients [29, 30]. Nevertheless, the clinical trials for neurotrophins have been disappointing due to the poor permeability through the blood brain barrier and a short serum half-life. Thus as a small molecule TrkB agonist, 7,8-DHF has a better chance to mimic the profound neuroprotective effects of BDNF in a translational point of view. In our current study, pretreatment with 7,8-DHF preserved DA-mediated behaviors in rat 6-OHDA model and improved the expression of phospho-TrkB in the SN. These results indicated a correlation between TrkB activation and the neuroprotective effects of 7,8-DHF. However, since there were greater neurons survived in DHF pretreatment group, the elevated expression of phospho-TrkB may also be due to the preserved cell quantity. Future studies, such as the detecting of the TrkB downstream signalings and using of transgenic tools, could be performed to further confirm the molecular mechanisms of the neuroprotective effects of 7,8-DHF. 5. Conclusion The TrkB agonist 7,8-DHF protects the loss of DA neurons induced by 6-OHDA and MPTP in rodents. Pretreatment with 7,8-DHF improves the behavioral performances in rat 6-OHDA model, which is probably 5
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Figure legends Figure 1. Effects of 7,8-DHF on locomotor function in 6-OHDA model. A. Experimental design and 7,8-DHF treatment schedule. B. Cylinder test. 6-OHDA significantly reduced the right forepaw use in rats, while it was improved by 7,8-DHF pretreatment (* p=0.0243 compared with the right side of control group). n=9-10. C. Apomorphine induced rotation. The number of contralateral rotation for pretreatment group was significantly decreased compared with control. n=22. D & E. Adjusting step test. 6-OHDA damaged the right forelimb stepping compared with its left side in the forehand direction. 7,8-DHF pretreatment significantly reversed the right forelimb dysfunction (* p=0.0084 compared with the right side of control group). n=9-10. None of the rats showed deficit in the backhand direction. Data were expressed as mean ± SEM.
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Figure 2. TH staining of the SN and striatum of 6-OHDA model. Rats were treated as described in Fig. 1A. Brain sections containing the SN (A-C) and striatum (E-G) were immunostained with TH antibody. The cell counting data of the SN revealed that 7,8-DHF pretreatment prevented 6-OHDA-induced DA neurons loss in the left SN (D). n=9-10. Scale bar in 2C = 200μm and in 2G = 1mm. Arrows in E-F directed the lesioned area of the striatum.
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Figure 3. Phosphorylated-TrkB immunostaining of the SN. Rats were treated as described in Fig. 1A. SN sections were immunostained with phosphor-Y816-TrkB antibody. Representative sections of the left SN were shown for each group. A: control; B: post-treatment; C: pretreatment. Scale bar = 100μm. Data were expressed as mean ± SEM. n=9-10. The result revealed that TrkB phosphorylation was significantly increased in 7,8-DHF pretreated animals.
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Figure 4. Neuroprotective effects of 7,8-DHF in acute MPTP mouse model. Mice were treated as described in the Methods section 2.5. Pole test was performed and the SN sections were stained with TH antibody. A: normal; B: MPTP + regular water; C: MPTP + 7,8-DHF. D: MPTP significantly degenerated the DA neurons of the SN, which was rescued by 7,8-DHF pretreatment. Data were expressed as mean ± SEM. n=5-6. Scale bar = 100 μm. E. The time to orient downward (Tturn) and total time to descend (TLA) in pole test. There was no significant difference between groups.
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