J Mol Neurosci DOI 10.1007/s12031-015-0566-x

A Potent Multi-functional Neuroprotective Derivative of Tetramethylpyrazine Hai-Yun Chen 1 & Da-Ping Xu 2 & Guo-Lian Tan 1 & Wei Cai 1 & Gao-Xiao Zhang 1 & Wei Cui 2 & Jin-Zhao Wang 3 & Cheng Long 3 & Ye-Wei Sun 1 & Pei Yu 1 & Karl Wahkeung Tsim 4 & Zai-Jun Zhang 1,2 & Yi-Fan Han 2,5 & Yu-Qiang Wang 1

Received: 15 March 2015 / Accepted: 14 April 2015 # Springer Science+Business Media New York 2015

Abstract Neurodegenerative disorders are one of the leading causes of death among the elderly. Therapeutic approaches with a single target have proven unsuccessful in treating these diseases. Structural combination of multi-functional compounds may lead to a molecule with multiple properties. In this study, we designed and synthesized T-006, a novel analog derived from two multi-functional neuroprotective chemicals, tetramethylpyrazine and J147. The methoxyphenyl group of J147 was replaced by tetramethylpyrazine. Bioactivity evalu-

Hai-Yun Chen and Da-Ping Xu contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12031-015-0566-x) contains supplementary material, which is available to authorized users. * Zai-Jun Zhang [email protected] * Yi-Fan Han [email protected] 1

Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Huangpu Road, Guangzhou 510632, China

2

Department of Applied Biology and Chemical Technology, Institute of Modern Chinese Medicine, Hong Kong Polytechnic University, Hong Kong, China

3

School of Life Sciences, South China Normal University, Guangzhou 510631, China

4

Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China

5

State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China

ation showed that T-006 at very low concentrations had multifunctional neuroprotective effects including rescuing iodoacetic acid-induced neuronal loss, preventing oxidative stress-induced neurotoxicity and reducing glutamate-induced excitotoxicity in vitro. Most importantly, T-006 significantly ameliorated memory impairments in APP/PS1 transgenic mice. These multiple functions of a single molecule suggest that T-006 is a promising novel neuroprotective agent for treating various neurodegenerative disorders, including and in particular Alzheimer’s disease. Keywords Neurodegenerative disorders . Multi-functional neuroprotection . Tetramethylpyrazine . J147

Abbreviations AD Alzheimer’s disease TMP Tetramethylpyrazine CGNs Cerebellar granule neurons PC12 Pheochromocytoma cell DIV Days in vitro ROS Reactive oxygen species RNS Reactive nitrogen species IAA Iodoacetic acid

Introduction Neurodegenerative disorders including Alzheimer’s disease (AD) are among the leading causes of death and disability in the elderly (Marx 2006). These medical problems are becoming much more serious with the rapid increase in the aging populations. Neuronal loss plays a central role in the development and process of these diseases (Lin and Beal 2006). However, the detailed mechanisms underlying the neuronal

