Neuroprotection of Brain-Targeted Bioactive Dietary Artoindonesianin

Mar 30, 2015 - The novel effects of artoindonesianin O, a dietary phenolic compound from mulberry, were investigated on oligomer Aβ42-, NMDA- or okad...
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Neuroprotection of Brain-Targeted Bioactive Dietary Artoindonesianin O (AIO) from Mulberry on Rat Neurons as a Novel Intervention for Alzheimer’s Disease Aimin Qiao,† Yihai Wang,§ Wanwan Zhang,† and Xiangjiu He*,§ †

School of Biosciences and Biopharmaceuticals, Guangdong Pharmaceutical University, Guangzhou 510006, China School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China

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ABSTRACT: The novel effects of artoindonesianin O, a dietary phenolic compound from mulberry, were investigated on oligomer Aβ42-, NMDA- or okadaic-acid-induced neurotoxicity and the restorative effect on the oligomer Aβ42-induced synapses dysfunction using rat hippocampus neuron cells in vitro. The phenolic compound of AIO can exert neuroprotection by blocking oligomer Aβ42- or NMDA-induced neurotoxicity and okadaic-acid-induced tau protein hyperphosphorylation through inhibiting the expression of kinase p-ERK1/2. Meanwhile, it is also beneficial to synaptic plasticity. These interesting results strongly suggest that AIO, which is rich in abundant sources of mulberry and other fruits, is suitable and possible candidate for the development of general food type neuroprotection on AD by protecting against brain damage and memory impairment. KEYWORDS: Alzheimer’s disease, artoindonesianin O, hyperphosphorylated tau, mulberry, synaptic plasticity

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INTRODUCTION

Mulberries (Morus alba L.) are delicious and nutritious berries, which are low in calories and contain health-promoting phytonutrients like polyphenols and vitamins. In oriental medicine, especially in traditional Chinese medicine, the mulberry has long been used to treat and prevent diabetes, and as a general tonic to enhance health. Consumption of mulberry has been linked to the prevention of various chronic diseases. Mulberry has been found to have potent antioxidant activity,9 antitumor activity,10 hypolipidemic effect,11 and neuroprotective activity.12,13 Many of these bioactivities were linked to the presence of phenolics in mulberry fruit. Artoindonesianin O (AIO, Figure 1), isolated from the

Alzheimer’s disease (AD) continues to be the leading cause of dementia in the elderly. It is a progressive neurodegenerative disorder. This disease is characterized by extracellular accumulation of senile plaques composed of insoluble βamyloid (Aβ) peptides and intracellular accumulation of neurofibrillary tangles formed of hyperphosphorylated microtubule-associated protein tau.1 In fact, numerous other structural and functional alterations ensue, including neuroinflammatory responses,2 oxidative stress,3 mitochondrial dysfunction,4 and disturbances of cholesterol and lipid metabolism.5 The combined consequences of all the pathological changes, including the effects of the Aβ and tau pathologies, are severe neuronal and synaptic dysfunction and memory loss. Early studies show that Aβ40 and Aβ42 are two major forms to produce Aβ plaque. In contrast to Aβ40, Aβ42 is more neurotoxic as a result of its higher hydrophobicity, which leads to faster oligomerization. Aβ oligomers can cause features of AD pathology even in absence of senile plaques in transgenic mice lacking the ability of Aβ fibrillization. Tau protein was first known for its function as a microtubule stabilizer. Although basal levels of phosphorylated tau promotes axonal stability, enabling synaptic plasticity and axonal transport of organelles to and from cell bodies to nerve terminals,6 its hyperphosphorylation at certain epitopes in the adult brain is pathological and specifically related to degeneration, cognitive impairment, and dementias. In familial and sporadic tauopathies, there is clear evidence showing that tau pathology alone can cause neurodegeneration.7 The kinases that can hyperphosphorylate tau are GSK3, CDK5, p38 MAP kinase, JNK, ERK, protein kinase A, protein kinase C, and calmodulin kinase II.8 Inhibition of tau kinase will show promising effects to block the progression of AD. © 2015 American Chemical Society

