Catalpol attenuates MPTP induced neuronal degeneration of nigral-striatal dopaminergic pathway in mice through elevating glial cell derived neurotrophic factor in striatum

Neuroscience 167 (2010) 174 –184

CATALPOL ATTENUATES MPTP INDUCED NEURONAL DEGENERATION OF NIGRAL-STRIATAL DOPAMINERGIC PATHWAY IN MICE THROUGH ELEVATING GLIAL CELL DERIVED NEUROTROPHIC FACTOR IN STRIATUM G. XU,a1 Z. XIONG,a1 Y. YONG,b Z. WANG,a Z. KE,b Z. XIAa AND Y. HUa*

locomotor ability by attenuating the neuronal degeneration of nigral-striatal dopaminergic pathway, and this attenuation is at least partially through elevating the striatal GDNF expression. Crown Copyright © 2010 Published by Elsevier Ltd on behalf of IBRO. All rights reserved.

a Research Laboratory of Cell Regulation, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, PR China b Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, PR China

Key words: MPPⴙ intoxication, dopamine transporter (DAT), tyrosine hydroxylase (TH), MPTP chronic Parkinsonism model, rotarod performance, glial cell derived neurotrophic factor (GDNF).

Abstract—The protective effect of an iridoid catalpol extracted and purified from the traditional Chinese medicinal herb Rehmannia glutinosa on the neuronal degeneration of nigral-striatal dopaminergic pathway was studied in a chronic 1-methyl-4phenyl-1,2,3,4-tetrahydropyridine (MPTP)/probenecid C57BL/6 mouse model and in 1-methyl-4-phenylpyridimium (MPPⴙ) intoxicated cultured mesencephalic neurons. Rotarod performance revealed that the locomotor ability of mice was significantly impaired after completion of model production and maintained thereafter for at least 4 weeks. Catalpol orally administered for 8 weeks (starting from the second week of model production) dose dependently improved the locomotor ability. HPLC revealed that catalpol significantly elevated striatal dopamine levels without changing the metabolite/ dopamine ratios. Nor did it bind to dopamine receptors. Therefore it is unlikely that catalpol resembles any of the known compounds for treating Parkinsonism. Instead, catalpol dose dependently raised the tyrosine hydroxylase (TH) neuron number in substantia nigra pars compacta (SNpc), the striatal dopamine transporter (DAT) density and the striatal glial cell derived neurotrophic factor (GDNF) protein level. Linear regression revealed that both the TH neuron number and DAT density were positively correlated to the GDNF level. In the cultured mesencephalic neurons, MPPⴙ decreased the dopaminergic neuron number and shortened the neurite length, whereas catalpol showed protective effect dose dependently. Furthermore, the expression of GDNF mRNA was up-regulated by catalpol to a peak nearly double of normal control in neurons intoxicated with MPPⴙ for 24 h but not in normal neurons. The GDNF receptor tyrosine kinase RET inhibitor 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP1) abolished the protective effect of catalpol either partially (TH positive neuron number) or completely (neurite length). Taken together, catalpol improves

Parkinson’s disease (PD) is a chronic neurodegenerative disease, the main pathological changes of which involve selective degeneration of dopaminergic neurons, reduced expression of tyrosine hydroxylase (TH) of substantia nigra pars compacta (SNpc) and decreased density of dopamine transporter (DAT) and dopamine (DA) content in the striatum (Kish et al., 1988; Nutt et al., 2004; Nagatsu and Sawada, 2007). The effects of current drugs are mostly palliative, and may gradually decrease after prolonged use or cause side effects such as dyskinesia (Olanow and Tatton, 1999; Savit et al., 2006). New therapeutic approaches aimed at delaying or reversing the neurodegenerative process have attracted the attention of many investigators in recent years (Silverdale et al., 2003; Savit et al., 2006; Peterson and Nutt, 2008). Many studies showed that neurotrophic factors such as the brain derived neurotrophic factor (BDNF) and the glial cell derived neurotrophic factor (GDNF) have powerful neuroprotective effects (Murer et al., 2001; Dawson and Dawson, 2002; Cass et al., 2006; Evans and Barker, 2008). Recently, it has been hypothesized that neurotrophic factors, most notably GDNF, might have the ability to restore function of dopaminergic neurons (Hong et al., 2008; Yang et al., 2009). However, these factors cannot reach their target areas in brain by systematic administration. Therefore, an important new research field involves the search for small molecules that can enter the brain tissue and then trigger the endogenous neuroprotective mechanisms. The iridoid catalpol (Fig. 1) is an active component in some important medicinal herbs such as Rehmannia glutinosa (Oshio and Inouye, 1981), that are frequently used for treating neurodegenerative diseases in traditional Chinese medicine, but the underlying mechanisms are mostly unclear. Preliminary data suggest that it can improve the memory of animal models by raising the choline acetyltransferase activity in brain (Wang et al., 2006) and can ameliorate beta amyloid-induced degeneration of cholin-

1 Contributed equally to the work. *Corresponding author. Tel: ⫹8621-64671552; fax: ⫹8621-64671552. E-mail address: [email protected] (Y. Hu). Abbreviations: AUC, area under the curve; DA, dopamine; DAT, dopamine transporter; DOPAC, dihydroxy-phenyl acetic acid; FP-CIT, 2␤-carbomethoxy-3␤-(4-iodophenyl)-N-(3-fluoropropyl) nortropane; GDNF, glial cell derived neurotrophic factor; HVA, homovanillic acid; MPP⫹, 1-methyl-4-phenylpyridimium; MPTP, 1-methyl-4-phenyl-1,2,3,4tetrahydropyridine; PD, Parkinson’s disease; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase.

