The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 μM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca2+ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca2+ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca2+-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca2+-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca2+-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.


Introduction
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS); however, excess levels may cause significant oxidative glutamate toxicity and nerve cell injury, which are closely related to the pathogenesis of many CNS diseases, such as ischemic stroke, epilepsy, and neurodegenerative diseases [1,2]. Therefore, the level of glutamate must be strictly maintained within the nervous system, and agents that modulate its release have potential as therapeutics [3][4][5]. Notably, the use of natural plants in the treatment of neurological diseases has increased considerably [6,7], and many plant-derived compounds are effective in inhibiting presynaptic glutamate release [8][9][10]. Wasabi (Wasabia japonica), a perennial plant that is native to Japan and belongs to the Brassicaceae family, is commonly used in food as a pungent spice [11]. Wasabi has numerous health-promoting effects, such as antioxidant, anti-inflammatory, anti-obesity, anti-cancer, antiviral, and neuroprotective activities [12][13][14][15][16]. Previous studies have shown that wasabi leaves contain flavonoids [11], phenylpropanoids [17], terpenoids [18], and carotenoids [18]. In addition, functional studies have demonstrated biological activity, especially antioxidative effects [17]. Numerous flavonoids with antioxidative activity are known to reduce glutamate release and counteract glutamate-induced oxidative damage to neurons [19][20][21]. In the present study, isosaponarin, a 4 -O-glucosyl-6-C-glucosyl apigenin ( Figure 1A), was chosen because it is one of the flavonoids found in wasabi leaves [11,22]; however, its role in the regulation of glutamate release has not yet been clarified. Therefore, in this study we used isolated nerve terminals (synaptosomes) prepared from the rat cortex to evaluate the effect of isosaponarin on glutamate release, and clarified the related mechanisms that contribute to its effect. Synaptosomes are isolated nerve endings that carry the structural features of neuronal terminals in vivo and are, therefore, extensively used as an in vitro model to evaluate presynaptic effects on neurotransmitter release [23].

Introduction
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS); however, excess levels may cause significant oxidative glutamate toxicity and nerve cell injury, which are closely related to the pathogenesis of many CNS diseases, such as ischemic stroke, epilepsy, and neurodegenerative diseases [1,2]. Therefore, the level of glutamate must be strictly maintained within the nervous system, and agents that modulate its release have potential as therapeutics [3][4][5]. Notably, the use of natural plants in the treatment of neurological diseases has increased considerably [6,7], and many plantderived compounds are effective in inhibiting presynaptic glutamate release [8][9][10]. Wasabi (Wasabia japonica), a perennial plant that is native to Japan and belongs to the Brassicaceae family, is commonly used in food as a pungent spice [11]. Wasabi has numerous health-promoting effects, such as antioxidant, anti-inflammatory, anti-obesity, anti-cancer, antiviral, and neuroprotective activities [12][13][14][15][16]. Previous studies have shown that wasabi leaves contain flavonoids [11], phenylpropanoids [17], terpenoids [18], and carotenoids [18]. In addition, functional studies have demonstrated biological activity, especially antioxidative effects [17]. Numerous flavonoids with antioxidative activity are known to reduce glutamate release and counteract glutamate-induced oxidative damage to neurons [19][20][21]. In the present study, isosaponarin, a 4′-O-glucosyl-6-C-glucosyl apigenin ( Figure 1A), was chosen because it is one of the flavonoids found in wasabi leaves [11,22]; however, its role in the regulation of glutamate release has not yet been clarified. Therefore, in this study we used isolated nerve terminals (synaptosomes) prepared from the rat cortex to evaluate the effect of isosaponarin on glutamate release, and clarified the related mechanisms that contribute to its effect. Synaptosomes are isolated nerve endings that carry the structural features of neuronal terminals in vivo and are, therefore, extensively used as an in vitro model to evaluate presynaptic effects on neurotransmitter release [23].  (C) Inhibition of 4-AP-evoked glutamate release by isosaponarin in a dose-dependent manner; this inhibition was prevented in the absence of extracellular Ca 2+ or in the presence of the vesicular transporter inhibitor bafilomycin A1. Isosaponarin was added 10 min before depolarization, and bafilomycin A1 was added 10 min before this. Data are presented as mean ± SEM (n = 5-14 per group). *** p < 0.001 vs. control group.

