European Journal of Pharmacology 734 (2014) 67–76

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Dimebon, an antihistamine drug, inhibits glutamate release in rat cerebrocortical nerve terminals Che-Chuan Wang a,b, Jinn-Rung Kuo a,b, Su-Jane Wang c,d,n a

Department of Neurology, Chi Mei Medical Center, Tainan, Taiwan Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan c Graduate Institute of Basic Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang District, New Taipei 24205, Taiwan d School of Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang District, New Taipei 24205, Taiwan b

art ic l e i nf o

a b s t r a c t

Article history: Received 3 December 2013 Received in revised form 5 March 2014 Accepted 26 March 2014 Available online 12 April 2014

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of dimebon, an antihistamine with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. Dimebon inhibited the release of glutamate that was evoked by exposing the synaptosomes to the K þ channel blocker 4-aminopyridine, and this effect was prevented by chelating extracellular Ca2 þ ions, and the vesicular transporter inhibitor bafilomycin A1. Dimebon inhibited depolarization-evoked increase in cytosolic free Ca2 þ concentration, and the dimebon-mediated inhibition of glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC. The inhibitory action of dimebon on glutamate release was not due to its decreasing synaptosomal excitability, because dimebon did not alter the resting synaptosomal membrane potential or 4-aminopyridine-mediated depolarization. Furthemore, the dimebon effect on 4-aminopyridine-evoked glutamate release was prevented by the protein kinase C inhibitor, and dimebon substantially reduced the 4-AP-induced phosphorylation of protein kinase C. However, the dimebon-mediated inhibition of glutamate release was unaffected by the N-methyl-Daspartate receptor agonist or antagonist. These results suggest that dimebon inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic voltage-dependent Ca2 þ entry and protein kinase C activity. This implies that the inhibition of glutamate release is an additional pharmacological activity of dimebon that may play a critical role in the apparent clinical efficacy of this compound. & 2014 Elsevier B.V. All rights reserved.

Keywords: Dimebon Glutamate release Cerebrocortical nerve terminals Voltage-dependent Ca2 þ channels Protein kinase C

1. Introduction Dimebon is a non-selective antihistamine drug that has been suggested as a therapeutic application for treating neurodegenerative disorders, including Alzheimer's disease and Huntington's disease (Doody et al., 2008; Kieburtz et al., 2010; Sabbagh and Shill, 2010). Dimebon exhibits numerous benefits for the central nervous system, specifically, neuroprotective effects. For example, dimebon stabilizes neuronal Ca2 þ signals and mitochondrial function (Wu et al., 2008; Zhang et al., 2010), attenuates glutamate-, methamphetamine- and β-amyloid-induced neurotoxicity (Bachurin et al., 2001; Geldenhuys et al., 2012; Lermontova et al., 2001; Perez et al., 2012; Wu et al., 2008), and protects against n Corresponding author at: School of Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang Dist., New Taipei, Taiwan, 24205. Tel.: þ 886 2 29053465; fax: þ886 2 29052096. E-mail address: [email protected] (S.-J. Wang).

http://dx.doi.org/10.1016/j.ejphar.2014.03.052 0014-2999/& 2014 Elsevier B.V. All rights reserved.

N-methyl-D-aspartate (NMDA)-induced seizures (Bachurin et al., 2001). Furthermore, dimebon has been reported to improve cognitive function and delay the progress of brain degeneration in experimental models and clinical trials (Bachurin et al., 2001; Lermontova et al., 2000; Steele et al., 2013; Vignisse et al., 2011; Wang et al., 2011; Webster et al., 2011). However, the precise mechanism of action underlying these neuroprotective effects is unclear. In the brain, glutamate is a major excitatory neurotransmitter that plays an important role in many brain functions such as synaptic plasticity, learning, and memory (Fonnum, 1984). However, in addition to the physiological role of glutamate, excessive glutamate release and activation of the glutamate receptors induce an increase in intracellular Ca2 þ levels, which subsequently triggers a cascade of cellular responses, including enhanced oxygen free radical production, disturbed mitochondrial function, and protease activation, which ultimately kill the neurons (Coyle and Puttfarcken, 1993; Schinder et al., 1996). This process has been

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implicated as a pathophysiological factor in multiple neurological disorders, both acute, such as stroke and head trauma, and chronic, such as neurodegenerative disorders (Meldrum and Garthwaite, 1990; Meldrum, 2000). Modulation of glutamate neurotransmission, such as inhibition of glutamate release at nerve terminals, may provide a potential target for neuroprotective action. Indeed, some neuroprotective drugs (e.g. riluzole, memantine, and minocycline) have been revealed to decrease glutamate release in rat brain tissues (González et al., 2007; Lu et al., 2010; Wang et al., 2004). Because dimebon has a neuroprotective profile, and the excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders, an assessment of the effects of dimebon on glutamate release is warranted. The purpose of the present study was to use isolated nerve terminals (synaptosomes) purified from the rat cerebral cortex to determine whether dimebon affects glutamate release. By using this model, we examined the effects of dimebon on the levels of released glutamate, the synaptosomal plasma membrane potential, and the activation of voltage dependent Ca2 þ channels. In addition, because the role of protein kinase C in presynaptic modulation has been demonstrated (Coffey et al., 1993; Vaughan et al., 1998), we also evaluated whether this signaling pathway is involved in the regulation of glutamate release by dimebon. Finally, because dimebon is known to be an NMDA receptor antagonist (Grigorev et al., 2003; Wu et al., 2008), we examined whether the regulation of glutamate release by dimebon is linked to antagonism at the NMDA receptor.