0306-4522/10 $ - see front matter. Crown Copyright © 2010 Published by Elsevier Ltd on behalf of IBRO. All rights reserved. doi:10.1016/j.neuroscience.2010.01.048

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Fig. 1. Chemical structure of catalpol.

ergic neurons by elevating BDNF (Wang et al., 2009), yet it is neither a cholinesterase inhibitor nor a muscarinic receptor (M receptor) agonist (Li et al., 2004; Liu et al., 2007a,b). It has also reported that catalpol could elevate the activity of mitochondria complex 1 in the 1-methyl-4phenylpyridimium (MPP⫹) intoxicated cultured mesencephalic neurons (Tian et al., 2007). However, several important questions have not yet been addressed. Can catalpol improve locomotor ability in animal models of Parkinsonism? Can catalpol attenuate the neurodegenerative changes of dopaminergic pathway such as DAT and DA in striatum? Can catalpol really attenuate the loss of TH positive cells in SNpc if a realible method is used to count the cells (for example, the unbiased stereological counting)? Is the improvement of locomotor ability closely related to the neuroprotection effect? Furthermore, what happens to be most interested to us is whether the neuroprotective effect is related to GDNF which has been said to be the most potent neurotrophic factor for Parkinsonism? In an attempt to answer these questions, we first carried out in vivo experiments to study the effect of catalpol on the locomotor deficit. For this purpose, the chronic 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP) mouse model (Petroske et al., 2001; Meredith et al., 2008; Schintu et al., 2009) was chosen since the locomotor deficit in Parkinsonism is a chronic progressive process and according to the experience of traditional Chinese medicine, the effect of catalpol is also a chronic progressive process. When the test of locomotor ability was completed, dopamine and its principal metabolites in striatum were measured to examine whether catalpol is different from the known drugs currently in clinical use for treating Parkinsonism. Subsequently, another set of in vivo experiments using the chronic MPTP model was carried out to study the effect of catalpol on the neurodegenerative changes of dopaminergic neurons, involving the transporter density in striatum, the TH positive neuron number in SNpc, and the striatal GDNF content. Finally, in vitro experiments using the cultured mesencephalic neurons intoxicated with MPP⫹ were carried out to study the molecular mechanism of the neuroprotection of catalpol, especially the role played by GDNF synthesis.

EXPERIMENTAL PROCEDURES Production of an animal model for in vivo experiments Male C57BL/6 mice (21.2⫾2.7 g, 10 weeks old), purchased from Shanghai SIPPR-BK Laboratory Animal Company, were housed

175

five per cage with room temperature and relative humidity set at 22⫾2 °C and at 55%⫾15% respectively, and lit by artificial light for 12 h each day. Sterilized drinking water and standard chow diet were supplied ad libitum. The animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023), revised in 1996, and approved by the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine. The chronic model was produced as described (Petroske et al., 2001) with slight modifications. In short, 10 doses of probenecid (Sigma, St. Louis, MO, USA) plus MPTP (Sigma, St. Louis, MO, USA) were given to each mouse over 5 weeks (two doses per week). For each dose, an i.p. injection of 250 mg/kg of probenecid in DMSO was given 0.5 h prior to the s.c. injection of 15 mg/kg of MPTP in saline. The normal control group was injected simultaneously with solvents.

Drug administration for in vivo experiments Experiment 1. Three groups of mice (seven in each group), the normal control, the model control, and model treated with catalpol (50 mg/kg dissolved in saline) for 8 weeks once daily through a gastric tube starting from the third dose of model production. Catalpol was extracted and purified in our laboratory from fresh roots of the traditional Chinese medicinal herb Rehmannia glutinosa and identified with MS and NMR. Its purity was ⬎97% as determined by HPLC. The locomotor ability was tested in the last 3 days of drug administration and the animals were sacrificed for HPLC assay of striatal DA and its metabolites (dihydroxy-phenyl acetic acid (DOPAC) and homovanillic acid (HVA)). Experiment 2. Six groups of mice (eight in each group) were involved: the normal control, the normal control treated with catalpol (50 mg/kg), the model control, and models treated with catalpol at doses of 5, 15 and 50 mg/kg per day. Catalpol or vehicle was administrated by the same way as in experiment 1. On the last 3 days of drug/vehicle administration, locomotor ability was tested. On the next day (4 weeks after the last injection of MPTP), five mice were taken from each group, sacrificed, and their brains were rapidly taken and kept at ⫺70 °C until assayed for DAT and GDNF. The other three mice of each group were prefixed with 4% paraformaldehyde for immunohistochemical examination of TH-positive cells in SNpc.