Isosaponarin Does Not Change the Synaptosomal Membrane Potent
Since neurotransmitter release can be modulated by regula brane potential and consequently altering the Ca 2+ influx [27], we u tential-sensitive dye DiSC3(5) to determine whether isosaponarin a membrane potential. The dye became incorporated into the synap brane lipid bilayer. Upon depolarization with 4-AP, the release of (1 mM) in the absence (control) or presence of isosaponarin that was added 10 min before stimulation. (B) Effect of isosaponarin on 4-AP-evoked glutamate release in the presence of the Ca 2+ channel toxins ω-conotoxin GVIA or ω-agatoxin IVA, which was added either alone or in combination. Data are presented as mean ± SEM (n = 5 per group). *** p < 0.001 vs. control group; # p < 0.001 vs. ω-conotoxin GVIA-or ω-agatoxin IVA-treated group.

Isosaponarin Does Not Change the Synaptosomal Membrane Potential
Since neurotransmitter release can be modulated by regulating the plasma membrane potential and consequently altering the Ca 2+ influx [27], we used the membrane potential-sensitive dye DiSC 3 (5) to determine whether isosaponarin affects the synaptosomal membrane potential. The dye became incorporated into the synaptosomal plasma membrane lipid bilayer. Upon depolarization with 4-AP, the release of the dye from the membrane bilayer is indicated as an increase in fluorescence [28]. As shown in Figure 3

Isosaponarin Decreases SNAP-25 and MARCKS Phosphorylation Evoked by 4-AP in Synaptosomes
PKC has multiple targets within the exocytotic machinery, including SNAP-25 and MARCKS, which are thought to interfere with synaptic vesicle function [29,31]. We next investigated whether isosaponarin can alter the phosphorylation levels of SNAP-25 and MARCKS. As shown in Figure 5

Isosaponarin Decreases SNAP-25 and MARCKS Phosphorylation Evoked by 4-AP in Synaptosomes
PKC has multiple targets within the exocytotic machinery, including SNAP-25 and MARCKS, which are thought to interfere with synaptic vesicle function [29,31]. We next investigated whether isosaponarin can alter the phosphorylation levels of SNAP-25 and MARCKS. As shown in Figure 5

Isosaponarin Decreases the Release of FM1-43 from Synaptosomes
In order to confirm that isosaponarin affects synaptic vesicle release during 4-AP stimulation, we performed an experiment measuring the release of FM1-43, which tracks actively recycling synaptic vesicles [32]. FM1-43 is incorporated into synaptic vesicles during endocytosis. Accumulated FM1-43 is released during exocytosis when the lumen of the synaptic vesicle is exposed to the extracellular medium. The release of FM1-43 from synaptic vesicles is detected as a decrease in fluorescence [33]. As shown in Figure 6A In order to confirm that isosaponarin affects synaptic vesic

Isosaponarin Inhibits the 4-AP-Induced Decrease in the Number of Synaptic Vesicles in Synaptosomes
In Figure 7A, the synaptosome ultrastructure and the number of synaptic vesicle were observed with transmission electron microscopy (TEM). In the control group, no mal synaptosome ultrastructure was observed: a clear synaptic cleft, postsynaptic densit (PSD), and certain numbers of synaptic vesicles in the presynaptic membrane. In 4-AP treated synaptosomes, the number of synaptic vesicles decreased. However, in the isosa ponarin-pretreated synaptosomes, the 4-AP-caused synaptic vesicle reduction was atten uated. Figure 7B shows significant differences in the numbers of synaptic vesicles betwee control conditions, following treatment with 4-AP, or after treatment with isosaponari and 4-AP (p < 0.05).

Isosaponarin Inhibits the 4-AP-Induced Decrease in the Number of Synaptic Vesicles in Synaptosomes
In Figure 7A, the synaptosome ultrastructure and the number of synaptic vesicles were observed with transmission electron microscopy (TEM). In the control group, normal synaptosome ultrastructure was observed: a clear synaptic cleft, postsynaptic density (PSD), and certain numbers of synaptic vesicles in the presynaptic membrane. In 4-AP-treated synaptosomes, the number of synaptic vesicles decreased. However, in the isosaponarinpretreated synaptosomes, the 4-AP-caused synaptic vesicle reduction was attenuated. Figure 7B shows significant differences in the numbers of synaptic vesicles between control conditions, following treatment with 4-AP, or after treatment with isosaponarin and 4-AP (p < 0.05).

Discussion
Excess glutamate exocytosis from nerve terminals alters synaptic plasticity, contributing to the pathogenesis of most neurological diseases [1,2]. In this context, drugs that inhibit synaptic glutamate release could be therapeutic for disease progression, and natural compounds represent potential candidates for such a therapeutic approach [8][9][10]. Here, we studied the effect of isosaponarin, a flavone glycoside natural product derived from wasabi leaf, on glutamate release in rat cortex nerve terminals (synaptosomes).