Figure 1. Chemical structure of AIO.

mulberry, was reported to inhibit nitric oxide production in RAW264.7 cells and has significant inhibitory activity toward the differentiation of 3T3-L1 adipocytes. It is also used as an antiobesity and antidiabetic agent.14 However, little is known about its function on neuroprotection. Increasing evidence has shown that dietary factors improve neuronal function and synaptic plasticity. Mechanisms underlying such actions have primarily been characterized through antioxidant and antiinflammatory bioactivities and signaling regulation at the molecular level.15,16 Received: Revised: Accepted: Published: 3687

January 22, 2015 March 23, 2015 March 30, 2015 March 30, 2015 DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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Journal of Agricultural and Food Chemistry In our pilot study, we first find that AIO can exert neuroprotection by blocking oligomer Aβ42- or NMDAinduced neurotoxicity and OA-induced tau protein hyperphosphorylation through inhibiting the expression of kinase pERK1/2. Meanwhile, it may reflect the potential activation of the Aβ-decomposing system, and this is also beneficial to synaptic plasticity. Consequently, AIO, which is abundant in mulberry and other fruits, is a suitable and promising candidate for the development of general food type neuroprotection on AD.

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Oligomer Aβ42 Preparation. Briefly, Aβ42 peptide was dissolved in cold hexafluoro-2-propanol (HFIP) to a final concentration of 1 mM. The peptide was aliquoted and dried under vacuum. The aliquoted peptides were stored at −80 °C until use. For use in cell experiments, the peptide was dissolved in DMSO to a final concentration of 5 mM and then diluted to 100 μM in Ham’s/F12 media. Cell Viability Assay. Neuron cells(DIV14) in culture medium were pretreated by 10 μM AIO for 1 h before incubated by 10 μM oligomer Aβ42 for another 24 h or pretreated by 100 μM NMDA for 24 h then the AIO was used. After 24 h of treatment, 0.5 mg/mL MTT was subsequently added to each well. After 4 h of additional incubation, 100 μL of DMSO was added to dissolve the crystals. The absorption values at 570 nm were determined with a microplate reader. Experiments were performed in triplicate for each sample. ATP Assay. ATP luminescence were detected followed the manufactured information with modification to evaluate the cells viability after Aβ42 or NMDA treatment. Neuron cells in culture medium were pretreated by 10 μM AIO for 1 h before treatment with 10 μM oligomer Aβ42 for another 24 h, or cells were pretreated by 100 μM NMDA for 24 h, and then the AIO was used. The plates and its contents were equilibrated at room temperature for approximately 30 min after 24 h of treatment. Cell titer reagent (100 μL) was added to each well in a 96-well plate, and the plate was shaken gently for 2 min to induce cell lysis. The plate was allowed to incubate at room temperature for 10 min to stabilize the luminescent signal, and then the luminescence was recorded. OA and AIO Treatment. To investigate the effects of OA-induced neurotoxicity, the cultured neurons (DIV14) were preincubated with different concentrations of AIO (0, 0.1, 0.3, 1, 3, 10 μM) for 1 h, and then the neurons were treated with10 nM of OA for 24 h at 37 °C. Western Blot Analysis. Neuron cells treated by AIO and OA or treated by AIO and oligomer Aβ42 were washed in cold PBS and then were lysed in a RIPA buffer that contained a mixture of protease inhibitors. Fifteen micrograms of total protein from each sample were run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with appropriately diluted primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies against the corresponding species. Labeling was detected using the ECL system (Amersham Biosciences). Reported values are representative of three independent experiments. Electroperation Transfection. The hippocampus neuron cells were counted, and the required number of cells was centrifuged (4 × 106 cells per sample) at 800 rpm for 5 min at room temperature. The supernatant was removed completely, and the cell pellet was resuspended carefully in 100 μL of room-temperature nucleofector solution per sample with 4 μg GFP plasmid DNA. The cell/DNA suspension was transferred into a certified cuvette (sample must cover the bottom of the cuvette without air bubbles). The cuvette with cell/ DNA suspension was inserted into the Nucleofector Cuvette Holder, and the appropriate Nucleofector program was selected to finish the transfection. Immunofluorescence and Confocal Microscopy. The transfected cells pretreated by 10 μM AIO 1 h before 10 μM oligomer Aβ42 treatment were washed with Dulbecco’s phosphate buffered saline (DPBS, pH 7.4), fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min, and then imaged with a confocal laser-scanning microscope (LSM510, Zeiss) using the 63× oil-immersion objective. The selected areas are zoomed according to requirement. Spine density was expressed as the number of spines per 10 μm of dendritic length. Statistical Analysis. Data are expressed as the mean ± SD. Analyses were performed using Prism 5 software with a two-way ANOVA to identify significant effects. Differences were considered significant at p < 0.05, n = 3.