Primary culture of mesencephalic cells and treatment of the cultured neurons Sprague–Dawley (SD) pregnant rats were acquired from Shanghai SIPPR-BK Laboratory Animal Company and the rat mesencephalic neurons were cultured as described previously (Zhang et al., 2008). In brief, freshly dissected ventral mesencephalons from brains of rat embryos (E14 –15 days) were dispersed by trypsin digestion and trituration. After centrifugation, cells were resuspended in DMEM/F12 containing 10% fetal calf serum and 5% horse serum, seeded on poly-D-lysine-coated 96-well plates at 1⫻105 cells/cm2 and cultured at 37 °C in 5% CO2. The medium was changed to DMEM/F12 plus 2% B27 on the next day and was then renewed every 3– 4 days. In order to study the effect of catalpol against MPP⫹ (Sigma, St. Louis, MO, USA) induced toxicity, the culture medium was replaced with fresh medium containing various concentrations of catalpol (10⫺6 M to 10⫺4 M) or vehicle on the 6th day. Twenty four hours later, MPP⫹ (10⫺5 M) was added to the medium and the cells were further cultured for 48 h. For studying the effect of catalpol (10⫺5 M) on the expression of GDNF mRNA, cells were harvested at 0 h, 6 h, 24 h, 48 h after the administration of MPP⫹(10⫺5 M). For blocking the GDNF receptor tyrosine kinase RET, 4-amino-5-(4-methyphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]pyrimidine (PP1) (Biomol, Alexis Corp., UK) was added to the medium 10 min before administration of catalpol

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(10⫺5 M) (Knowles et al., 2006). After 24 h, MPP⫹ (10⫺5 M) was added to the medium and the cells were further cultured for 48 h. Then the cells were fixed by 4% paraformaldehyde for immunocytochemical examination.

Rotarod test for locomotor ability The method described by Rozas (Rozas et al., 1998) was used with some modifications. The locomotor ability was examined 4, 6 and 8 weeks after the start of catalpol or vehicle administration with a rotarod apparatus (IITC life science, USA). All mice were trained on the rod with low speed rotation (8 rpm) on the day before test. Then the mice were tested on the revolving rods at six incremental speeds (8, 12, 16, 20, 24, 28 rpm) for up to 150 s per speed once per day in the last three consecutive days of drug administration. The time that the animal remained on the rod at each speed was recorded. To represent the overall locomotor ability of each mouse, the mean time that each mouse remained on the rod in the 3 days at each speed was plotted against the corresponding revolving speed and the area under curve (AUC) was calculated. A resting period (5 min minimum) was provided between successive speeds to minimize stress and fatigue.

HPLC analysis of DA and its metabolites The method described by Liang was followed with some modifications (Liang and Tang, 2006). In brief, striatum was rapidly dissected, weighed, homogenized and centrifuged. The final supernatant was stored at ⫺70 °C until HPLC assay. The concentrations of dopamine and its metabolites (DOPAC and HVA) were determined by a Waters 515 HPLC system (Milliford, MA, USA) with an electrochemical detector (HPLC-ECD) and a microbore ODS column (100 mm⫻1 mm i.d.). The mobile phase consisted of 0.1 M NaH2PO4, 0.5 mM EDTA, 10 mM NaCl, 1 mM octylsodium sulfate, pH 3.1 and 5% acetonitrile, and was pumped at 65 ␮l/min. The sample was diluted 1:4 with the mobile phase to neutralize the pH before chromatography. The calibration materials were assayed in parallel.

Competitive binding assay for testing whether catalpol binds to dopamine receptor The procedure described by Burt was followed with some modifications (Burt et al., 1975). In brief, striatum was homogenized in ice-cold Tris buffer, pH 7.7, centrifuged at 50,000⫻g for 10 min, and the pellet was homogenized in the same buffer and recentrifuged. Then the pellet was suspended in Tris buffer, pH 7.0, with 0.1% ascorbic acid and 10 ␮M selegiline and 10 ␮M isoniazid, and incubated at 37 °C for 5 min. Two hundred microliters of the suspension was added to 200 ␮l of the same buffer containing 2 pmol 3H-dopamine (specific activity 1.8 TBq/mmol, Amersham, Buckinghamshire, UK) with or without various concentrations (10⫺9 M to 10⫺4 M) of catalpol or unlabeled dopamine (serving as positive control). The binding was carried out at 25 °C for 15 min and the bound portion was suctioned onto glass fiber filter and measured with a liquid scintillation counter (Beckman LS6500, Fullerton, CA, USA). The measured cpm of each tube was finally converted to % of cpm of the tube without competitor, and a competitive curve was drawn.

TH immunohistochemical examination A free-floating immunohistochemical method described by Brouard et al. (1992) was followed with modifications. Briefly, Coronal frozen sections (50 ␮m) or cultured cells were treated with 3% H2O2 followed by sequential incubation with 0.4% Triton X-100, and 0.5% BSA. Then the samples were incubated with a rabbit anti-mouse TH antibody (Chemicon, Temecula, CA, USA, 1:500 dilution at 4 °C overnight for the brain sections or 1:2000 dilution

at 37 °C for 2 h for the cultured cells). Subsequently, the sections or cells were incubated with sheep anti-rabbit IgG, color developed with the ABC kit and 3,3=-diaminobenzidine (DAB). The cultured cells were examined with a Nikon TE300 inverted microscope. For counting TH-positive neurons in the cultured cells, two or three wells of each sample, with ten randomly selected optic fields from each well, were counted to obtain the average number of neurons per optic field. For measuring the neurite outgrowth length, the longest neurite of each neuron (excluding those having neurite(s) ending outside the optic field or having neurite connection(s) with other neurons) was measured for the above optic fields with the aid of Image J software (segmented line selection) to give the average longest neurite length of each sample. The brain slices were examined with an Olympus BX61 microscope and the total number of TH-positive neurons in the SNpc was counted by the unbiased stereological counting method as described in the next section.