Isosaponarin Decreases 4-AP-Evoked Vesicular Glutamate Release by Blocking N-and P/Q-Type Ca 2+ Channels
We observed that isosaponarin inhibited 4-AP-triggered glutamate release from synaptosomes. The release of glutamate triggered by 4-AP from neurons is mediated by Ca 2+dependent vesicular exocytosis and reverse transport by glutamate transporters [34,35]. In this study, the inhibitory effect of isosaponarin on 4-AP-triggered glutamate release was not observed in extracellular Ca 2+ -free solution. The fraction of glutamate release by 4-AP in the absence of external Ca 2+ is completely dependent on the entrance of Na + into the nerve ending, and involves reversal of the glutamate transporters [34]. Therefore, the involvement of decreased reverse transport through glutamate transporters in the isosaponarin-mediated inhibition of 4-AP-triggered glutamate release from synaptosomes is not included in our observation. In addition, the isosaponarin effect was completely

Discussion
Excess glutamate exocytosis from nerve terminals alters synaptic plasticity, contributing to the pathogenesis of most neurological diseases [1,2]. In this context, drugs that inhibit synaptic glutamate release could be therapeutic for disease progression, and natural compounds represent potential candidates for such a therapeutic approach [8][9][10]. Here, we studied the effect of isosaponarin, a flavone glycoside natural product derived from wasabi leaf, on glutamate release in rat cortex nerve terminals (synaptosomes).

Isosaponarin Decreases 4-AP-Evoked Vesicular Glutamate Release by Blocking N-and P/Q-Type Ca 2+ Channels
We observed that isosaponarin inhibited 4-AP-triggered glutamate release from synaptosomes. The release of glutamate triggered by 4-AP from neurons is mediated by Ca 2+dependent vesicular exocytosis and reverse transport by glutamate transporters [34,35]. In this study, the inhibitory effect of isosaponarin on 4-AP-triggered glutamate release was not observed in extracellular Ca 2+ -free solution. The fraction of glutamate release by 4-AP in the absence of external Ca 2+ is completely dependent on the entrance of Na + into the nerve ending, and involves reversal of the glutamate transporters [34]. Therefore, the involvement of decreased reverse transport through glutamate transporters in the isosaponarin-mediated inhibition of 4-AP-triggered glutamate release from synaptosomes is not included in our observation. In addition, the isosaponarin effect was completely prevented by the vesicular transport inhibitor bafilomycin A1. This finding supports the conclusion that isosaponarin inhibits 4-AP-evoked Ca 2+ -dependent vesicular glutamate release from synaptosomes. In addition, an intracellular Ca 2+ rise in nerve terminals, mainly mediated via N-and P/Q-type Ca 2+ channels, is crucial for efficient vesicular neurotransmitter release [26,36,37]. Here, we found that an elevation of [Ca 2+ ] C resulting from nerve terminal depolarization with 4-AP was reduced by isosaponarin. Furthermore, although isosaponarin-induced inhibition of glutamate release persisted after an individual blockade of N-or P/Q-type Ca 2+ channels, a combined blockade of both channel types prevented the effect of isosaponarin. These results suggest that the inhibition of N-and P/Q-type Ca 2+ channel activities together potentially underlies the influence of isosaponarin on vesicular glutamate release from nerve terminals. In addition, neurotransmitter release can be modulated by regulating the plasma membrane potential, which consequently alters Ca 2+ influx [27]. However, the suppression of VGCCs by isosaponarin is not because of an indirect effect through modulation of membrane potential changes, and hence synaptosomal excitability, as we observed that 4-AP-evoked membrane potential depolarization measured with a membrane-potential sensitive dye DiSC 3 (5) was unaffected by isosaponarin. Whether the inhibition of VGCCs by isosaponarin reflects a direct interaction with VGCCs requires further investigation.