2. Material and methods 2.1. Chemicals Dimebon, ω-conotoxin MVIIC, dantrolene, bisindolylmaleimide I (GF109203X), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran4-one (PD98059), 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), N-methyl-D-aspartate (NMDA), D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), and DL-threobeta-benzyl-oxyaspartate (DL-TBOA) were obtained from Tocris Cookson (Bristol, UK). 30 ,30 ,30 -dipropylthiadicarbocyanine iodide [DiSC3(5)], and fura-2-acetoxymethyl ester (Fura-2-AM) were obtained from Invitrogen (Carlsbad, CA, USA). The anti-phosphoprotein kinase C (pan) rabbit polyclonal antibody directed protein kinase C-α, -βI, -βII, -ζ, -ε, -η and -δ, phosphorylated at a carboxyterminal residue homologous to Ser-660 of protein kinase C-βII was from Novus (Littleton, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were from BioRad (Milan, Italy). Ethylene glycol bis (β-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), sodium dodecyl sulfate (SDS), and all other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

The tissue was homogenized in a medium containing 320 mM sucrose, pH 7.4. The homogenate was centrifuged at 3000 g (5000 rpm in a JA 25.5 rotor; Beckman Coulter, Inc., USA) for 10 min at 4 1C, and the supernatant was centrifuged again at 14,500 g (11,000 rpm in a JA 25.5 rotor) for 12 min at 4 1C. The pellet was gently resuspended in 8 ml of 320 mM sucrose, pH 7.4. Two milliliters of this synaptosomal suspension was placed into 3 ml Percoll discontinuous gradients containing 320 mM sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3%, 10% and 23% Percoll, pH 7.4. The gradients were centrifuged at 32,500 g (16,500 rpm in a JA 20.5 rotor) for 7 min at 4 1C. Synaptosomes sedimenting between the 10 and the 23% Percoll bands were collected and diluted in a final volume of 30 ml of HEPES buffer medium consisting of 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2  6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES (pH 7.4). Protein concentration was determined using the Bradford assay. Synaptosomes were centrifuged in the final wash to obtain synaptosomal pellets containing 0.5 mg of protein. Synaptosomal pellets were stored on ice and used within 4–6 h. 2.4. Glutamate release Glutamate release from purified cerebrocortical synaptosomes was monitored online, with an assay that employed exogenous glutamate dehydrogenase (GDH) and NADP þ to couple the oxidative deamination of the released glutamate to the generation of NADPH detected fluorometrically (Nicholls et al., 1987). Synaptosomal pellets were resuspended in HEPES buffer medium that contained 16 μM bovine serum albumin and incubated in a stirred and thermostatted cuvette maintained at 37 1C in a Perkin-Elmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA). NADP þ (2 mM), GDH (50 units/ml) and CaCl2 (1 mM) were added after 3 min. In experiments that investigated Ca2 þ -independent efflux of glutamate, EGTA (200 μM) was added in place of CaCl2. Other additions before depolarization were made as described in the figure legends. After a further 10 min of incubation, 4-aminopyridine (1 mM), or KCl (15 mM) was added to stimulate glutamate release. Glutamate release was monitored by measuring the increase of fluorescence (excitation and emission wavelengths of 340 nm and 460 nm, respectively) caused by NADPH being produced by oxidative deamination of released glutamate by GDH. Data were accumulated at 2 s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment, and the fluorescence response used to calculate released glutamate was expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg). Values quoted in the text and depicted in bar graphs represent the levels of glutamate cumulatively released after 5 min of depolarization, and are expressed as nmol/mg/5 min. Estimation of the IC50 was based on a one-site model [Inhibition¼(InhibitionMAX  [dimebon]/(IC50 þ [dimebon])], and calculated using the nonlinear curve-fitting function provided in MicroCal Origin. Cumulative data were analyzed using Lotus 1-2-3.

2.2. Animals 2.5. Cytosolic free Ca2 þ concentration ([Ca2 þ ]C) Adult male Sprague-Dawley rats (150–200 g) were used in this study. All of the animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and were approved by the Fu Jen Institutional Animal Care and Utilization Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.3. Synaptosomal preparation Synaptosomes were prepared as described previously (Chiu et al., 2011; Nicholls et al., 1987). Briefly, the animals were killed by decapitation and the cerebral cortex were rapidly removed at 4 1C.