MATERIALS AND METHODS

Plant Material and Reagents. Dried mulberries (Morus alba L.) were purchased from Wuhan Qiangkang Pharmaceutical Co. Ltd. (Wuhan, China). We also used the following in our procedures: poly-D-Lysine (Sigma), N-methyl-D- aspartate (Sigma), Neurobasal (Gibco), B27 (Invitrogen), Aβ42 peptide (American peptide), rabbit polyclonal antip-tau(Ser396) (1:500; Invitrogen), p-CDK5(1:500; Santa Cruz), PGSK3α/β(1:500; Santa Cruz) rabbit polyclonal anti-p-tau(Ser235) (1:500; Santa Cruz), mouse monoclonal anti-AT270 (1:500; Santa Cruz), mouse monoclonal anti-tau5 (1:500; Millipore Biotechnology), rabbit polyclonal anti-ERK, JNK, p-ERK1/2, p-JNK1/2 (1:500; Cell Signaling), 6E10(1:1000; Signet), MTT(Sigma-Aldrich Chemical Co). Amaxa Rat Neuron Nucleofector Kit (Lonza), CellTiter-Glo Luminescent Cell Viability Assay (Promega), Microplate reader (IMark, Bio-Rad), AmaxaNucleofector (Lonza), GloMax Luminometer (Promega), ChemiDoc XRS ECL system (Bio-Rad). Extraction and Purification of AIO from Mulberry. Dried mulberries (15.0 kg) were extracted three times using 5× volumes of 70% ethanol for 2 h at reflux. The filtrate was concentrated to dryness under vacuum at 50 °C to afford 70% ethanol extract. The 70% ethanol extract was suspended in 10 L of distilled water and successively partitioned three times with the same volume of hexane, CHCl3, EtOAc, and n-butanol, which yielded hexane (123.0 g), CHCl3 (148.0 g), EtOAc (78.0 g), n-BuOH (246.0 g) and water-soluble extracts, respectively, after organic solvent was removed under vacuum at 50 °C. The chloroform-soluble fraction (148.0 g) was further purified by silica gel chromatography and eluted with a CHCl3/MeOH gradient elution (the ratios of CHCl3/MeOH were from 100:0 to 0:100). The CHCl3/MeOH (20:1) elution (4.1 g) was subjected to a silica gel column, followed by a Sephadex LH-20 column and a semipreparative HPLC column (Cosmosil 5C18-MS- II, 10 ID × 250 mm), resulting in a yield of 27.0 mg for AIO. Primary Culture of Rat Hippocampus Neurons. Primary hippocampus neurons were prepared from Sprague-Dawley rats at 18 embryonic days according to a previously established method with few modifications.17 Briefly, hippocampus from fetal rat brains (free of meninges, olfactory bulbs, and striata) were pooled, chopped in Ca2+and Mg2+-free HBSS, and incubated with 0.025% trypsin for 10 min at 37 °C. The enzymatic digestion was terminated by mixing the suspension with an equal volume of DMEM supplemented with 10% FBS. The cells were passed through 70 μm cell strainers (BD Falcon, Bedford, MA) and collected by brief centrifugation. The hippocampus neuron cells are resuspended in neurobasal medium with 2% B27, 0.5 mM glutamine, 25 μM glutamate, 50 units/mL penicillin, and 50 μg/ mL streptomycin or transfected by GFP plasmid DNA using electroperation method. The dissociated cells were plated on 96 well plates or 6 well plates (precoated with 10 μg/mL poly-D-lysine) in seeding medium consisting of neurobasal medium supplemented with 2% B27. The cultures were maintained at 37 °C in a humidified incubator with 5% CO2. One day following plating, the seeding medium was removed and replaced with maintenance medium (seeding medium without glutamate) and refreshed twice a week. Neurons are the principal surviving cell type under these serum-free culture conditions.

