Calmodulin-dependent regulation of neurotransmitter release differs in subsets of neuronal cells

brain research 1535 (2013) 1–13

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Research Report

Calmodulin-dependent regulation of neurotransmitter release differs in subsets of neuronal cells Kosuke Andoa,1, Yoshihisa Kudoa, Kyota Aoyagib,2, Ryoki Ishikawac, Michihiro Igarashid, Masami Takahashib,n a

School of Life Sciences, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Biochemistry, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan c Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Showamachi, Maebashi, Gunma 371-8511, Japan d Department of Neurochemistry and Molecular Cell Biology, Niigata University Graduate School of Medical and Dental Sciences, Asahi-machi, Chuo-ku, Niigata 951-8510, Japan b

art i cle i nfo

ab st rac t

Article history:

The purpose of this study was to determine whether calmodulin (CaM) plays a role in

Accepted 8 August 2013

neurotransmitter release by examining the effect that ophiobolin A (OBA), a CaM antagonist,

Available online 23 August 2013

on neurotransmitter release from clonal rat pheochromocytoma PC12 cells, primary cortical neurons, and primary cerebellar granule cells. OBA inhibited Ca2þ/CaM-dependent phos-

Keywords:

phorylation of cAMP response element binding protein in all cell types tested. Moreover,

CaM

Ca2þ-dependent release of dopamine and acetylcholine from PC12 cells were remarkably

Neurotransmitter release

reduced by OBA in a dose-dependent and temporal manner, but neurotransmitter release

Myosin Va

partially recovered with the addition of CaM in membrane permeabilized PC12 cells. OBA

PC12 cells

and several synthetic CaM antagonists suppressed Ca2þ-dependent glutamate release from

Cerebral cortical neurons

cerebral cortical neurons, but not from cerebellar granule cells. Myosin Va, a CaM binding

Cerebellar granule cells

protein, localized to synaptic vesicles of PC12 cells and cerebral cortical neurons, but not in cerebellar granule cells. OBA suppressed Ca2þ-induced myosin Va dissociation from secretory vesicles, and inhibited secretory vesicle motility in PC12 cells. These results

Abbreviations: ACh, protein; DA, Glu, OBA,

acetylcholine; BSA,

bovine serum albumin; CaM,

calmodulin; CREB,

dopamine; DMEM, Dulbecco’s modified Eagle’s medium; DNase,

glutamate; hGH,

human growth hormone; HRP,

ophiobolin A; PAGE,

sulphate; SNAP-25,

horseradish peroxidase; MEM,

polyacrylamide gel electrophoresis; PBS,

cAMP response element binding

deoxyribonuclease; FCS, fetal calf serum; minimum essential medium;

Dulbecco’s phosphate buffered saline; SDS,

synaptosomal-associated protein of 25 kDa; SNARE,

sodium dodecyl

soluble N-ethylmaleimide-sensitive fusion protein receptor;

VAMP-2, vesicle-associated membrane protein-2 n Corresponding author. Fax: þ81 42 778 8441. E-mail address: [email protected] (M. Takahashi). 1 Present address: Japan Patent Office, Medical Science Division, Kasumigaseki, Chiyoda-ku, Tokyo 100–8915, Japan. 2 Department of Biochemistry, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo 181–8611, Japan. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.08.018