The [Ca2 þ ]C was measured using the Ca2 þ indicator Fura-2. Synaptosomes (0.5 mg/mL) were resuspended in HEPES buffer medium containing 0.1 mM CaCl2 and loaded with 5 μM Fura-2 for 30 min at 37 1C. The synaptosomes were washed with HEPES buffer medium by being centrifuged, resuspended in 2 mL of HEPES buffer medium containing bovine serum albumin, and placed in a PerkinElmer LS-55 spectrofluorometer at 37 1C with stirring in the presence of 1.2 mM CaCl2. The synaptosomes were incubated for 10 min in the presence of dimebon (10 μM) prior to being depolarized with 4-aminopyridine (1 mM). Fura-2-Ca fluorescence was determined at excitation wavelengths of 340 nm and 380 nm (emission wavelength, 505 nm), and data were accumulated at 2 s intervals. [Ca2 þ ]C (nM)

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was calculated by using calibration procedures (Sihra et al., 1992) and equations described previously (Grynkiewicz et al., 1985). Cumulative data were analyzed using Lotus 1-2-3. 2.6. Synaptosomal plasma membrane potential The plasma membrane potential was determined using a membrane-potential-sensitive dye, DiSC3(5) (Akerman et al., 1987). Synaptosomes were resuspended in HEPES buffer medium and incubated in a stirred and thermostated cuvette at 37 1C in a Perkin-Elmer LS-55 spectrofluorimeter. After 3 min of incubation, 5 μM DiSC3(5) were added and allowed to equilibrate before the addition of CaCl2 (1 mM) after 4 min of incubation. 4-aminopyridine was then added to depolarize the synaptosomes for 10 min, and DiSC3(5) fluorescence was monitored at excitation and emission wavelengths of 646 nm and 674 nm, respectively. Cumulative data were analyzed using Lotus 1-2-3 and expressed in fluorescence units. 2.7. Western blotting Synaptosomes (0.5 mg protein/ml) from control and drug-treated groups were lysed in ice-cold Tris–HCl buffer solution, pH 7.5, that contained 20 mM Tris–HCl, 1% Triton, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate and 1 μg/ml leupeptin. The lysates were sonicated for 10 s and then centrifuged at 13,000 g at 4 1C for 10 min. Equal amounts of samples were separated by electrophoresis on 7.5% SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline that contained 5% low-fat milk and incubated with appropriate primary antibodies (anti-phospho-protein kinase C (pan), 1:1000). After incubation with appropriate peroxidase-conjugated donkey anti-rabbit IgG secondary antibodies (1:3000), protein bands were detected by using the enhanced chemiluminescence method. An aliquot of samples was loaded and probed with anti-β-actin antibody for detection of β-actin as a loading control. Films were scanned using a scanner and the level of expression or phosphorylation was assessed by band density, which was quantified by densitometry. 2.8. Data analysis Data were expressed as mean 7SEM. The data reported were analyzed by using the unpaired Student's t test or by using two-way ANOVA accompanied by Tukey's test for multiple

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comparisons. Analysis was completed via software SPSS (17.0; SPSS Inc., Chicago, IL). A value of P o0.05 was considered to be significant.

3. Results 3.1. Dimebon inhibits 4-aminopyridine-evoked glutamate release from rat cerebrocortical synaptosomes; this effect is due to a decrease in vesicular exocytosis To investigate the influence of dimebon on glutamate release, isolated nerve terminals were depolarized with the K þ -channel blocker 4-aminopyridine. 4-aminopyridine destabilizes the membrane potential and is thought to cause repetitive spontaneous Na þ channel-dependent depolarization that closely approximates in vivo depolarization of the synaptic terminal that leads to the activation of voltage-dependent Ca2 þ channels and neurotransmitter release (Tibbs et al., 1989). In synaptosomes incubated in the presence of 1 mM CaCl2, 4-aminopyridine (1 mM) evoked a glutamate release of 7.970.1 nmol/mg/5 min. Application of dimebon (10 μM) reduced the amount of 4-aminopyridine-evoked glutamate release to 4.170.2 nmol/mg/5 min [t(8)¼14.34, P¼0.000001) without altering the basal release of glutamate (Fig. 1A). The dimebon-induced inhibition of 4-aminopyridine-evoked glutamate release was concentration-dependent, and produced an IC50 value of approximately 6 μM, which was derived from a dose-response curve (Fig. 1B). We next investigated whether the inhibition of 4-aminopyridineevoked glutamate release by dimebon was mediated by an effect on exocytotic vesicular release, or on Ca2 þ -independent release attributable to cytosolic efflux via reversal of the glutamate transporter (Nicholls et al., 1987). In Fig. 2A, the Ca2 þ -independent glutamate efflux was measured by depolarizing the synaptosomes with 1 mM 4-aminopyridine in extracellular-Ca2 þ -free solution that contained 300 μM EGTA. Under these conditions, the release of glutamate evoked by 4-aminopyridine (1 mM) was 2.470.3 nmol/mg/5 min. This Ca2 þ -independent release evoked by 4-aminopyridine was, however, not affected by 10 μM dimebon (2.070.4 nmol/mg/ 5 min) [t(6)¼ 0.95, P¼0.38] (Fig. 2A). In Fig. 2B and C, we investigated the action of dimebon in the presence of DL-TBOA, a nonselective inhibitor of all excitatory amino acid transporter (EAAT) subtypes, or bafilomycin A1, which causes the depletion of glutamate in synaptic vesicles. Fig. 2B shows that 4-aminopyridine-evoked glutamate release was increased by 10 μM DL-TBOA (because of inhibition of reuptake of released glutamate) [F(1,15)¼ 112.4, P¼0.000]. In the presence of DL-TBOA, dimebon was able to produce

Fig. 1. Dimebon inhibits 4-aminopyridine-evoked release of glutamate in rat cerebrocortical nerve terminals. (A) Glutamate release was evoked by the addition of 1 mM 4-aminopyridine in the abscence (control) and in the presence of dimebon (10 μM), added 10 min prior to the addition of 4-aminopyridine. (B) Dose–response curve for dimebon inhibition of 4-aminopyridine-evoked glutamate release. Results are mean 7 SEM of 5 independent experiments. ***P o0.001 versus control group.