Stereology of TH-positive neurons in SNpc Stereological analysis was performed according to the previous paper (Ke et al., 2003). Briefly, an Olympus BX61 microscope with a monitored x-y-z stage linked to the StereoInvestigator software (version 8, MicroBrightField, USA) following the user’s guide. According to the mouse brain atlas (Franklin and Paxinos, 1997), anatomical boundaries were delineated. The medial border followed a vertical line passing through the medial tip of the cerebral peduncle to exclude the neurons in ventral tegmental area (VTA), the dorsal border was defined by a line passing between the vental margin of the medial lemniscus and pars compacta. The ventral border followed the dorsal boundary of the cerebral peduncle. Every fourth section was used in each animal for cell counting. The SNpc was outlined using a 4⫻ objective on each THimmunostained section and dopaminergic neurons were sampled using a 40⫻ objective with a counting frame. The section thickness determined before counting at different position throughout the area of SNpc showed that immunohistochemically stained sections with an initial thickness of 50 ␮m shrink to a thickness of approximately 16 ␮m following dehydration and coverslipping, allowing us to use a 12 ␮m-high desector to ensure adequate guard zones on both sides of the dissector. Optical dissector counting rules were used to count the TH-positive neurons in each dissector volume, the total number of TH-positive neurons was calculated by the formula: N⫽兺 Q⫺

t 1 1 h asf ssf

Where ⌺Q⫺ is the total number of TH-positive neurons counted, t is the mean section thickness, h is the height of the optical dissector, asf is the area sampling fraction, and ssf is the section sampling fraction. The estimated CE values of all samples were given by the StereoInvestigator software (using the Gundersen’s estimation) and were less than 0.1.

Autoradiographic detection of DAT density in striatum 125 I-FP-CIT was prepared by reaction of unlabeled FP-CIT (Jiangsu Institute of Nuclear medicine, Wuxi, China.) and Na125I (carrier free, Chengdu Gaotong Isotope Corporation, Chengdu, China) in an iodogen precoated tube and purified by paper chromatography. The radiochemical purity of 125I-FP-CIT was ⬎95%. The method of DAT binding described by Gunther (Günther et al., 1997) was followed with some modifications. Frozen sections containing the striatum (bregama 1.0⫾0.2 mm) (Franklin and Paxinos, 1997) were incubated in the binding buffer containing 50 pM 125I-FP-CIT and 100 nM fluoxetine (to inhibit serotonine bind-

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ing, Sigma, St. Louis, MO, USA) for 2 h at room temperature. Nonspecific binding was measured in parallel slices with the addition of 100 ␮M GBR 12909 (Sigma, St. Louis, MO, USA). After being rinsed and air-dried, the slices were exposed to X-ray film (Kodak, Rochester, NY, USA) at 4 °C for 48 h. Then the film was developed, fixed, and finally analyzed (Biorad Gel Doc 2000).

ELISA assay of GDNF Striatum of the right hemisphere was dissected, weighed and homogenized in lysis buffer at 4 °C. The supernatants were then assayed with the GDNF Emax Immunoassay System (Promega, Madison, WI, USA) following the user’s guide. In brief, 96-well plates were coated with anti-GDNF monoclonal antibody overnight, 100 ␮l sample supernatants (diluted 1:4) were added, incubated for 6 h at room temperature, parallel tubes with serial GDNF protein standards (0 –125 pg/ml) were used to establish the calibration curve. Followed by polyclonal anti-human GDNF antibody, secondary antibody, TMB One solution, and the color development was terminated by 1 M HCl. The absorbance was measured at 450 nm. Data were expressed as pg/mg of total protein which was measured by the micro-Lowry method.

RT-PCR analysis of GDNF mRNA Total RNA was isolated by acid guanidium thiocyanate-phenolchloroform extraction method (Zhang et al., 2008). The cDNA reverse-transcribed from 250 ng of total RNA was amplified using the following primer (Ledda et al., 2007) sets: GDNF: forward, 5=-ATG TCA CTG ACT TGG GTT TGG G-3=; reverse, 5=-GCT TCA CAG GAA CCG CTA CAA-3=; GAPDH: forward, 5=-TGG GTG TGA ACC ACG AGA AAT A-3=; reverse, 5=-GCT AAG CAG TTG GTG GTG CAG-3=. Real-time PCR using the SYBR Green PCR Master Mix kit was performed with an ABI Prisma 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Data were normalized by respective GAPDH values. The value of normal control at 0 h was taken as 100 in each run.

Statistical analysis Data were expressed as mean⫾standard error (SEM). The results of different groups were compared by one way ANOVA followed by post hoc Newman–Keuls test using the SAS software package. When only two sets of data were compared, the Student’s t-test was used. Linear regression was applied for testing the correlation between two parameters.