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suggest that CaM, although not essential, regulates neurotransmitter release in a subset of neurons and secretory cells, and myosin Va is a possible target of OBA in this process. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Synaptic transmission is mediated by neurotransmitters released from nerve terminals. Neurotransmitters are stored in synaptic vesicles and released into the synaptic cleft by exocytosis of vesicle contents, which involves docking and fusion of the vesicle membrane with the presynaptic plasma membrane (Jahn and Fasshauer, 2012; Südhof and Rizo, 2013). Recent studies have shown that soluble N-ethylmaleimidesensitive fusion protein receptor (SNARE) proteins, including syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane and vesicle-associated membrane protein-2 (VAMP-2, also called synaptobrevin 2) in the synaptic vesicle membrane, play essential roles in neurotransmitter release (Jahn and Scheller, 2006; Hussain and Davanger, 2011; Rizo and Südhof, 2012). Neurotransmitter release is triggered by Ca2þ, and C2domain containing proteins including synaptotagmin are believed to function as Ca2þ sensors for membrane fusion (Pang and Südhof, 2010; Walter et al., 2011). Accumulating evidence suggests that there are several steps prior to membrane fusion and that some of these steps are likely regulated by Ca2þ. However, Ca2þ sensor proteins regulating these steps are not well understood (Neher and Zucker, 1993; Neher and Sakaba, 2008). Calmodulin (CaM) is the most ubiquitous Ca2þ binding protein in cells and has four EFhand motifs with high Ca2þ affinity (Jurado et al., 1999). SNARE-mediated vacuole fusion in yeast is activated by Ca2þ efflux from the vacuole lumen and uses CaM as a Ca2þ sensor (Peters and Mayer, 1998). CaM is also essential for Ca2þ-dependent exocytosis in Paramecium (Kerboeuf et al., 1993). In mammalian cells, vesicular Ca2þ and CaM have been implicated in intra-Golgi and endosome fusion (Colombo et al., 1997; Porat and Elazar, 2000; Pryor et al., 2000). Early studies using CaM inhibitors and anti-CaM antibodies have demonstrated the involvement of CaM in neurotransmitter and hormone release in mammalian neurons and endocrine cells (Baker and Knight, 1981; Burgoyne et al., 1982; Kenigsberg et al., 1982; Wilson and Kirshner, 1983; Sasakawa et al., 1983; Burgoyne and Norman, 1984; Brooks and Treml, 1984; Kenigsberg and Trifaro, 1985; Ahnert-Hilger and Gratzl, 1987; Matthies et al., 1988; Courtney et al., 1991; Reig et al., 1993; Hens et al., 1996; Igarashi and Watanabe, 2007). In past studies, CaM was identified as a candidate Ca2þ sensor for membrane fusion (Okabe et al., 1992; Chamberlain et al., 1995; Kibble and Burgoyne, 1996; Chen et al., 1999). However, this hypothesis has been refuted, since (1) the off-rate for Ca2þ dissociation is too slow to account for the transient nature of the response to elevated Ca2þ in nerve terminals, and (2) CaM does not bind Ba2þ, which could substitute for Ca2þ in triggering regulated exocytosis (Burgoyne and Clague, 2003). From other recent studies, it is currently thought that CaM plays a regulatory role, not in the fusion step, but rather in the steps prior to Ca2þ-induced membrane fusion (Sakaba and Neher, 2001;

Junge et al., 2004; Neher, 2006; Zikich et al., 2008). In the calyx of Held synapse, CaM promotes refilling of the rapidly releasing synaptic vesicle pool. In another study, CaM functioned as a regulator acting antagonistically to synaptotagmin in SNARE-mediated membrane fusion (Di Giovanni et al., 2010). A number of possible candidates exist for CaM binding proteins involved in CaM-dependent regulation of neurotransmitter release, including Ca2þ/CaM-dependent protein kinase II (Ohyama et al., 2002; Nomura et al., 2003), calcineurin (Hens et al., 1998), actin (Sullivan et al., 2000), synaptotagmin (Perin, 1996; Fournier and Trifaro, 1988), Munc13 (Junge et al., 2004; Zikich et al., 2008), Myosin V (Watanabe et al., 2005), syntaxin 1 (Di Giovanni et al., 2010), VAMP-2 (Quetglas et al., 2000, 2002), and rab3A (Park et al., 1997; Coppola et al., 1999). Given the variation in exocytic control mechanisms between adrenal chromaffin cells and glutamatergic synapses (Neher, 2006), CaM-mediated regulation of exocytotic release appears to differ considerably between neurons and secretory cells. In the present study, we examined the effect of ophiobolin A (OBA), a potent CaM inhibitor (Au et al., 2000), on neurotransmitter release in PC12 cells, cerebral cortical neurons, and cerebellar granule cells in vitro. Our results reveal striking differences on how OBA regulates these cells, and that OBA may exert its inhibitory effect through the suppression of myosin Va dissociation from secretory vesicles.