J Mol Neurosci

loss of these diseases are largely unknown. Many studies have shown that neuronal loss might be attributed to multiple causes, including genetic, environmental, and endogenous factors (Zolezzi et al. 2013; Cao et al. 2010). Therefore, therapeutic approaches with a single target could not effectively modify the processes of these diseases. Their results are limited to relieving the symptoms of these diseases. Recent studies have demonstrated that some common pathways, such as the reduction of cellular energy metabolism, the increase of oxidative stress, and the induction of excitotoxicity, are involved in the neuronal loss during these brain disorders (Jove et al. 2014; Radi et al. 2014). Hence, it is important and necessary to design and identify multiple functional drug candidates which could inhibit these common pathways leading to neuronal loss. Traditional drug development approach requires identification of a specific disease target and screening of high affinity ligands for this disease target (Koehn and Carter 2005). However, such approach is proven unsuccessful in treating complex brain disorders that involve multiple factors (Citron 2010). A relatively simple strategy for developing novel multiple functional drug candidates starts from chemical modification of compounds with some desirable biological properties. Structural modification and/or combination of such compounds may lead to novel molecule with multiple properties derived from the original compounds (Liu et al. 2008). It is worth noting that this novel molecule may also possess unique properties that are distinct from the original compounds. Tetramethylpyrazine (TMP) is one of the most important active ingredients of a Chinese herb Ligusticum wallichii Franch. TMP has diverse biological activities. It is found to inhibit platelet aggregation, lyse blood clot, block calcium entry, increase cerebral blood flow, inhibit cell apoptosis, and scavenge free radicals. However, the therapeutic efficacy of TMP is limited due to its weak activities. Structural modifications need to be done to increase the bioactivities of TMP. Previously, we had reported two TMP nitrone compounds, termed TBN and TN-2. Both TBN and TN-2 displayed multiple functions and conferred neuroprotective effects in neuronal cell cultures and rat models of ischemic stroke (Xu et al. 2014a; Sun et al. 2008). J147 (Fig. 1) is a novel compound derived from curcumin and cyclohexyl-bisphenol A (CBA) (Chen et al. 2011). Previous studies have shown that J147 has broadly

Fig. 1 Structures of TMP, J147, and T-006

neuroprotective effects, including inhibiting oxidative stress, reducing trophic factor withdrawal-induced neurotoxicity, and preventing the reduction of energy metabolism (Chen et al. 2011; Prior et al. 2013). To augment the therapeutic effect of TMP and discover novel multi-functional molecule for the treatment of neurodegenerative disease, in the current work, we designed and synthesized a new TMP derivative, termed T-006 (Fig. 2), in which the methoxyphenyl group of J147 was replaced with TMP. The methoxyphenyl group has no bioactivities, while TMP has diverse pharmacological functions. We investigated its protective effects against different insult factor-induced neuronal damage in vitro and memory impairments in APP/PS1 transgenic mice.

Materials and Methods Chemicals and Reagents Unless otherwise noted, all media and supplements used for cell cultures were purchased from Invitrogen (Carlsbad, CA, USA). Cytotoxicity detection kit was purchased from Roche (Indianapolis, IN, USA). Hydroxyphenyl fluorescein (HPF), 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA), 3-amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM), and dihydrorhodamine 123 (DHR123) were from Molecular Probes (Invitrogen, Carlsbad, CA, USA). All other reagents were purchased from Sigma (St Louis, MO, USA). T-006 and J147 were synthesized in our laboratory.

Fig. 2 T-006 prevents glutamate-induced excitotoxicity in CGNs. CGNs were pre-incubated with various agents at the indicated concentrations for 2 h and exposed to 75 μM glutamate. Cell viability was measured at 24 h post t-BHP challenge by using the MTT assay. Data, expressed as percentage of control, were the mean ± SD of three separate experiments; ###p < 0.001 versus control group; **p < 0.01 versus glutamate group

J Mol Neurosci

Chemical Synthesis

Primary Cortical Neurons Cultures

Compound 1 (3,5,6-trimethylpyrazine-2-carbaldehyde) was synthesized using a method we previously reported (Sun et al. 2008). T-006 ((E)-N-(2,4-dimethylphenyl)-2,2,2-trifluoro-N′-((3, 5,6-trimethylpyrazin-2-yl)methylene) acetohydrazide): to compound 1 (1.50 g, 10 mmol) in ethanol (10 mL), the commercially available 2,4-dimethylphenylhydrazine hydrochloride (1.72 g, 10 mmol) was added. The resulting reaction mixture was stirred at room temperature under N2 atmosphere for 10 min and a brick red solid was precipitated. The mixture was filtered. The solid was washed with ethanol and methyl butyl ether, dried in vacuum to afford compound 3 as a brick red solid. The unstable compound 3 was used directly in the next step without further purification. To a solution of compound 2 (3.04 g, 10 mmol, 1.0 eq) in a solution of triethylamine (1.68 mL) and methanol (25 mL), trifluoroacetic anhydride (1.7 mL, 12 mmol) was added. The mixture was stirred at 0 °C for 30 min. Solvent was removed in vacuo. 1 H NMR spectra were recorded at ambient temperature on a 300-MHz spectrometer (AV-300, Bruker) in CDCl3. The chemical shift values were expressed in parts per million relative to tetramethylsilane as an internal standard. Electrospray ionization mass spectra (ESI-MS) was obtained in the positive ion detection mode on a Finnigan LCQ Advantage MAX mass spectrometer (Applied Biosystems, 4000 Q TRAP). Elemental analysis was performed at the experimental center of Jinan University, Guangzhou, China, and the results were within ±0.4 % of the theoretical values unless otherwise noted.