RESULTS Catalpol improves rotarod performance of chronic MPTP mouse model When the revolving speed of the rods on the rotarod apparatus increased, the time that the mice remained on the rod decreased. Thus, the relationship of the time that the mice remained on the rod versus the revolving speed was a declining curve. Fig. 2A shows the curves of various groups after catalpol or vehicle administered for 8 weeks. The curve dropped more rapidly in the MPTP model than in normal mice. Catalpol prolonged the time that the model mice remained on the rod, but showed no effect on normal control mice. Statistical results of AUCs are shown in Fig. 2B. The rotarod performance of model mice was significantly worse than normal by the end of model production and remained worse for at least 4 weeks. The action of catalpol was slow and progressive. The improvement of locomotor ability became significant (P⬍0.01) after catal-

Fig. 2. Effect of catalpol on the rotarod performance of chronic MPTP model mice. (A) The curves of the average time that the mouse remained on the rod versus revolving speed of rods in six groups of animals. Each point is the average of eight animals. (B) Statistical data showing the relative AUCs of various groups after administration of catalpol or vehicle for 4, 6 and 8 wk. Data are expressed as mean⫾SEM, n⫽8. AUCs were first calculated from the curves of each animal as absolute values and then converted to relative values taking the respective value of the normal control group as 100. * and ** indicate P⬍0.05 and P⬍0.01 versus the vehicle treated model group.

pol was administered for 8 weeks, but was not significant after 4 or 6 weeks. Catalpol elevates striatal DA concentration in chronic MPTP mouse model but does not inhibit the catabolism of DA In the chronic MPTP model, the average concentration of DA in striatum was only 35.8% of normal controls (P⬍0.01). After administration of catalpol for 8 weeks at 50 mg/kg/d, DA concentration increased to 49% of normal (P⬍0.05) (Fig. 3). On the other hand, the concentrations of the metabolites DOPAC and HVA, which were decreased in

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A

Control catalpol(50mg/kg)

Normal control

Catalpol does not occupy the ligand binding site of the dopamine receptor As shown in Fig. 4, unlabeled dopamine showed typical competitive binding whereas catalpol did not show any competition with the binding of 3H-dopamine to the striatal membranous sample. This implies that catalpol is neither an agonist nor an antagonist of the dopamine receptor. Catalpol increases the number of TH immunoreactive neurons in SNpc Fig. 5A shows the representative micro-images of the TH immunoreactive neurons in SNpc of various groups of animal models. Fig. 5B shows the statistical results of unbiased stereological counting. The number of TH-positive neurons of SNpc in MPTP model was markedly decreased compared with normal control. Catalpol dosedependently increased the number of TH-positive neurons

B TH-Positive neurons number in SNpc

the MPTP model, were also elevated by catalpol so that the ratios of DOPAC/DA and HVA/DOPAC were not significantly changed by catalpol. For DOPAC/DA, the ratios were 0.149⫾0.004 and 0.158⫾0.007 respectively for model and model treated with catalpol, P⬎0.1. For HVA/ DOPAC, the ratios were 7.19⫾0.34 and 8.40⫾0.47 respectively, P⬎0.05. These data implicate that catalpol did not inhibit or stimulate the catabolism of DA.

Model Model Model catalpol(5mg/kg) catalpol(15mg/kg) catalpol(50mg/kg)

8000 6000

**

5684

**

5705

*

4000 2458

**

**

4893

4200

3215

2000 0

C

0

50 0 5 15 50 Dose of catalpol ( mg/kg per day ) MPTP Vehicle

2500

AUC in rotarod test

Fig. 3. Results of HPLC analysis of DA. Data are expressed as mean⫾SEM, n⫽7. * and ** indicate P⬍0.05 and P⬍0.01 versus saline-treated MPTP group.

Model control

R=0.74

p<0.01 n=15

2000 1500 1000 500 1000

3000

5000

7000

TH-Positive neurons number in SNpc

Fig. 4. Competitive binding assay between catalpol and 3H-dopamine. Unlabeled dopamine showed a typical competitive binding curve against the binding of 3H-dopamine to the membranous portion of striatal homogenate, whereas catalpol did not show any competition even at a concentration as high as 10⫺4 M.

Fig. 5. The effect of catalpol on TH-positive neuron numbers in SNpc. (A) Representative micro images (⫻100) of substantia nigra compacta in chronic MPTP mouse model showing that TH immunoreactivity was reduced in MPTP model mice and was raised by catalpol in a dose dependent manner(⫻100, scale bar⫽100 ␮m). (B) Statistical results of the relative number of TH-positive neurons in various groups. Data were obtained by unbiased stereological analysis and were expressed as mean⫾SEM, n⫽3 mice for each group, 10 slices of each mouse were counted so that to yield the total count of TH positive neurons in SNpc of that mouse with the coefficient of error for all individual optical fractional estimates less than 0.1. * and ** indicates P⬍0.05 and P⬍0.01 as compared with vehicle-treated MPTP group. (C) Linear regression of the rotarod performance versus TH positive cell number of individual mice showing a strong positive correlation between the two parameters. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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of the MPTP model. In the MPTP model mice, the number of TH-positive neurons was only 43% of the average normal control (P⬍0.01). Catalpol at doses of 5, 15 and 50 mg/kg per day significantly raised the average number of TH-positive neurons to 57% (P⬍0.05), 74% (P⬍0.01) and 86% (P⬍0.01) of normal control. Linear regression showed that there was a strong positive correlation between the AUC of rotarod performance and the number of TH-positive neurons in SNpc (Fig. 5C).