2.

Results

2.1. OBA suppresses CaM-dependent CREB phosphorylation in vitro Ca2þ/CaM-dependent protein kinase IV phosphorylates cAMP response element binding protein (CREB) at Ser133. To assess the effective inhibitory concentration of OBA on PC12 cells and neurons, the effect of OBA on Ca2þ/CaM-dependent CREB phosphorylation was examined by Western blot analysis. As shown in Fig. 1, ionomycin treatment markedly enhanced CREB phosphorylation in PC12 cells, cerebral cortical neurons, and cerebellar granule cells. This effect was suppressed by pretreating cells with 3 μM OBA for 30 min. This suggests that OBA effectively inhibited ionomycin-induced CREB phosphorylation in PC12 cells and neurons at a concentration of 3 μM.

2.2. OBA suppresses both ACh and DA release from PC12 cells PC12 cells have two morphologically distinct types of secretory vesicles: one is a large dense-core vesicle containing dopamine (DA) and the other is a small synaptic microvesicle containing acetylcholine (ACh). OBA suppresses exocytotic release of exogenously expressed hGH in the micromolar range (Quetglas et al., 2002). To determine whether OBA

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1 μM. These results suggest that neurotransmitter release is positively regulated by CaM in PC12 cells.

2.4. Partial recovery of DA release from OBA-treated PC12 cells by exogenous addition of CaM We next determined whether the suppression of DA release by OBA can be attenuated by the exogenous addition of CaM to membrane-permeabilized PC12 cells. PC12 cells were permeabilized with 8 μM digitonin and incubated for 1 min with or without 100 μM Ca2þ in the absence of ATP. DA release was enhanced noticeably in the presence of Ca2þ. Addition of recombinant CaM to the extracellular solution slightly increased Ca2þ-dependent release. OBA prevented DA release from permeabilized PC12 cells; DA release was partially restored by exogenous addition of recombinant CaM into the extracellular solution (Fig. 4). These results indicate that OBA prevents DA release from PC12 cells by inhibiting CaM activity.

Fig. 1 – Suppression of Ca2þ-dependent CREB phosphorylation at Ser133 by OBA in PC12 cells and neurons. PC12 cells (top), rat cerebral cortical neurons (middle), and rat cerebellar granule cells (bottom) were preincubated in low-Kþ solution in the presence (þ) or absence () of 3 μM OBA for 30 min at 37 1C. The cells were further treated in the same solution in the presence (þ) or absence () of ionomycin (PC12 cells, 1 μM for 1 min; cerebral cortical neurons, 10 μM for 1 min; cerebellar granule cells, 10 μM for 5 min). Cellular proteins were solubilized in SDS sample buffer and analyzed by immunoblotting with an antiphospho-CREB (Ser133) antibody (arrowhead). In cerebellar granule cells, this antibody also recognized phospho-ATF1 (asterisk). Immunoblot of synaptotagmin (Stg) is shown as a loading control.

suppresses Ca2þ-dependent release of endogenous neurotransmitters, we evaluated the effect of OBA on Ca2þ/ionomycin-induced DA and ACh release from PC12 cells. As shown in Fig. 2A, pretreating cells with 1 μM OBA for 30 min suppressed the release of DA and ACh. OBA inhibited DA release in a temporal manner, and a 10 min pre-incubation with 3 μM OBA suppressed Ca2þ-dependent DA release by 96.272.5% (Fig. 2B). The inhibition was also concentrationdependent, with 1 μM OBA suppressing the release by 91.2þ14.2% (Fig. 2C). ACh release was also suppressed by OBA in a temporal and concentration-dependent manner (data not shown).