Suppression of the Ca 2+ -Dependent PKC/SNAP-25 and MARCKS Pathways and the Consequent Reduction in Available Synaptic Vesicles May Account for Isosaponarin-Induced Inhibition of Vesicular Glutamate Release
PKC, a presynaptically enriched multifunctional enzyme, is known to be a crucial modulator of the exocytotic pathway, where it is involved in the enhancement of both the priming and fusion steps of vesicle exocytosis through phosphorylation of several proteins of the exocytotic machinery [38,39]. The phosphorylation targets of PKC include SNAP-25 and MARCKS [29,31]. SNAP-25 is a component of the SNARE complex, which together with syntaxin-1 and synaptobrevin, mediates synaptic vesicle fusion with the plasma membrane. SNAP-25 is phosphorylated by PKC at Ser187, which is known to enhance Ca 2+dependent release by regulating the refilling of synaptic vesicles [40,41]. MARCKS, another prominent substrate of PKC, regulates F-actin dynamics. Phosphorylation of MARCKS by PKC at Ser152/156 results in F-actin disassembly and increases vesicle recruitment to the plasma membrane [42][43][44]. Therefore, decreased phosphorylation of SNAP-25 or MARCKS by PKC reduces the number of available synaptic vesicles as well as their release probability [3,4]. Based on our finding that isosaponarin inhibits glutamate release through a mechanism supported by N-and P/Q-type Ca 2+ channels, we infer that the decreased Ca 2+ level upon isosaponarin treatment has an impact on Ca 2+ -dependent PKC activity, and consequently on PKC-mediated synaptic vesicle fusion. Several lines of evidence support this hypothesis. Firstly, in synaptosomes treated with the Ca 2+ -dependent PKC inhibitor Go6976, the inhibitory effect of isosaponarin on 4-AP-evoked glutamate release was completely blocked. However, the inhibition of release by isosaponarin was unaffected in the presence of rottlerin, an inhibitor of Ca 2+ -independent PKC. Secondly, isosaponarin reduced the 4-AP-induced phosphorylation levels of PKC, SNAP-25, and MARCKS. Thirdly, in the FM1-43 analysis, the observed decay in FM1-43 fluorescence is a measure of activitydependent synaptic vesicular uptake and release for neurons [32,45]. We found that isosaponarin markedly reduces the release of FM1-43 from synaptosomes during 4-AP stimulation, which indicates that the delivery of synaptic vesicles to the active zone is inhibited. Fourthly, using TEM, we also observed that the 4-AP-caused synaptic vesicle reduction was markedly inhibited in isosaponarin-treated synaptosomes. These results indicate that isosaponarin inhibits 4-AP-evoked glutamate release from synaptosomes by reducing the number of available synaptic vesicles through Ca 2+ -dependent PKC/SNAP-25 and MARCKS mechanisms. In addition to SNAP-25 and MARCKS, however, the possible involvement of other molecules should be considered. For example, Munc13-1 and Munc13-8 have been reported to be phosphorylated by PKC and involved in the regulation of synaptic vesicle recycling [46,47]. Therefore, reduced phosphorylation levels of Munc13-1 and Munc13-8 in isosaponarin-treated synaptosomes may also contribute to the reductions in available synaptic vesicles and release probability.
In the present study, isosaponarin inhibited 4-AP-evoked vesicular glutamate release from rat cortical nerve terminals at concentrations ranging from 20 to 100 µM. Although the dose of isosaponarin used in our study to produce the effect was higher, the action of isosaponarin was specific. For example, isosaponarin reduced the 4-AP-induced increase in [Ca 2+ ] C , whereas it did not affect 4-AP-mediated membrane potential depolarization. Furthermore, the isosaponarin-mediated inhibition of glutamate release was significantly blocked by Ca 2+ -dependent but not Ca 2+ -independent PKC inhibitors. In addition, although we demonstrated that isosaponarin has an inhibitory effect on glutamate release from nerve terminals, the relevance of our findings to in vivo glutamate homeostasis and glutamate excitotoxicity requires further exploration.
Taken together, we report that the isosaponarin-mediated decrease in 4-AP-evoked glutamate release may be explained through the inhibition of N-and P/Q-type Ca 2+ channels, which interrupts SNAP-25 and MARCKS phosphorylation by PKC, and leads to a subsequent reduction in available synaptic vesicles in nerve terminals ( Figure 8). These data shed light on the mechanism of action of isosaponarin in the brain, which could have implications for the role of isosaponarin in neurological diseases associated with glutamate excitotoxicity.
in the regulation of synaptic vesicle recycling [46,47]. Therefore, reduced phosphorylation levels of Munc13-1 and Munc13-8 in isosaponarin-treated synaptosomes may also contribute to the reductions in available synaptic vesicles and release probability.
In the present study, isosaponarin inhibited 4-AP-evoked vesicular glutamate release from rat cortical nerve terminals at concentrations ranging from 20 to 100 μM. Although the dose of isosaponarin used in our study to produce the effect was higher, the action of isosaponarin was specific. For example, isosaponarin reduced the 4-AP-induced increase in [Ca 2+ ]C, whereas it did not affect 4-AP-mediated membrane potential depolarization. Furthermore, the isosaponarin-mediated inhibition of glutamate release was significantly blocked by Ca 2+ -dependent but not Ca 2+ -independent PKC inhibitors. In addition, although we demonstrated that isosaponarin has an inhibitory effect on glutamate release from nerve terminals, the relevance of our findings to in vivo glutamate homeostasis and glutamate excitotoxicity requires further exploration.
Taken together, we report that the isosaponarin-mediated decrease in 4-AP-evoked glutamate release may be explained through the inhibition of N-and P/Q-type Ca 2+ channels, which interrupts SNAP-25 and MARCKS phosphorylation by PKC, and leads to a subsequent reduction in available synaptic vesicles in nerve terminals ( Figure 8). These data shed light on the mechanism of action of isosaponarin in the brain, which could have implications for the role of isosaponarin in neurological diseases associated with glutamate excitotoxicity.