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RESULTS Structure Identification of AIO. AIO was obtained as a white powder. The ESI-MS showed the [M − H]− ion at m/z 3688

DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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Journal of Agricultural and Food Chemistry

Figure 2. Neuroprotection of AIO on cultured neuron cells treated by oligomer Aβ42 or NMDA. (A) Neuron cells (DIV14) were pretreated with or without 10 μM AIO for 1 h before 10 μM oligomer Aβ42 treatment; or neurons were pretreated by 100 μM NMDA before treatment with or without 10 μM AIO; or neurons were treated by only AIO, then the cells were cultured for another 24 h to detect cell viability using MTT method. (B) Neuron cells (DIV14) were pretreated by 10 μM AIO for 1 h before 10 μM oligomer Aβ42 treatment, then the cells were cultured for another 24 h to detect the ATP level. (C) Neuron cells (DIV14) were pretreated by 100 μM NMDA for 1 h before different does of AIO treatment, and then the cells were cultured for another 24 h to detect the ATP level. * P < 0.05, **P < 0.01, ***p < 0.001, n = 3.

Figure 3. Effect of AIO on Aβ-decomposition system in cultured neurons. After pretreatment without or with 10 μM AIO for 1 h, rat hippocampus neurons (DIV 14) were treated by 10 μM oligomer Aβ42 for 24 h. After being washed with cold PBS, Aβ oligomers in the whole cell lysates were detected by Western blot analysis (12% sodium dodecyl sulfate polyacrylamine gel; a mouse anti-Aβ monoclonal antibody 6E10) and actin was used as a loading control in the same membrane. ***p < 0.001, n = 4.

oligomer Aβ42 treatment compared with negative control. To our surprise, the ATP level of neuron in AIO pretreatment group was enhanced compared with the 10 μM oligomer Aβ42 treatment group (Figure 2B). Excitotoxicity is a key mechanism of cell death following acute neuronal injury that involves deregulation of intracellular calcium. To model excitotoxicity, we used the agonist NMDA to activate NMDA receptors in primary neuron cells.19 The cells at DIV 14 were pretreated with 100 μM NMDA for 1 h, except for the control group, followed by returning to conditioned media without or with different concentrations of AIO. The ATP level was detected after 24 h treatment. The result clearly showed that NMDA can reduce the cell ATP level, but in the 3 μM and 10 μM AIO groups, there is a remarkable reverse in the reduction (Figure 2C). AIO May Reflect the Potential Activation of AβDecomposing System. Because AIO could protect the neurons treated by oligomer Aβ42, we wanted to know whether AIO could partly activate the intracellular Aβdecomposing system to exert its neouroprotection. Interestingly, AIO could significantly decrease cellular Aβ42 tetramer and trimer compared with no AIO treatment group (Figure 3A,B).