2.3. A synthetic CaM antagonist inhibits DA release from PC12 cells DY-9760e is a synthetic CaM antagonist that acts on cells in the micromolar range (Sugimura et al., 1997). As shown in Fig. 3, DY-9760e and W-7, a classical CaM antagonist, effectively suppressed Ca2þ/ionomycin-induced DA release from PC12 cells. Consistent with previous reports, DY-9760e was more potent than W-7, with 68.6% suppression achieved at

2.5. CaM antagonists inhibit glutamate (Glu) release from cerebral cortical neurons Cerebral cortical neurons release Glu in a Ca2þ-dependent manner. As shown in Fig. 5, 3 μM OBA suppressed Ca2þdependent Glu release by 72.671.6%. W-7 and DY-9760e also suppressed Glu release at 3 μM, although the inhibitory effects were smaller than that of OBA. These results suggest that CaM positively regulates Ca2þ-dependent Glu release from cerebral cortical neurons.

2.6. OBA does not suppress Ca2þ-dependent Glu release from cerebellar granule cells We also examined whether OBA would suppress Ca2þ-dependent Glu release from cerebellar granule cells. As with cerebral cortical neurons, Ca2þ/ionomycin treatment induced Glu release. Surprisingly, as shown in Fig. 6, OBA minimally inhibited Glu release at concentrations sufficient to suppress Ca2þ-dependent CREB phosphorylation (Fig. 1). Furthermore, OBA stimulated Ca2þ-dependent Glu release when the concentration was increased to 10 μM (data not shown). Neither W7 nor DY-9760e inhibited Glu release in cerebellar granule cells. These results suggest that CaM does not positively regulate Glu release from cerebellar granule cells.

2.7. Myosin Va specifically associates with synaptic vesicles in PC12 cells and cerebral cortical neurons Myosin Va is a motor protein that transports synaptic vesicles to release sites (Trybus, 2008; Rudolf et al., 2011; Hammer and Sellers, 2012). Myosin Va associates with synaptic and secretory vesicles in a Ca2þ-dependent manner (Prekeris and Terrian, 1997; Rose et al., 2003). Myosin Va has IQ-motifs, which are CaM binding regions, and its motor activity is regulated by CaM (Nguyen and Higuchi, 2005). Interestingly, myosin Va expression varies in different brain regions (Tilelli et al., 2003), suggesting that myosin Va is not essential but acts as a regulator of neurotransmitter release in a subset of synapses. To determine whether myosin Va associates with

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Fig. 2 – Effect of OBA on DA and ACh release from large dense-core vesicles and small synaptic microvesicles, respectively, from PC12 cells. (A) PC12 cells were preincubated in low-Kþ solution in the presence (triangles) or absence (circles) of 1 μM OBA for 30 min at 37 1C. The solution was then replaced every 1 min, and the amounts of DA and ACh released were quantified as described in the Experimental procedures section. Ca2þ-induced release was initiated by changing the solution from low-Kþ solution (open symbols) to low-Kþ solution containing 1 μM ionomycin (closed symbols) in the presence (triangles) or absence (circles) of 1 μM OBA. (B) PC12 cells were preincubated in low-Kþ solution with 3 μM OBA for the indicated durations at 37 1C. Cells were further incubated in low-Kþ solution containing 1 μM ionomycin for 1 min and the amount of DA released was quantified as described in the Experimental procedures section. (C) PC12 cells were preincubated in low-Kþ solution in the presence or absence of various concentrations of OBA as indicated for 30 min at 37 1C. Cells were further incubated in the same solution containing 1 μM ionomycin for 1 min, and the amount of DA released was quantified as described in the Experimental procedures section. The amounts of DA and ACh released were expressed as percentages of the total DA and ACh content at the beginning of the experiments. Values are presented as mean7S.E. (n¼ 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (5, 12)¼338.8, po0.0001 and F (6, 14)¼22.38, po0.0001 for B and C, respectively. nnnpo0.001 compared to control (0 min for B and 0 M or C).