Fetal rat brains from Sprague-Dawley pregnatal rats (The Experimental Animal Center of Sun Yat-sen University) of 16–18 days of gestation were used. Cortical neurons were obtained as described previously with minor modifications (Fu et al. 2006). Following enzymatic treatment (TrypLE Express) for 15 min at 37 °C, isolated neurons were suspended in commercial EMEM containing 0.3 g/L glutamine, 4 g/L glucose, 10 % heat-inactivated iron-supplemented calf serum, and 1 % (v/v) penicillin/streptomycin mixture. Cells were placed on 96-well at a density of 4–5×104 cells/well and were placed on 12-well at 3–4×105 cells/well. The plates were precoated with 10 μg/mL of poly-L-lysine, to allow the attachment of neurons themselves to the plates. Cortical neurons were grown at 37 °C in a humidified incubator with 5 % CO2/95 % air atmosphere. After 4–6 h, unattached cells and debris were removed by replacing the initial medium with fresh Neurobasal medium containing B27 supplements. Subsequent partial medium replacement was carried out twice a week, and cultures at 10–12 DIV were used for the experiments.

Ethics Statement All animal studies were conducted according to the guidelines of the Experimental Animal Care and Use Committee of Jinan University. The experimental protocols were approved by the Ethics Committee for Animal Experiments of Jinan University. Primary Cerebellar Granule Neuron Cultures Cerebellar granule neurons (CGNs) were prepared from 8day-old Sprague-Dawley rats (The Experimental Animal Center of Sun Yat-sen University) as described in our previous publication (Cui et al. 2013b). Neurons were seeded at a density of 1.0–1.5 × 106 cells/mL in basal modified Eagle’s (BME) medium containing 10 % fetal bovine serum, 25 mM KCl, 2 mM glutamine, and penicillin (100 units/mL)/streptomycin (100 μg/mL). Cytosine arabinoside (10 μM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. Cultures at 8 days in vitro (DIV) were used for the experiments.

PC12 Cell Cultures PC12 cells (American Type Culture Collection, Manassas, VA, USA) were grown in Ham’s F12K medium supplemented with 12.5 % (v/v) horse serum, 2.5 % (v/v) fetal bovine serum, and 1 % (v/v) penicillin/streptomycin mixture. Cells were seeded in 96-well plates (100 μL/well) at a concentration of 1×105 cells/mL. Cultures were maintained at a 37 °C humidified atmosphere containing 5 % CO2. Experiments were carried out 24 h after the cells were seeded. MTT Assay The percentage of surviving neurons was determined by the activity of mitochondrial dehydrogenases with 3(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. The assay was performed according to the procedure described in our previous publication (Xu et al. 2014b). Cell viability was expressed as a percentage of the value of the cells without stimuli treatment. Lactate Dehydrogenase (LDH) Release LDH leakage is an indicator of cell membrane integrity in neuronal injury. Total and released LDH activities were determined according to the instructions of the cytotoxicity detection kit (Roche Applied Science). Absorbance at 490 nm was measured using a microplate reader.