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Fig. 2. Dimebon effects on the Ca2 þ -dependent, exocytotic component of 4-aminopyridine-evoked glutamate release. (A) Ca2 þ -independent release was assayed by omitting CaCl2 and adding 300 μM EGTA 10 min before depolarization, and was evoked by 1 mM 4-aminopyridine in the abscence (control) and in the presence of 10 μM dimebon, added 10 min before the addition of 4-aminopyridine. (B, C) Glutamate release was evoked by 1 mM 4-aminopyridine in the absence (control) or presence of 10 μM dimebon, 10 μM DL-TBOA, 10 μM DL-TBOA þ10 μM dimebon, 0.1 μM bafilomycin A1, or 0.1 μM bafilomycin A1 þ 10 μM dimebon. (D) Quantitative comparison of the extent of glutamate release by 4-aminopyridine in the absence or presence of dimebon, and absence and presence of DL-TBOA or bafilomycin A1. Dimebon was added 10 min before depolarization and, DL-TBOA or bafilomycin A1, 10 min prior to this. Results are mean 7SEM of 4–5 independent experiments. ***Po 0.001 versus control group. #Po 0.05 versus the DL-TBOA-treated group.

a 41.573.8% inhibition on 4-aminopyridine-evoked glutamate release, which was similar to that observed for the 45.172.5% inhibition produced by dimebon (10 μM) alone [F(1,15)¼ 3.92, P¼0.53; Fig. 2B and D). In contrast to DL-TBOA, bafilomycin A1 (0.1 μM) reduced 4-aminopyridine-evoked glutamate release [F(1,15)¼554.95, P¼ 0.000, Fig. 2C]. In the presence of bafilomycin A1, however, dimebon (10 μM) only produced a 4.772.3% decrease in the 4-aminopyridineevoked glutamate release, which was less than that the inhibition produced by dimebon alone (45.172.5%) [F(2,23)¼12.28, Po0.05, Fig. 2C and D]. These results suggest that the inhibition of glutamate release by dimebon is mediated by a reduction in the Ca2 þ -dependent exocytotic component of glutamate release. 3.2. Dimebon reduces 4-aminopyridine-induced increase in intraterminal Ca2 þ levels but does not change the synaptosomal membrane potential Transmitter release can be modulated by regulating the plasma membrane potential, and consequently altering the calcium influx. To investigate the potential mechanisms underlying the dimebonmediated inhibition of glutamate release, the effect of dimebon on

intrasynaptosomal Ca2 þ levels was determined by using the Ca2 þ indicator Fura-2. In Fig. 3A, 4-aminopyridine (1 mM) caused a rise in cytosolic Ca2 þ concentration ([Ca2 þ ]C) from 145.471.5 nM to a plateau level of 202.774.5 nM (Po0.001). Applying dimebon (10 μM) did not affect basal Ca2 þ levels, but caused an approximately 20% decrease in the 4-aminopyridine-evoked rise in [Ca2 þ ]c (173.775.8 nM; t(8)¼53.5, P¼0.001; Fig. 3A). The inhibition of the [Ca2 þ ]C elevation by dimebon might be attributed either to a direct reduction in the amount of Ca2 þ entering through voltagedependent Ca2 þ channels, or to secondary effects caused by, for example, the modulation of potassium channels and the consequently altered plasma membrane potential. To discern between these two possibilities, the effect of dimebon on the synaptosomal plasma membrane potential under resting conditions and on depolarization was examined using membrane potential-sensitive dye DiSC3(5). Fig. 3B shows that 4-aminopyridine (1 mM) caused an increase in DiSC3(5) fluorescence of 9.170.5 fluorescence units/ 5 min. Preincubation of the synaptosomes with dimebon (10 μM) for 10 min before adding 4-aminopyridine did not alter the resting membrane potential, and produced no substantial change in the 4-aminopyridine-mediated increase in DiSC3(5) fluorescence (9.270.4 units/5 min; t(8)¼  1.56, P¼0.82). This suggests that the

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Fig. 3. Dimebon reduces the 4-aminopyridine-induced increase in cytosolic Ca2 þ concentration ([Ca2 þ ]C) but fails to alter the synaptosomal membrane potential. (A) The [Ca2 þ ]C (nM) was monitored using Fura-2 in the absence (control) and in the presence of 10 μM dimebon, added 10 min before depolarization with 1 mM 4-aminopyridine. (B) Synaptosomal membrane potential was monitored with DiSC3(5) in the absence (control) and in the presence of 10 μM dimebon, added 10 min before depolarization with 1 mM 4-aminopyridine. Inset. Glutamate release was induced by 15 mM KCl in the absence (control) or presence of 10 μM dimebon, added 10 min before depolarization. Results are mean 7 SEM of 5 independent experiments. *Po 0.05 versus control group.