secretory and synaptic vesicles, we isolated vesicles from PC12 cells, cerebral cortical neurons, and cerebellar granule cells by immunoprecipitation using anti-synaptophysin antibody-conjugated magnetic beads. Immunoblotting analysis revealed that the beads effectively immunoprecipitated vesicular proteins, including synaptotagmin, synaptophysin, and VAMP-2 (Fig. 7). The immunoprecipitation was specific, since these proteins were not immunoprecipitated with control IgG antibody-conjugated beads. Interestingly, myosin Va was coimmunoprecipitated with vesicles from both PC12 cells and cerebral cortical neurons, but not cerebellar granule cells. These results raise the possibility that CaM regulates neurotransmitter release in PC12 cells and cerebral cortical neurons via a mechanism distinct from that in cerebellar granule cells, possibly through an association with myosin Va. Myosin Va associates with synaptic vesicles through Ca2þdependent binding to synaptophysin in synaptic vesicles (Prekeris and Terrian, 1997; Rose et al., 2003). Thus, we examined the effect of OBA on the association of myosin Va with secretory vesicles. PC12 cells were homogenized and cell lysates were incubated in the presence or absence of 10 μM OBA for 30 min on ice. Cell lysates were further incubated

with or without Ca2þ for 10 min at 37 1C, and secretory vesicles were immunoisolated using either control IgG antibody- or anti-synaptophysin antibody-conjugated magnetic beads. As shown in Fig. 8, myosin Va disassociated from secretory vesicles in a Ca2þ-dependent manner. OBA partially, but significantly, suppressed the Ca2þ-dependent dissociation of myosin Va from secretory vesicles. These results suggested that OBA inhibits neurotransmitter release by suppressing myosin Va dissociation from vesicles.

2.8. OBA suppresses the movement of secretory vesicles near the plasma membrane in PC12 cells Previous studies reported that myosin Va plays important roles in the maturation and movement of secretory vesicles in adrenal chromaffin cells and PC12 cells (Rose et al., 2003; Rudolf et al., 2003; Kögel et al., 2010). Thus, we were interested in determining how OBA would affect vesicular movement. Exogenously expressed hGH is stored in large dense-core vesicles and is co-released with DA from PC12 cells in a Ca2þ-dependent manner (Wick et al., 1993). We transfected a gene encoding a fusion protein of hGH and EGFP (hGH-EGFP) in

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Fig. 3 – Inhibition of DA release from PC12 cells with synthetic CaM antagonists. PC12 cells were preincubated in low-Kþ solution in the presence (þ) or absence () of 1 μM CaM antagonists, (A) W-7 and (B) DY-9760e, for 30 min at 37 1C. Cells were further incubated in the same solution in the presence (þ) or absence () of 1 μM ionomycin for 1 min and the amount of DA released was quantified as described in the Experimental procedures section. The amount of DA released was expressed as a percentage of the total DA content at the beginning of the experiments. Values are presented as mean7S.E. (n¼ 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (3, 8) ¼436.2, po0.0001 and F (3, 8) ¼48.5, po0.0001 for A and B, respectively. nnnpo0.001.

Fig. 4 – Effect of OBA on DA release from permeabilized PC12 cells. PC12 cells were preincubated in low-Kþ solution in the presence or absence of 3 μM OBA for 30 min at 37 1C. Cells were then permeabilized by incubation in KGEP solution containing 8 μM digitonin, 5 mM ATP, and 5 mM Mg2þ for 5 min. Permeabilized cells were further incubated in KGEP solution in the presence of either 100 μM Ca2þ (closed bars) or 5 mM EGTA (open bars) with (þ) or without () 5 μg/ml recombinant CaM for 5 min, and the amount of DA released was quantified as described in the Experimental procedures section. The amount of DA released was expressed as a percentage of the total DA content at the beginning of the experiments. Values are presented as mean7S.E. (n¼ 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (7, 16)¼ 147.2, po0.0001. np ¼0.0103.