in the chronic MPTP model mice and was increased markedly by catalpol at doses of 15 and 50 mg/kg/d. Fig. 6B shows that the average value of the untreated model mice was 54% of the normal controls (P⬍0.01) and was raised to 80% and 90% of the normal controls by catalpol at 15 and 50 mg/kg/d (P⬍0.05 and P⬍0.01, respectively). As shown in Fig. 6C, a strong positive correlation was demonstrated by linear regression of the AUC obtained from the rotarod test against DAT density in striatum.

Catalpol elevates the striatal DAT in chronic MPTP mouse model

Striatal GDNF concentration in chronic MPTP mouse model was elevated by catalpol and was closely related to the elevation of TH positive neurons in SNpc and striatal DAT

As shown in Fig. 6A, autoradiograms revealed that the binding of 125I-FP-CIT to striatum significantly decreased

As shown in Fig. 7A, there was no significant difference among the striatal GDNF concentrations in normal controls, untreated model mice and normal control treated with catalpol (50 mg/kg/d) mice at 4 weeks after the last dose of MPTP. In contrast, a dose dependent increase in GDNF occurred in model mice treated with catalpol. The concentration was raised to approximately 150% of normal control at 5 mg/kg/d, to more than 220% and 240% of normal control at 15 and 50 mg/kg/d, respectively (P⬍0.01 for all the three doses). Linear regressions showed that there was a strong positive correlation between the striatal GDNF concentration and TH positive neurons in SNpc (R⫽0.835, P⬍0.01, n⫽12) (Fig. 7B) and a strong positive correlation between the striatal DAT and the striatal GDNF concentration (R⫽ 0.666, P⬍0.01, n⫽20) (Fig. 7C). Effect of catalpol on GDNF mRNA levels in mesencephalic neurons treated or untreated with MPPⴙ As shown in Fig. 8, Quantitative RT-PCR revealed that there was no significant difference among the GDNF mRNAs in the mesencephalic neurons of normal control, normal control treated with catalpol, and MPP⫹ intoxicated but untreated with catalpol. However, if the neurons were pretreated with catalpol for 24 h and then intoxicated with MPP⫹, the GDNF mRNA expression in mesencephalic cells was elevated significantly. The amount determined by quantitative RT-PCR was 152%, 196% and 162% (relative to respective normal control as 100%) of the MPP⫹ group pretreated with vehicle at 6 h (P⬍0.1), 24 h (P⬍0.01) and 48 h (P⬍0.1) after addition of MPP⫹ respectively. Effect of catalpol on the number of TH-positive neurons and their neurite outgrowth length in MPPⴙ intoxicated mesencephalic cultures and the blocking effect of PP1 Fig. 6. Effect of catalpol on striatal DAT in MPTP chronic mouse model. (A) Representative autoradiograms of striatal DAT binding showing that the density of striatal DAT was decreased in vehicle treated model mice and was significantly increased by catalpol. (B) Statistical results showing the relative gray values of striatal DAT in various groups. Data are expressed as mean⫾SEM, n⫽5. * and ** indicate P⬍0.05 and P⬍0.01 compared with untreated model group. (C) Linear regression showing that there was a very significant positive correlation between the AUC in rotarod test versus the striatal DAT.

Fig. 9A shows the representative micro-images of the cultured mesencephalic TH positive neurons under various treatments. Fig. 9B shows the protective effect of 1⫻10⫺5 and 1⫻10⫺4 M catalpol on neuron numbers, raising the density from 64⫾5% to 86⫾2% (P⬍0.01) and 91⫾1% (P⬍0.01) of normal control respectively. Fig. 9C shows the protective effect of 1⫻10⫺5 and 1⫻10⫺4 M catalpol on

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On the other hand, the blocking effect of PP1 on neuron density seems to be incomplete (Fig. 9D). With the same concentration of PP1 and catalpol, the density dropped from 88⫾6% of normal (without PP1) to 77⫾8% of normal (with PP1) whereas the model control (without catalpol) was 67⫾5% of normal.