observed inhibition of 4-AP-evoked glutamate release by dimebon is unlikely to be due to a hyperpolarizing effect of the drug on the synaptosomal plasma membrane potential or an attenuation of depolarization produced by 4-aminopyridine. Confirmation that the dimebon effect did not impinge on synaptosomal excitability was obtained with experiments using high external [K þ ]-mediated depolarization. Elevated extracellular KCl depolarizes the plasma membrane by shifting the K þ equilibrium potential above the threshold potential for activation of voltage-dependent ion channels. While Na þ channels are inactivated under these conditions, voltagedependent Ca2 þ channels are activated nonetheless to mediate Ca2 þ entry, which supports neurotransmitter release (Barrie et al., 1991). In Fig. 3B (inset), KCl (15 mM) evoked a glutamate release of 8.070.4 nmol/mg/5 min, which was reduced to 5.170.9 nmol/mg/ 5 min in the presence of 10 μΜ dimebon [t(8)¼3.1, P¼0.02]. 3.3. A reduction of calcium influx through Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels Is involved in the action of dimebon on glutamate release In the adult rat cerebrocortical nerve terminals, the release of glutamate evoked by depolarization is supported by Ca2 þ influx through Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels (Millan and Sanchez-Prieto, 2002; Vazquez and Sanchez-Prieto, 1997).

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To determine whether the decrease in Ca2 þ channel activity was involved in the effect of dimebon on 4-AP-evoked glutamate release, we examined the effect of dimebon in the presence of ω-conotoxin MVIIC, a wide spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2 þ channels. In Fig. 4A, 4-aminopyridine (1 mM)-evoked glutamate release was substantially reduced in the presence of ω-conotoxin MVIIC (2 μM) [F(1,13) ¼229.58, P ¼0.000]. Although the 4-aminopyridine-evoked glutamate release was considerably reduced in the presence of dimebon (10 μM), this effect was prevented by the presence of ω-conotoxin MVIIC (Fig. 4A). On average, the inhibitory effect of dimebon on 4aminopyridine-evoked glutamate release was reduced to 5.1 71.9% in the presence of ω-conotoxin MVIIC, which was significantly different from the effect of dimebon alone (44.1 73.4%) [F(3,22) ¼19.18, P o0.05, Fig. 4D]. In addition to the Ca2 þ influx through voltage-dependent 2þ Ca channels, the release of glutamate evoked by depolarization was reported to be caused by a Ca2 þ release from intracellular stores such as endoplasmic reticulum and mitochondria (Berridge, 1998). Thus, we tested the effect of dantrolene, an inhibitor of intracellular Ca2 þ release from endoplasmic reticulum, and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin2(3H)-one (CGP37157), a membrane-permeant blocker of mitochondrial Na þ /Ca2 þ exchange, on the dimebon-mediated inhibition of glutamate release. In Fig. 4B and C, 4-aminopyridine (1 mM)-evoked glutamate release was reduced by dantrolene (100 μM) [F(1,14) ¼6.53, P ¼0.02] or CGP37157 (100 μΜ) [F (1,12) ¼ 11.91, P ¼0.005]. In the presence of either dantrolene or CGP37157, dimebon (10 μΜ) still effectively inhibited 4aminopyridine-evoked glutamate release by 39.5 73.3% and 44.7 76.3%, respectively, which was not significantly different from the inhibition of dimebon alone (44.1 73.4; P 40.05; Fig. 4D). These results suggest that decrease in the Ca2 þ release from intracellular stores appears not to mediate the inhibitory effect of demebon on glutamate release. 3.4. Involvement of protein kinase C in the dimebon-mediated inhibition of glutamate release Since the protein kinase C signaling cascade is known to be present at the presynaptic level and has a crucial role in neurotransmitter exocytosis (Coffey et al., 1993; Hung et al., 2009; Vaughan et al., 1998; Wang and Sihra, 2004), we investigated whether the cascade participated in dimebon-mediated inhibition of glutamate release. To this end, we studied the effect of dimebon following inhibition of protein kinase C. Consistent with previous studies, the protein kinase C inhibitor GF109203  (10 μM) reduced 4-aminopyridine (1 mM)-evoked glutamate release [F(1,12)¼43.59, P¼ 0.000, Fig. 5A]. In the presence of GF109203  , dimebon only reduced glutamate release by 1.775.9%, indicating significant reduction compared with that obtained when dimebon was applied alone (45.172.9%) [F(2,21)¼32.05, Po0.05] (Fig. 5A and C). In contrast to the effect of protein kinase C inhibitor, the mitogen-activated protein kinase inhibitor PD98059 (50 μM) reduced 4-aminopyridine (1 mM)-evoked glutamate release [F (1,10)¼24.81, P¼ 0.001], but it failed to influence the ability of dimebon to inhibit 4-aminopyridine-evoked release of glutamate (Fig. 5B). In the presence of PD98059, dimebon was still able to reduce the 4-aminopyridine-evoked glutamate release by 45.973.3%, which was not significantly different from the inhibition of dimebon alone (45.172.9%; P40.05; Fig. 5C). To confirm that the protein kinase C signaling pathway was suppressed by dimebon during its inhibition of 4-aminopyridine-evoked glutamate release, we determined the effect of dimebon on the phosphorylation of protein kinase C in cerebrocortical synaptosomes. Fig. 5D shows that depolarization of synaptosomes with 4-aminopyridine (1 mM)