PC12 cells and observed hGH-EGFP-containing vesicles by total internal reflection fluorescence microscopy (Stout and Axelrod, 1989; Aoyagi et al., 2005). In the absence of OBA,

hGH-EGFP-containing large dense-core vesicles showed two types of movement. One is a small movement near the plasma membrane (i.e., fluctuations). As shown in Fig. 9A, we took two images, 10 s apart. The first image (taken at t¼ 0) was assigned a red color, while the second one (t¼10) was green. Superimposing the two images yields yellow, which is due to the overlap of the green and red (and thus indicative of no vesicle movement), or green and red (indicative of some vesicle movement). Many red and green spots were present in the superimposed image, indicating that many vesicles moved slightly in one location in 10 s. In contrast, most signals taken in the presence of OBA overlapped and appeared yellow. These results indicate that small fluctuations of vesicles were suppressed by OBA, but not by KN-62, a specific Ca2þ/CaMdependent protein kinase II inhibitor (Fig. 9A and B). The second type of movement is the long distance movement observed in the cytoplasm (Fig. 9C, arrowhead). As shown in Fig. 9D, the number of vesicles showing long distance migration was markedly reduced in the presence of OBA. These results indicate that OBA inhibits both types of vesicular movement in PC12 cells.

3.

Discussion

In the present study, we examined the effects of OBA, a potent CaM inhibitor, on neurotransmitter release in PC12 cells, cerebral cortical neurons, and cerebellar granule cells. We found that: (1) OBA suppressed DA and ACh release from PC12 cells and Glu release from cerebral cortical neurons, whereas it had no effect on Glu release from cerebellar granule cells, (2) OBA reduced the Ca2þ-induced dissociation of myosin Va from secretory vesicles, and (3) unlike in PC12

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Fig. 5 – Effect of CaM antagonists on Glu release from cerebral cortical neurons. Cells were preincubated in low-Kþ solution in the presence (þ) or absence () of either 3 μM OBA, 3 μM W-7, or 3 μM DY-9760e for 30 min at 37 1C. Cells were further incubated in the same solution in the presence (þ) or absence () of 10 μM ionomycin for 1 min, and the amount of Glu released was quantified as described in the Experimental procedures section. Values are presented as mean7S.E. (n ¼ 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (7, 16)¼ 302.6, po0.0001. nnnpo0.001.

Fig. 6 – Effect of CaM antagonists on Glu release from cerebellar granule cells. Cells were preincubated in low-Kþ solution in the presence (þ) or absence () of either 3 μM OBA, 3 μM W-7, or DY-9760e for 30 min at 37 1C. Cells were further incubated in the same solution in the presence (þ) or absence () of 10 μM ionomycin for 1 min, and the amount of Glu released was quantified as described in the Experimental procedures section. Values are presented as mean7S.E. (n ¼3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (7, 16) ¼66.1, po0.0001. nnnp¼ 0.0073.

cells and cerebral cortical neurons, myosin Va was not associated with synaptic vesicles in cerebellar granule cells. These results suggest that CaM is not essential, but rather

Fig. 7 – Association of myosin Va with secretory vesicles in PC12 cells and synaptic vesicles in cerebral cortical neurons and cerebellar granule cells. Vesicles were purified from cellular homogenates by immunoprecipitation with either anti-synaptophysin antibody- or control IgG antibodyconjugated magnetic beads as described in the Experimental procedures section. Proteins in immunoisolated vesicles were solubilized in SDS sample buffer and analyzed by immunoblotting with the indicated antibodies.

plays a regulatory role in neurotransmitter release in a cell type-dependent manner, and that OBA may inhibit neurotransmitter release by suppressing Ca2þ-induced dissociation of myosin Va from vesicles. Several CaM inhibitors, including trifluoperazine, W-7, and calmidazolium, have been used to study the mechanisms of neurotransmitter release, but with inconsistent results. For example, these CaM antagonists were reported to suppress catecholamine release from chromaffin cells and PC12 cells (Baker and Knight, 1981; Kenigsberg et al., 1982; Burgoyne et al., 1982; Burgoyne and Norman, 1984; Courtney et al., 1991; Reig et al., 1993), but not in other studies (Wilson and Kirshner, 1983; Sasakawa et al., 1983; Brooks and Treml, 1984; Ahnert-Hilger and Gratzl, 1987; Matthies et al., 1988). These discrepancies may have resulted from differences in the specificity and membrane permeability of these drugs. OBA is a very potent CaM inhibitor and inhibits CaM by covalently bonding to Lys75 in the central helical region (Au et al., 2000). We found that OBA effectively inhibited the release of endogenous neurotransmitters from PC12 cells. The inhibitory action of OBA on neurotransmitter release appeared specific because the suppression was partially reversed by the exogenous addition of CaM in PC12 cells, and similar effects were also observed with other synthetic CaM inhibitors, such as W7 and DY-9760e. Thus, our findings