323, and its molecular formula was drawn as C20H20O4 from its 1 H and 13C NMR. In the 1H NMR (400 MHz, acetone-d6), an ABX spin-coupling system was ascribed to the three protons of the benzene ring, with signals at 7.43 (H, d, J = 8.4 Hz), 6.99 (H, d, J = 2.4 Hz), and 6.81 ppm (H, dd, J = 8.4, 2.4 Hz), respectively. The proton signals at 5.13 (H, tq, J = 6.5, 1.4 Hz), 3.47 (2H, d, J = 6.4 Hz), 1.67 (3H, s), and 1.64 ppm (3H, d, J = 1.2 Hz) were ascribed to a prenyl group in the molecule. The signal at 3.83 ppm (3H, s) was ascribed to a methoxy group. In the 13C NMR, there were 20 carbons, including a benzofuran, a prenyl, and a benzene ring. Compared to the reported data,18 it was identified as artoindonesianin O (AIO), the chemical structure of which was given in Figure 1. AIO Can Exert Neuroprotection after Oligomer Aβ42 or before NMDA Treatment on Hippocampus Neuron Cells. Previous study has demonstrated that oligomer Aβ42 is toxic to a neuron and that NMDA can induce excitotoxicity. In this study, we first investigated the cell viability using the MTT method. The result clearly showed that AIO can significantly enhance the cell viability after oligomer Aβ42 or before NMDA treatment; however, AIO alone has no impact on cell viability even at the concentration of 10 μM (Figure 2A). At the same time, we also detected the ATP level of neurons. We found that the ATP level of neuron was remarkably reduced after 10 μM 3689

DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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Journal of Agricultural and Food Chemistry

Figure 4. Expressions of okadamic acid (OA)-induced hyperphosphorylated tau-related proteins on neuron cells treated by AIO. Rat hippocampus neurons (DIV 14) were pretreated by various concentrations of AIO (0−10 μM) for 1 h, and then the cells were continuously treated by 25 nM OA for 24 h. Western blot analysis was used to detect the expressions of hyperphosphorylated tau, total tau, and its kinase (panel A). The optical density of the each band was as 1 arbitrary densitometry unit, normalized to actin for the same membrane (panels B−F). * P < 0.05, **P < 0.01, ***p < 0.001 vs Control.

Figure 5. Morphological change on cultured neuron cells transfected by GFP after oligomer Aβ42 and AIO treatment. Neuron cells were transfected by GFP plasmid DNA on the cultured day, and then images of the cells were taken using a confocal microscope (63×) after 21 days of culture (panel A), in which cells were pretreated by 10 μM AIO for 1 h before 10 μM oligomer Aβ42 treatment. Graphical result of dendritic spines on cultured neuron cells (panel B). * P < 0.05, **P < 0.01 vs control. Scale bar represents 10 μM.

AIO Attenuates OA-Induced Tau Hyperphosphorylation and Protects the Neuron from Neurotoxicity. On the

basis of the above result, we wondered whether or not AIO could impact the tau hyperphosphorylation on cultured neuron 3690

DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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Journal of Agricultural and Food Chemistry cells. OA was used to induce tau hyperphosphorylation.20,21 Interestingly, we found that AIO could significantly reduce the expressions of tau hyperphosphorylation at Ser396 and Ser235 from dosage 1 to 10 μM. But it was almost no action about AT270 at Thr181 site (Figure 4A−D). AIO Has No Obvious Effect on Total Tau (Tau5) Level. Because of the reduction of tau hyperphosphorylation at Ser396 and Ser235, we further investigated the expression of total tau using the same samples. As we expected, total tau did not change (Figure 4A). The quantity result of total tau was shown in Figure 4E. AIO Inhibits the Expression of Hyper-Phosphorylated Tau Kinase on Neuron Cells. Tau hyperphosphorylation is mediated by several kinases, many of which show large overlap in their ability to phosphorylate tau at the same epitopes in AD.22 It has been reported that the inhibition of PP2A by OA induced a dramatic increase in the phosphorylation/activation of ERK1/2, and thus, OA could promote the activity of ERK1/ 2 to phosphorylate the tau in vitro at many abnormal hyperphosphorylatation sites and cause neurotoxicity.23 Our result showed that the expression of p-ERK1/2 was reduced after AIO treatment in a dose over 1 μM. At the same time, the total ERK did not change. Another kinase, p-JNK1/2, did not change (Figure 4A, 4F). Other related kinases, including pCDK5 and p-GSK3α/β, remained unchanged (data not shown). AIO Elevates the Number of Dendritic Spines on Neurons Treated with Oligomer Aβ42. Dendritic spines are related with synaptic plasticity and memory loss in AD. On the basis of the above result, we were curious to know that whether AIO can exert its neuroprotection to improve the synaptic plasticity. Our methods helped to find a compelling result. The numbers of dendritic spines were significantly reduced in the oligomer Aβ42-treated group compared with the control group. Moreover, in the combined AIO oligomer and Aβ42 treatment, the dendritic spines numbers were remarkably increased as expected (Figure 5A,B).