J Mol Neurosci

Hoechst Staining To observe nuclear changes occurring in apoptosis, the chromatin-specific dye Hoechst 33342 was used. Primary cortical neurons were seeded on 12-well plates. After 10 DIV, neurons were pretreated with T-006 or TMP for 2 h. After subsequent incubation with 100 μM tert-butyl hydroperoxide (t-BHP) for 24 h, cortical neurons were washed with ice-cold PBS, fixed with 4 % paraformaldehyde at room temperature for 15 min, and membrane-permeabilized in 0.1 % Triton X100 for 15 min. Then, cells were washed again with PBS three times and exposed to 10 μg/mL Hoechst 33342 at room temperature in the dark for 10 min. Samples were observed and photos were taken under a fluorescence microscope. Measurement of Mitochondrial Membrane Potential The dye JC-1 was used as a molecular probe to measure mitochondrial membrane potential (Δψm). Primary cortical neurons were placed on 12-well plates at a density of 3–4× 105 cells/well. After 10 DIV, neurons were pretreated with T006 or TMP at the indicated concentrations for 2 h. After subsequent incubation with 100 μM t-BHP for 24 h, the cells were washed in JC-1 buffer and stained with 2 μM JC-1 for 10 min. Fluorescence intensity was measured on a microplate reader using 488 nm excitation and 529/590 nm dual emissions. The mitochondrial accumulation of JC-1 is dependent on Δψm and is reflected by a shift in 529 and 590 nm emissions. Mitochondrial membrane depolarization is indicated by a decrease in the ratio of 590 to 529 nm emissions. Measurement of Intracellular Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) PC12 cells were pretreated with T-006 or TMP at indicated concentrations for 2 h followed by 100 μM t-BHP stimulation for 24 h. Intracellular ROS and RNS, such as hydrogen peroxide, hydroxyl radical, nitric oxide, and peroxynitrite were detected using H2DCF-DA (10 μM), HFP (5 μM), DAF-FM (10 μM), and DHR123 (5 μM), respectively. The fluorescence intensity was acquired using a multi-detection microplate reader. The fluorescence values of the treated group were normalized to the fluorescence of the control cells. HuAPPswe/PS1ΔE9 Transgenic Mice The APP/PS1 transgenic mice (line 85) carrying two transgenes, the mouse/human chimeric APP/Swe which are linked to Swedish FAD and human PS1ΔE9, were characterized by PCR before the experiment. At 5–6 months of age, 12 transgenic mice and 4 wild type littermates, 4 mice of each group, were selected and subjected to the study. Transgenic mice of T-006

treatment group were orally given 2 mg/kg T-006, once daily for 14 days. Control transgenic mice and wildtype mice orally were given an equal volume of vehicle (saline containing 3 % DMSO). Spatial memory was determined using an electric YMaze (Sanxin Laboratory Instrument Company, Zhangjiagang, China) according to the procedure described by Ye et al. with minor modifications (Ye et al. 2010). A Y-Maze test was carried out 1 h after drug administration on days 1, 2, and then every other day. Data are expressed as the average correct ratio and average training time of each group. The Y-Maze apparatus consisted of three identical arms (40 cm length× 10 cm width × 20 cm height, dark opaque Plexiglas) with a conductive grid floor. The three arms were symmetrically oriented at 120° to one another. During the test, electric shocks were administered within two of the arms that are referred to as Bnon-safety zones.^ The third arm was a safety zone in which no electric shocks were administered when a light turned on at the end of this arm. Mice were placed randomly in a non-safety zone, and a fixed resistance shock source was connected to an automatically operated switch (30 V; 5 s delayed). Once a shock was applied, a mouse could escape from further shocks by entering the safety zone. This was counted as one correct response. If the mouse instead entered the other non-safety zone, the response was recorded as an error. The animals were repeatedly trained in this procedure, 30 times per day. By using a ceilingmounted CCD camera, all trials were analyzed for the number of entries the mice made into each arm. The correct ratio and the total time spent in the training were recorded. If the criterion with the correct ratio ≥0.9 and training time ≤300 s was achieved for two consecutive days (24 h between test days), then the animal was regarded as having sufficient spatial learning and memory to be used in the study. Data Analysis and Statistics The experimental data are expressed as the mean±SD. Oneway analysis of variance (ANOVA) and SNK-q test were used to make comparison among the groups using Prism 6 program (GraphPad Software, Inc.). Differences of p