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Fig. 4. Blockade of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels prevents the inhibitory effect of dimebon on 4-aminopyridine-induced glutamate release. (A–C) Glutamate release was evoked by 1 mM 4-aminopyridine in the absence (control) or presence of 10 μM dimebon, 2 μM ω-conotoxin MVIIC, 2 μM ω-conotoxin MVIIC þ 10 μM dimebon, 100 μM dantrolene, 100 μM dantroleneþ10 μM dimebon, 100 μM CGP37157, or 100 μM CGP37157þ 10 μM dimebon. (D) Quantitative comparison of the extent of glutamate release by 4-aminopyridine in the absence or presence of dimebon, and absence and presence of ω-conotoxin MVIIC, dantrolene, or CGP37157. Dimebon was added 10 min before depolarization, whereas the other drugs were added 30 min before depolarization. Results are mean 7 SEM of 3–6 independent experiments. ***Po 0.001 versus control group. *Po 0.05 versus control group. #P o0.05 versus the dantrolene- or CGP37157-treated group.

markedly increased the phosphorylation of protein kinase C (120.573.2%; t(4)¼2.1, P¼0.002). When synaptosomes were pretreated with dimebon (10 μM) for 10 min before depolarization with 1 mM 4-aminopyridine, 4-aminopyridine-enhanced phosphorylation of protein kinase C was markedly decreased to 102.772.4% [F(2,6)¼ 23.29, P¼0.001; Fig. 5D]. 3.5. Dimebon-inhibited glutamate release Is unlikely to involve interaction with NMDA receptors Previous reports suggested that dimebon may act as an inhibitor of NMDA receptor (Grigorev et al., 2003; Wu et al., 2008). NMDA receptor activation has been shown to regulate glutamate release from nerve terminals (Breukel et al., 1998; Luccini et al., 2007; Sequeira et al., 2001). Thus, the final experiment was to examine whether NMDA receptor was involved in the observed dimebonmediated inhibition of glutamate release. In Fig. 6A, the effect of dimebon on 4-aminopyridine-evoked glutamate release in the absence or presencr of NMDA (a agonist of NMDA receptor) was compared. 4-aminopyridine-evoked glutamate release was increased by NMDA (100 μM) [F(1,16)¼ 25.92, P¼0.000]. In the presence of NMDA, dimebon was still able to reduce the 4-aminopyridine-evoked

glutamate release by 46.373.2%, which was not significantly different from the inhibition of dimebon alone (40.876.6%) [F(2,24)¼2.01, P¼0.16, Fig. 6C and D]. Next, the NMDA receptor antagonist D-AP5 (100 μM) had no effect on either 4-aminopyridine-evoked glutamate release or inhibition thereof by dimebon (Fig. 6B). On average, dimebon resulted in a 36.873.6% inhibition on 4-aminopyridineevoked glutamate release after treatment with D-AP5, which was similar with the inhibition produced by dimebon alone (40.876.6%) [F(2,24)¼ 2.01, P¼0.16, Fig. 6C).

4. Discussion The release of glutamate from a presynaptic site is a possible target for the drug modulation of excitability and synaptic transmission in central neurons (Wu and Saggau, 1997). Therefore, the purpose of this study was to investigate the relationship between the antihistamine drug dimebon and the presynaptic modulation of glutamate release, and to determine the underlying molecular mechanisms. By preparing nerve terminals (synaptosomes) from rat cerebral cortex, this study is the first to report that dimebon preferentially inhibits depolarization-evoked glutamate release.

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Fig. 5. Protein kinase C pathway is involved in the inhibitory effect of dimebon on 4-aminopyridine-evoked glutamate release. (A, B) Glutamate release was evoked by 1 mM 4-aminopyridine in the absence (control) or presence of 10 μM dimebon, 10 μM GF109203  , 10 μM GF109203  þ 10 μM dimebon, 50 μM PD98059, or 50 μM PD98059 þ 10 μM dimebon. (C) Quantitative comparison of the extent of glutamate release by 4-aminopyridine in the absence or presence of dimebon, and absence and presence of GF109203  or PD98059. GF109203  or PD98059 was added 40 min before depolarization, whereas dimebon was added 10 min before depolarization. (D) Phosphorylation of protein kinase C was detected in the absence (control) or presence of 1 mM 4-aminopyridine, or 1 mM 4-aminopyridine þ10 μM dimebon. Data are expressed as a percentage of the control phosphorylation obtained in the absence of 4-aminopyridine stimulation. Results are mean 7 SEM of 3–7 independent experiments. ***P o0.001 versus control group. **Po 0.01 versus control group; #Po 0.05 versus the PD98059- or 4-aminopyridine-treated group.