oligomerization. Excitotoxicity is a distinct mode of neuronal death that is attributed to increased presynaptic glutamate release. Disturbance of extracellular glutamate levels acting on NMDA receptors (NMDAR) results in enhanced calcium influx and death of the cells. Overactivation of NMDAR is a major cause of cell death following acute neuronal injury and is also implicated in neurodegenerative diseases, such as Parkinson, Huntington, and Alzheimer’s disease.17 As we expected, AIO can reverse the neuronal cell death induced by NMDA. All of those data clearly show for the first time that AIO has neuroprotection on hippocampus neuron cells. Tau-based strategies have received little attention until recently despite that the presence of extensive tau pathology is central to Alzheimer’s disease. The discovery of mutations within the tau gene that causes front-temporal dementia demonstrated that tau dysfunction, in the absence of amyloid pathology, was sufficient to cause neuronal loss and clinical dementia. Abnormal levels and hyperphosphorylation of tau protein have been reported to be the underlying cause of a group of neurodegenerative disorders collectively known as “tauopathies”. The detrimental consequence is the loss of affinity between this protein and the microtubules, increased production of fibrillary aggregates, and the accumulation of insoluble intracellular neurofibrillary tangles in AD.25 OA can induce microtubule-associated protein tau hyperphosphorylation leading to the pathological process of analogical AD.23,26 OA selectively inhibits the serine/threonine phosphatases 2A thereby induces hyperphosphorylation of tau. Hyperphosphoryltion of tau is triggered by multiple mechanisms involving MAP kinases via ERK1/2, Jun N-terminal kinase and other cellular stress and so on. The consequence of tau hyperphosphorylation is neurotoxicity. Thus, the inhibitory effect on tau hyperphosphorylation may reduce the damage of axonal transport and thereby benefit cognitive function. In this study, we wonder whether AIO can impact the tau hyperphosphorylation induced by OA on neurons. Interestingly, we find that AIO significantly reduces the expression of hyperphosphorylation of tau at Ser396 and Ser235, which had been reported to have higher disease incidences in AD brain, but it remains unchanged at about AT270 at the Thr 181 site. Is the reduction of tau hyperphosphorylation at Ser396 and Ser235 followed by the reduction of total tau (tau5)? With this question, the level of total tau is evaluated using the same sample. As we expected, the total tau was not remarkably changed in this process. These studies provide evidence that targeting p-tau may represent an effective treatment strategy. It is well-known that phosphorylation of tau at certain sites was involved in AD pathogenesis. Some kinases, including cyclin-dependent kinase 5, GSK3α/β, casein kinase 1, protein kinase A, and MAPKs, have been implicated in tau phosphorylation.27 In this study, we want to know whether the decrease in hyperphosphorylated tau was due in part to an inhibition of tau kinases. The results showed that p-ERK1/2 was involved in the reduction of hyperphosphorylated tau after AIO treatment despite no change in the level of total ERK, but p-JNK1/2, p-CDK5, and p-GSK3α/β keep the basal level. AIO may possibly compete with OA to recover the activity of PP2A and reduce the ERK phosphorylation, but a detailed mechanism still needs to be clarified. It is known that both Aβ and phosphorylation tau are correlated with synaptic and cognitive deficits.28 Aβ toxicity is mediated not only by the fibrillar form of the protein but also by the soluble oligomers. The dendritic spines have been