Animals
Rats (male, 150−200 g) were obtained from BioLASCO (Taipei, Taiwan), and were housed in the animal center of Fu Jen Catholic University under environmentally controlled conditions (temperature = 21−25 °C, humidity = 40%) on a 12-hour light/dark cycle with food and water ad libitum. All the rats were euthanized by cervical dislocation, followed by decapitation, and the cortices were rapidly removed. The experimental procedures were approved by the Animal Ethics Committee of Fu Jen Catholic University (A11113), according to the Guidelines for Animal Care and Use of the National Institutes

Animals
Rats (male, 150−200 g) were obtained from BioLASCO (Taipei, Taiwan), and were housed in the animal center of Fu Jen Catholic University under environmentally controlled conditions (temperature = 21−25 • C, humidity = 40%) on a 12-hour light/dark cycle with food and water ad libitum. All the rats were euthanized by cervical dislocation, followed by decapitation, and the cortices were rapidly removed. The experimental procedures were approved by the Animal Ethics Committee of Fu Jen Catholic University (A11113), according to the Guidelines for Animal Care and Use of the National Institutes of Health. In line with the 3Rs rules (replacement, refinement, and reduction), every effort was made to reduce the number of animals required to obtain statistically reliable results. The total number of rats used in the study was 34; specifically, the measurements of glutamate release, Ca 2+ concentrations, membrane potential, and protein phosphorylation in nerve terminals-20 animals; FM1-43 release and image-5 animals; TEM-9 animals.

Isolation of Nerve Terminals (Synaptosomes) from the Cortex Regions of the Rat Brains
Rat cortical purified synaptosomes were prepared as previously described [23]. Briefly, the rat cerebral cortex was homogenized in 0.32 M sucrose (pH 7.4) and centrifuged 10 min at 3000× g. The supernatant was recovered and centrifuged again for 15 min at 14,000× g. After discarding the supernatant, the pellet was resuspended in HEPES buffered medium (mM: NaCl 128, KCl 2.4, MgSO 4 1.2, KH 2 PO 4 1.2, CaCl 2 1.0, HEPES 10, and glucose 10, pH 7.4) and then gently layered on a discontinuous Percoll gradient (3, 10, and 23% Percoll in Tris-buffered sucrose). After centrifugation at 32,500× g for 7 min, the layer between 10 and 23% Percoll (synaptosomal fraction) was collected in HEPES buffered medium.

Glutamate Release Assay
Glutamate release from synaptosomes was detected through a glutamate dehydrogenase reaction, as described previously [48,49]. Briefly, synaptosomes (0.5 mg/mL of final protein concentration) were incubated in HEPES-buffered medium containing glutamate dehydrogenase (20 U/mL) (Sigma, St. Louis, MO, USA), β-nicotinamide adenine dinucleotide (NAD + , 1 mM), and CaCl 2 (1.2 mM), at 37 • C for 5 min. In the presence of glutamate, glutamate dehydrogenase reduced NAD + to NADH, a product that fluoresces (excitation and emission wavelengths of 340 and 460 nm, respectively). Fluorescence intensity of NADH was measured in a stirred thermostated cuvette (37 • C) using a PerkinElmer LS55 spectrofluorimeter. Endogenous glutamate released from the synaptosomes to the incubation medium was detected as an increase in NADH fluorescence. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg protein). Values quoted in the text and depicted in bar graphs represent the levels of glutamate that were cumulatively released after 5 min of depolarization, and are expressed as nmol/mg protein/5 min.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.