However, dimebon did not affect the basal release of glutamate from the nerve terminals, suggesting that dimebon might reduce the release of glutamate when it is triggered by neuronal activation. The synaptosome was applied because it is capable of accumulating, storing, and releasing neurotransmitters, and is devoid of functional glial and nerve cell body elements that might cause the findings to be misinterpreted because of modulatory loci at the non-neuronal, postsynaptic, or network levels (Nicholls and Sihra, 1986). Therefore, the synaptosome provides a useful model for directly studying the specific presynaptic regulation of neurotransmitter release. 4.1. Mechanism of dimebon-mediated inhibition of glutamate release In nerve terminals, the inhibition of Na þ channels or activation of K þ channels stabilizes membrane excitability and, consequently, causes a reduction in the levels of Ca2 þ entry and neurotransmitter release (Li et al., 1993; Pongs et al., 1999; Rehm and Tempel, 1991). The observed inhibitory effect of dimebon on evoked glutamate

release could occur through a reduction of nerve terminal excitability, but this is unlikely because of the following 3 observations: First, 4-aminopyridine- versus KCl-evoked glutamate release were significantly inhibited by dimebon. Although 4-aminopyridineevoked glutamate release involves the activation of Na þ and Ca2 þ channels, 15 mM external KCl-evoked glutamate release involves only Ca2 þ channels (Barrie et al., 1991; Nicholls, 1998), and this indicates that Na þ channels are not involved in the inhibitory effect of dimebon on glutamate release; Second, no substantial dimebon effect on synaptosomal plasma membrane potential was observed, which indicated a lack of effect on the K þ conductance; Third, dimebon did not affect the 4-aminopyridine-evoked Ca2 þ -independent glutamate release, a component of glutamate release that is dependent only on membrane potential (Nicholls et al., 1987). This indicates that dimebon does not affect glutamate release by reversing the direction of the plasma membrane glutamate transporter. This suggestion was supported by the observation that the inhibitory effect of dimebon on 4-aminopyridine-evoked glutamate release was prevented by the vesicular transporter inhibitor

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Fig. 6. Nerve terminal NMDA receptor activity is not involved in the dimebon-mediated inhibition of evoked glutamate release. (A, B) Glutamate release was evoked by 1 mM 4-aminopyridine in the absence (control) or in the presence of 10 μM dimebon, 100 μM NMDA (an agonist of NMDA receptor), 100 μM NMDA þ10 μM dimebon, 100 μM D-AP5 (an antagonist of NMDA receptor), or 100 μM D-AP5þ 10 μM dimebon. (C) Quantitative comparison of the extent of glutamate release by 4-aminopyridine in the absence or presence of dimebon, and absence and presence of NMDA or D-AP5. Results are mean 7 SEM of 3–6 independent experiments. ***Po 0.001 versus control group. # P o0.05 versus the NMDA- or D-AP5-treated group.

bafilomycin A1, but not by the glutamate transporter inhibitor DLTBOA. Thus, these data suggest that the dimebon-mediated inhibition of 4-aminopyridine-evoked glutamate release is mediated by a decrease in the Ca2 þ -dependent exocytotic component of glutamate release. Moreover, this phenomenon is not the result of a reduction in synaptosomal excitability caused by ion channel (e.g., the Na þ or K þ channels) modulation. Therefore, if the effect is not caused by the modulation of synaptosomal excitability, then dimebon possibly inhibits evoked glutamate release by decreasing the levels of Ca2 þ entry through the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2 þ channels that are coupled to glutamate exocytosis in the nerve terminals (Millan and Sanchez-Prieto, 2002; Vazquez and Sanchez-Prieto, 1997). This hypothesis is plausible because the present study demonstrated that dimebon decreased the 4-aminopyridine-evoked increase in [Ca2 þ ]C. Furthermore, the inhibitory effect of dimebon on 4-aminopyridineevoked glutamate release was prevented by ω-conotoxin MVIIC, a wide spectrum blocker of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2þ channels. However, neither dantrolene, an inhibitor of intracellular Ca2 þ release from the endoplasmic reticulum ryanodine receptors, nor CGP37157, a mitochondrial Na þ /Ca2þ exchange blocker, affected the inhibitory effect of dimebon on glutamate release. Thus, the participation of reduced release of stored Ca2 þ from the endoplasmic reticulum and mitochondria during the dimebon-mediated inhibition of glutamate release could be excluded. Based on these results, we suggest that dimebon inhibits evoked glutamate release by