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DISCUSSION The past decade has witnessed an intense interest in herbal medicines, in which phytochemical constituents can have longterm health-promoting or medicinal qualities. Phytochemicals present in vegetables and fruits are believed to reduce the risk of several major diseases, including cardiovascular diseases, cancers, as well as Alzheimer’s disease. Identification and characterization of new medicinal plants to cure neurodegenerative diseases has been a major and increasing scientific interest in recent years. In this pilot study, we first evaluated the neuroprotective activity of AIO extracted from mulberry in rat primary hippocampus neurons. As we know, the formation of amyloid plaques is one of the major pathologies of AD. Aβ is produced by endoproteolysis of the parental amyloid precursor protein (APP), which is achieved by the sequential cleavage of APP by groups of enzymes or enzyme complexes termed β- and γ-secretases. Recent studies suggest that the buildup of oligomer Aβ may be an early event in the pathogenesis of AD.24 Also it is the trigger to kill the cells in the AD brain. In this study, the ATP level and MTT method were used to evaluate the cells viability after oligomer Aβ42 treatment. Interestingly, we found that AIO could exert neuroprotection against oligomer Aβ42-induced neurotoxicity partly through the activation of the Aβ decomposition system, resulting in the reduction of cellular 3691

DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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Journal of Agricultural and Food Chemistry

(8) Avila, J.; Lucas, J. J.; Perez, M.; Hernandez, F. Role of tau protein in both physiological and pathological conditions. Physiol. Rev. 2004, 84, 361−384. (9) Yang, X.; Yang, L.; Zheng, H. Hypolipidemic and antioxidant effects of mulberry (Morus alba L.) fruit in hyperlipidaemia rats. Food Chem. Toxicol. 2010, 48, 2374−2379. (10) Jeong, J. C.; Jang, S. W.; Kim, T. H.; Kwon, C. H.; Kim, Y. K. Mulberry fruit (Moris f ructus) extracts induce human glioma cell death in vitro through ROS-dependent mitochondrial pathway and inhibits glioma tumor growth in vivo. Nutr. Cancer 2010, 62, 402−412. (11) Liu, L. K.; Chou, F. P.; Chen, Y. C.; Chyau, C. C.; Ho, H. H.; Wang, C. J. Effects of mulberry (Morus alba L.) extracts on lipid homeostasis in vitro and in vivo. J. Agric. Food Chem. 2009, 57, 7605− 7611. (12) Kang, T. H.; Hur, J. Y.; Kim, H. B.; Ryu, J. H.; Kim, Y. K. Neuroprotective effects of the cyanidin-3-O-beta-D-glucopyranoside isolated from mulberry fruit against cerebral ischemia. Neurosci. Lett. 2006, 391, 122−126. (13) Kim, H. G.; Ju, M. S.; Shim, J. S.; Kim, M. C.; Lee, S. H.; Huh, Y.; Kim, S. Y.; Oh, M. S. Mulberry fruit protects dopaminergic neurons in toxin-induced Parkinson’s disease models. Br. J. Nutr. 2010, 104, 8− 16. (14) Yang, Z. G.; Matsuzaki, K.; Takamatsu, S.; Kitanaka, S. Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells. Molecules 2011, 16, 6010−6022. (15) Hwang, S. L.; Shih, P. H.; Yen, G. C. Neuroprotective effects of citrus flavonoids. J. Agric. Food Chem. 2012, 60, 877−885. (16) Ngoungoure, V. L.; Schluesener, J.; Moundipa, P. F.; Schluesener, H. Natural polyphenols binding to amyloid: A broad class of compounds to treat different human amyloid diseases. Mol. Nutr. Food Res. 2015, 59, 8−20. (17) Bhuiyan, M. I.; Islam, M. N.; Jung, S. Y.; Yoo, H. H.; Lee, Y. S.; Jin, C. Involvement of ceramide in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation in primary cultured cortical neurons of rats. Biol. Pharm. Bull. 2010, 33, 11−17. (18) Hakim, H.; NUlinnuha, U. Z.; Syah, Y. M.; Ghisalberti, E. L. Artoindonesianins N and O, new prenylated stilbene and prenylated arylbenzofuran derivatives from Artocarpus gomezianus. Fitoterapia 2002, 73, 597−603. (19) Jahani-AsI, A.; Pilon-Larose, K.; Xu, W.; MacLaurin, J. G.; Park, D. S.; McBride, H. M.; Slack, R. S. The mitochondrial inner membrane GTPase, optic atrophy 1(Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity. J. Biol. Chem. 2011, 286, 4772−4782. (20) Feng, L. J.; Shen, Y. X.; Sun, A. M.; Shen, Y. J.; Fang, S. Y.; Li, J. Endoplasmic reticulum stress in involved in okadaic acid-induced tau hyperphosphorylation and neurotoxic effects of okadaic acid. Neural Regener. Res. 2010, 5, 1534−1540. (21) Sun, A. M.; Wang, H. P.; Shen, Y. J.; Shen, Y. X.; Fang, S. Y. Differential induction of tau hyperphoephorylation by OA treatment in primary cultured neurons and SH-SY5Y cell. Chin. Pharmacol. Bull. 2010, 27, 41−45. (22) Duka, V.; Lee, J. H.; Credle, J.; Wills, J.; Oaks, A.; Smolinsky, C.; Shah, K.; Mash, D. C.; Masliah, E.; Sidhu, A. Identification of the sites of Tau hyperphosphorylation and activation of tau kinases in synucleinopathies and Alzheimer’s diseases. PLoS One 2013, 8, 1−11. (23) Kamat, P. K.; Shivika, R.; Suprida, S.; Rakesh, S.; Chandishwar, N. Molecular and cellular mechanism of okadaic acid(OKA)-induced neurotoxicity: A novel tool for Alzheimer’s disease therapeutic application. Mol. Neurobiol. 2014, 50, 852−865. (24) LaFerla, F. M.; Green, K. N.; Oddo, S. Intracellular amyloid-beta in Alzheimer’s disease. Nat. Rev. Neurosci. 2007, 8, 499−509. (25) Medina, M.; Avila, J. New perspectives on the role of Tau in Alzheimer’s disease. Implication for therapy. Biochem. Pharmacol. 2014, 88, 540−547. (26) Zhao, J. F.; Wang, D.; Li, L. M.; Zhao, W. H.; Zhao, C. Protective effects of humamin on okadaic acid-induced neurotoxicity in cultured cortical neurons. Neurochem. Res. 2014, 39, 2150−2159.