DISCUSSION In general, MPTP mouse models may be classified as acute, subacute and chronic according to the dosing regimens of MPTP. The chronic mouse model is produced by 10 repeated dose of MPTP/probenicid in 5 weeks so that the pathologic changes last for a much longer period, with evident decrease of DA and DAT concentrations in striatum and decrease of TH positive neurons in SNpc even at 6 months after completion of model production (Petroske et al., 2001; Potashkin et al., 2007; Meredith et al., 2008; Schintu et al., 2009). Therefore the chronic model seems to be more suitable for the studies of drugs that need long term administration to yield therapeutic effects. Catalpol is very probably such a drug, as Rehmannia glutinosa tonics used in traditional Chinese medicine usually require long term application to achieve their full effects. In fact, we found that the rotarod test revealed a significant effect only after application of catalpol for 8 weeks but not 4 or 6 weeks. One of the important causes of locomotor dysfunction induced by MPTP is the decreased concentration of DA in striatum, which is mainly due to lack of DA synthesis and decreased DA storage in dopaminergic nerve endings. It has been reported that in idiopathic PD patients, symptoms became apparent only when about 70% of striatal dopamine was lost (Bernheimer et al., 1973; Dunnett and Björklund, 1999; Von Bohlen und Halbach, 2005). In the chronic MPTP mouse model, the level of DA in striatum decreased to 30% of normal control (Petroske et al., 2001)

Fig. 7. Effect of catalpol on striatal GDNF contents in chronic MPTP model mice measured by ELISA. (A) Data in various groups are expressed as mean⫾SEM, n⫽5, ** indicates P⬍0.01 versus untreated model group. (B) Linear regression between TH positive neuron number and striatal GDNF. Correlation coefficient, P-value and number of individual pairs were shown in the figure. (C) Linear regression of striatal DAT versus striatal GDNF content. Correlation coefficient, P-value and number of individual pairs were shown in the figure.

neurite length, raising the length from 41⫾3% to 67⫾8% (P⬍0.01) and 60⫾6% (P⬍0.01) of normal control respectively (Fig. 9C). The blocking effect of PP1 (concentration selected according to the user’s guide) for neurite outgrowth seems to be complete (Fig. 9E). When the catalpol concentration was 10 ␮M, the length dropped from 62⫾2% of normal (without PP1) to 42⫾0.4% of normal (with PP1), whereas the model control (without catalpol) was 43⫾2%.

Fig. 8. Quantitative RT-PCR results showing the effect of catalpol on the GDNF mRNA expression in cultures of mesencephalic neurons. Data are expressed as means⫾SEM from three independent experiments. In each experiment, there is one sample from each group for each time point (0 h, 6 h, 24 h, 48 h). The value of normal control at 0 h in each run was taken as 100 for that run. The interassay variability CVs of ⌬Ct values calculated were between 3.4% and 11.3% in the 16 groups. ** indicate P⬍0.01 versus vehicle treated MPP⫹ group.

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A

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Model+catalpol(10 6M)

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Fig. 9. The effect of catalpol on TH-positive neuron density and neurite outgrowth length in cultured rat mesencephalic neurons. (A) Representative micro-images of primary cultured mesencephalic neurons (⫻200, scale bar⫽50 ␮m) showing that the changes of neuron numbers, neurite outgrowth length and interneuron neurite connections in various groups. Also shown is the blocking effect of PP1 on the protective action of catalpol. (B) Statistical results of the neuron number per field (left) and (C) Average neurite outgrowth length (right) showing the dose dependent manner of the effect of catalpol. Data of (B) and (C) were expressed as mean⫾SEM of four independent experiments. The mean and SEM were finally converted to relative values taking the respective normal controls as 100%. ** indicate P⬍0.01 compared with vehicle treated MPP⫹ model. (D) and (E) show the effect of PP1 on the action of 1⫻10⫺5 M catalpol. Comparisons between individual groups were indicated in the diagram. * indicate P⬍0.05, NS indicate difference not significant. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

at 3 weeks or 36% of normal control (Fig. 3 of this paper) at 4 weeks after the completion of model production. Furthemore, results of this paper also revealed that when the striatal DA was only partially elevated (to 49% of control) by catalpol treatment (Fig. 3), the locomotor disability almost completely disappeared (Fig. 4). This is probably because there is a threshold of striatal DA level, below which locomotor disability become apparent while above which the locomotor disability is only insidious (Dunnett and Björklund, 1999; Schintu et al., 2009).

The results of HPLC analysis demonstrated that the increase of DA concentration in striatum was not due to inhibition of the catabolism of DA. If the acitivity of monoamine oxidase was inhibited, the ratio of DOPAC/DA should be significantly decreased (Kupsch et al., 2001; Lamensdorf et al., 1996). If the activity of catechol-Omethyl transferase was inhibited, the ratio of HVA/DOPAC should be significantly decreased (Napolitano et al., 2003). Yet neither of these changes occurred after catalpol application. Moreover, competitive binding assay indicated that