reducing presynaptic Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2þ channel activity. Our finding is consistent with previous studies, which have shown that dimebon inhibits glutamate-induced Ca2 þ increase and high voltage-activated Ca2 þ channels in cultured neurons (Lermontova et al., 2001; Wu et al., 2008). A role for the protein kinase C pathway in the dimebon-mediated inhibition of glutamate release is suggested in this study, based on the following results: (1) the inhibitory effect of dimebon on 4-aminopyridine-evoked glutamate release was substantially prevented by the protein kinase C inhibitor GF109203  , but not by the mitogenactivated protein kinase inhibitor PD98059; and (2) dimebon significantly decreased 4-aminopyridine-induced phosphorylation of protein kinase C. Protein kinase C is one of the important intracellular signaling system at the presynaptic level, and plays a crucial role in neurotransmitter exocytosis (Coffey et al., 1993; Millan et al., 2003; Pittaluga et al., 2005; Sanchez-Prieto et al., 1996; Wang and Sihra, 2004). It has been shown that depolarization-stimulated Ca2þ entry enhances protein kinase C-dependent phosphorylation and glutamate release (Coffey et al., 1994). Thus, we suggest that the inhibitory effect of dimebon on Ca2þ entry observed here may decrease protein kinase C activity and, in turn, glutamate release. In addition, protein kinase C is capable of phosphorylating numerous synaptic proteins involved in the synaptic vesicle trafficking/recruitment and exocytosis, including MARCKS, SNAP-25, synapsin I, and munc/nSecl. The phosphorylation of these synaptic proteins promotes the dissociation of synaptic vesicles from the actin cytoskeleton. This in turn makes more vesicles

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available at the active zone for neurotransmitter exocytosis, resulting in an increased glutamate release (Craig et al., 2003; Jarvis and Zamponi, 2001; Vaughan et al., 1998). Therefore, it is possible that dimebon inhibits glutamate release by decreasing protein kinase C-dependent phosphorylation of synaptic proteins and the availability of synaptic vesicles. It has been suggested that dimebon may act by blocking NMDA receptors (Grigorev et al., 2003; Wu et al., 2008). NMDA receptors are expressed in the brain and localize both pre- and postsynaptically (Engelman and MacDermott, 2004; Pinheiro and Mulle, 2008). At the presynaptic level, activation of NMDA receptors modulates Ca2 þ influx and glutamate release (Breukel et al., 1998; Luccini et al., 2007; Sequeira et al., 2001). Therefore, the question arises as to whether NMDA receptors play any role in the dimebon-mediated inhibition of glutamate release. In the present study, the NMDA receptor agonist NMDA increased 4-AP-evoked glutamate release. This finding is consistent with previous study (Breukel et al., 1998; Luccini et al., 2007). However, the inhibitory effect of dimebon on 4-aminopyridine-evoked glutamate release does not seem to involve NMDA receptors because it was obtained in the presence of the NMDA receptor agonist and antagonist. Our finding, although contrasting with previous studies (Grigorev et al., 2003; Wu et al., 2008), is consistent with a report by Giorgetti et al. (2010), showed that dimebon fails to block NMDA-induced Ca2 þ influx in primary neuronal cells. The discrepancy is not clear, but may be related to the different experimental models applied and the expression of NMDA receptor subunits. In addition, the data in this study show that the NMDA receptor antagonist D-AP5 failed to affect the 4-aminopyridine-evoked glutamate release. Although the reason for this is unclear, probably because we depolarized synaptosomes with 1 mM 4-aminopyridine that closely simulates physiological stimulation (Sihra et al., 1992). In fact, under physiological conditions, glutamate acts on both the NMDA and nonNMDA receptors. However, the NMDA receptors are blocked by Mg2 þ , they only activated by the sustained depolarization. Consistent with this, our study and Breukel et al. (1998) had found that NMDA, at relatively high concentrations (100 μM), increases the release of glutamate evoked by 4-aminopyridine. Although the data presented here suggest that antagonism at the NMDA receptor is unlikely mechanism for the inhibition of glutamate release produced by dimebon, the possible involvement of other neurotransmitter receptors should be considered. Serotonin, dopamine, and norepinephrine receptors, for example, are reported to be inhibited by dimebon at a 10 μM concentration (Grigorev et al., 2003; Schaffhauser et al., 2009; Wu et al., 2008). Consistent with these reports, the concentration of dimebon used to inhibit glutamate release in our study was 10 μM. In this study, dimebon-mediated inhibition of glutamate release is dose dependent, maximal at 30 μM, with a IC50 of 6 μM. In addition, numerous pharmacological effects of dimebon are also observed in a higher concentration range (i.e., 50–100 μM), including inhibition of acetylcholinesterase, blockade of NMDA receptors and voltage-gated Ca2 þ channels, stabilization of mitochondrial function, as well as attenuating glutamate- or amyloid-induced neuronal death (Bachurin et al., 2001, 2003; Grigorev et al., 2003; Lermontova et al., 2001; Perez et al., 2012; Wu et al., 2008). These effects are likely to be associated with the neuroprotective activity of dimebon (Bachurin et al., 2001; Grigorev et al., 2003; Lermontova et al., 2001), although the exact mechanism for this benefit remains to be explored. In this work, the ability of dimebon to decrease glutamate release from nerve terminals may explain, in part, its neuroprotective mechanism. This is because the excessive release of glutamate is a critical factor in the neuropathology of acute and chronic brain disorders (Meldrum, 2000). Hence, it may be reasonable to assume that dimebon may have the potential to protect brain against glutamate-induced neurotoxicity. Future studies are needed to make a direct link between the inhibitory

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effect of dimebon on glutamate release and the compound's neuroprotective effect against exocytotoxic insults. 4.2. Conclusion The results of the current study indicate that dimebon inhibits glutamate release from rat cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca2 þ channels and protein kinase C activity. This implies that the inhibition of glutamate release is an additional pharmacological activity of dimebon that may play a critical role in the apparent clinical efficacy of this compound.

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