assigned critical roles in the form of synaptic plasticity and memory. Dendritic spine defects are found in a number of cognitive disorders, including AD. Therefore, we hypothesized that the AIO is critical to synaptic function. In this study, we examined the relationship between AIO and synaptic defects induced by oligomer Aβ42. The result surprised us. In comparison, AIO treatment enhances the density of dendritic spines on neurons induced by oligomer Aβ42. Therefore, AIO may be possibly involved in the synaptic plasticity and may prevent memory loss. Taken together, our results strongly suggest that AIO, a dietary phenolic compound from mulberry, is a potential nutritional candidate for protection against neurodegeneration through the reduction of tau hyperphosphorylation and improvements in the synaptic plasticity. More studies are still needed to elucidate the initial mechanisms that trigger the neuroprotection in order to design appropriate therapeutic interventions to block the progression of AD.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: 86-20-3935-2132. Funding

The present work was supported by the National Natural Science Foundation of China under Grant (U1203103) and the grant from Colleges and Universities in Guangdong province 1000 cultivation of talents in China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank for Dr. Yanfeng Zhao for her assistant in modification the manuscript. ABBREVIATIONS USED Aβ, β-amyloid; AIO, artoindonesianin O; AD, Alzheimer’s disease; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MAPKs, mitogen-activated protein kinases; CDK5, cyclin dependent kinase 5; OA, okadaic acid; NMDA, N-methyl-D- aspartate; GSK3, glycogen synthase kinase 3; DIV, days in vitro

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REFERENCES

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DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693

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DOI: 10.1021/acs.jafc.5b00396 J. Agric. Food Chem. 2015, 63, 3687−3693