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catalpol is not a DA analogue. Therefore, the mechanism by which catalpol elevates striatal DA and affects locomotor ability is quite different from that of the known compounds currently used in replacement therapy. On the other hand, the results of our in vivo experiments showed that catalpol treatment, starting at the early stage of the model production, could significantly and dose dependently attenuate the MPTP induced reduction of DAT in striatum and TH positive neurons in SNpc which are the two important hallmarks of the neurodegenerative changes of dopaminergic neurons in Parkinson’s disease. The positive correlation between AUC of the rotarod test and DAT density in striatum and between AUC of the rotarod test and TH-positive neuron number in SNpc were both very significant. Although the pathological implications of DAT in Parkinsonism are still under active investigation (Sossi et al., 2007; Salahpour et al., 2008; AfonsoOramas et al., 2009), it is generally agreed that DAT plays a key role in the reuptake and storage of dopamine and hence is very important in the maintenance of dopamine levels in striatum and in the secretion of dopamine during nerve impulse (Dunnett and Björklund, 1999; Bannon, 2005; Himeda et al., 2006). In recent years, there have been many studies about the neuropreotective and neurorestorative effect of neurotrophic factors, especially of GDNF, in the treatment of Parkinson’s disease (Deraerts et al., 2007; Deierborg et al., 2008; Hong et al., 2008; Yang et al., 2009). It has been shown that GDNF can delay or even restore the neurodegenerative changes in Parkinson’s disease. Price and colleagues hypothesized that there might be six different strategies of searching for small molecules that could promote neurotrophic function (Price et al., 2007). Yet, up to now there is no effective drug for clinical application. Smilagenin seems to be a hopeful candidate (Zhang et al., 2008; Visanji et al., 2008). However, the effect of smilagenin on locomotor ability has not yet been reported. In the present study, we found that catalpol could significantly attenuate the locomotor deficit and attenuate the neurodegenerative changes of nigral-striatal dopaminergic pathway in MPTP mouse model simultaneously. The neurodegenerative changes and locomotor deficit is not due to decrease of GDNF. Since, consistent with the report of Inoue et al. (1999), we found that in the MPTP model, the GDNF protein content in the striatum was not different from normal level and the GDNF mRNA level in the MPP⫹ intoxicated mesencephalic neurons was also in the normal range. Nevertheless, when the MPTP model or the MPP⫹ intoxicated neurons was treated with catalpol, the GDNF protein and GDNF mRNA were significantly elevated to levels much higher than normal. Since GDNF was mainly synthesized in astrocytes, the role played by astrocytes in the neuroprotective effect of catalpol is important. However, it should be pointed out that GDNF can also to be synthesized by neurons (for example, Ivanova et al., 2002). Furthermore, the stimulation effect of catalpol on the sysnthesis of GDNF needs the coordination of certain signal from the intoxicated dopaminergic neurons. Data presented in the present paper showed that in case the

dopaminergic neurons were normal control instead of intoxicated by MPP⫹, the GDNF protein in mouse brain and the GDNF mRNA in the cultured neurons were not elevated by catalpol. Therefore, the stimulation of GDNF production by catalpol needs the coordination of certain signal from the intoxicated dopaminergic neurons. Nagatsu and Sawada (2005) suggested that such signal may came from certain cytokine, and Saavedra et al. (2007) provided evidence showing that when the action of one of the interleukins, IL-1␤, was blocked by its antagonist, IL-1ra, the decrease of the cultured dopaminergic neuron number was enhanced. Whether the same or different small molecules were involved in the protective action of catalpol remains to be further studied. Data presented in this paper also showed a strong positive correlation between striatal DAT density and GDNF concentration. Although correlation test does not demonstrate causality between parameters, it is reasonable to hypothesize from the above analysis that catalpol attenuates locomotor dysfunction and neurodegenerative pathological changes through stimulating the GDNF production. In order to prove the key role played by GDNF, in vitro experiments using primary culture of rat mesencephalic neurons were carried out. While MPP⫹ significantly reduced the TH positive neuron numbers and shortened the neurite outgrowth length, catalpol pre-treatment increased the neuron numbers and lengthened the neurite outgrowth significantly. Applilcation of PP1 which can inhibit the RET tyrosine kinase of GDNF receptor (Encinas et al., 2001; Paveliev et al., 2004) partially abolished the effect of catalpol on TH positive neuron numbers and completely abolished the effect of catalpol on the neurite outgrowth length. In addition, it has been reported that catalpol could reduce the production of reactive oxygen species (ROS) and elevate the activities of glutathione peroxidase and superoxide demutase in MPP⫹ intoxicated mesencephalic neurons and astrocytes (Tian et al., 2007; Bi et al., 2008) and attenuate the apoptosis of rotenone intoxicated mesencephalic neurons (Bi et al., 2009) which was also believed to be closely related to oxidative stress (Rodad et al., 2006). Whether these actions of catalpol were related to the elevation of GDNF was unknown. However, it has been reported recently that increased expression of GDNF could protect the retinal cells from injury of oxidative stress (Dong et al., 2007). Further study about the relation of GDNF and the effect of catalpol on the injury of neurons by oxidative stress would be interesting. Taken together, the above results show that catalpol improves locomotor ability in the chronic MPTP mouse model through a pharmacological mechanism quite different from those of a dopamine precursor or an inhibitor of dopamine catabolism. It attenuates the neurodegenerative decline of striatal DAT, nigral TH positive neurons and their neurite outgrowth length through elevating GDNF, thereby attenuating the decline of striatal DA concentration and improving the locomotor ability of diseased mice. Since catalpol is a small molecule that can be administered orally, it is a good candidate for further exploration. Furthermore, as there are many iridoids in herbal drugs used

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in traditional Chinese medicine, the study of catalpol may facilitate the study of other iridoids for treating neurodegenerative diseases. Acknowledgments—This work was partly supported by The Chinese National Natural Science Foundation No.30873056, partly supported by The Shanghai Unilevel Research and Development Foundation 06SU07006 and partly supported by a grant from Shanghai Municipal Education Commission 09YZ97. The authors declare that they do not have any financial or other involvement that might bias the present work.

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(Accepted 23 January 2010) (Available online 1 February 2010)