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Fig. 8 – Effect of OBA on the Ca2þ-dependent dissociation of myosin Va from secretory vesicles in PC12 cells. (A) Cell lysates were preincubated in the presence (þ) or absence () of 10 μM of OBA for 30 min on ice. Cell lysates were further incubated in the presence of either 2.5 mM Ca2þ (þ) or 2.5 mM EGTA () for 10 min at 37 1C. Vesicles were purified from treated cell lysates by immunoprecipitation with either anti-synaptophysin antibody- or control IgG antibodyconjugated magnetic beads as described in the Experimental procedures section. Proteins in immunoisolated vesicles were solubilized in SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. (B) The amount of myosin Va bound to vesicles under the indicated conditions was quantified and presented as relative to control (without Ca2þ and OBA). Values are presented as mean7S.E. (n ¼3). Statistical analysis was performed using two-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. Main effects of Ca2þ; F (1, 8) ¼65.9, po0.0001; and main effects of OBA; F (1, 8) ¼6.6, po0.0336. Interaction effects between Ca2þ and OBA; F (1, 8) ¼ 0.789, p¼0.40. nnn, po0.001 and n, po0.05.

suggest that CaM positively regulates neurotransmitter release from PC12 cells and cerebral cortical neurons. We also found notable differences in the effects of OBA on neurotransmitter release and the association of myosin Va

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with secretory vesicles in PC12 cells and neurons. Importantly, while OBA suppressed neurotransmitter release from PC12 cells and cerebral cortical neurons, it had no major effect on cerebellar granule cells. OBA did, however, affect intracellular CaM in cerebellar granule cells, as evidenced by its suppression of Ca2þ/CaM-dependent CREB phosphorylation in these cells. There are differences in morphology and perhaps protein composition between catecholaminecontaining large dense-core vesicles of PC12 cells and glutamate-containing synaptic vesicles of cerebellar granule cells. Exogenously expressed hGH is stored in large densecore vesicles and is released by Ca2þ-induced exocytosis from PC12 cells, and OBA was previously reported to inhibit its release (Quetglas et al., 2000). In the present study, OBA suppressed the release of both DA stored in large densecore vesicles and ACh stored in small synaptic microvesicles. OBA also inhibited the release of Glu stored in small synaptic vesicles in cerebral cortical neurons. Thus, it is likely that the different effects of OBA can be attributed to differences in cell type, rather than vesicle type. Various CaM binding proteins, including syntaxin (Di Giovanni et al., 2010), VAMP-2 (Quetglas et al., 2000, 2002), Munc-13-1 (Junge et al., 2004), rab3A (Park et al., 1997; Coppola et al., 1999), and myosin Va, are expressed in neurons and endocrine cells. However, their relative contributions to neurotransmitter release are not well understood. Myosin Va is a motor protein involved in vesicular transport in various cells (Trybus, 2008; Rudolf et al., 2011; Hammer and Sellers, 2012). It has four major structural domains: (i) the motor domain contains actin-binding and nucleotide-binding sites, (ii) the neck region serves as a lever arm for the power stroke, (iii) the rod region is responsible for dimerization of the molecule, and (iv) the C-terminal globular tail domain binds to cargo. Six calmodulin and related light chains bind to the neck region. Myosin Va is abundantly expressed in neurons and endocrine cells and regulates vesicular traffic and exocytosis (Espreafico et al., 1992; Kögel et al., 2010, Igarashi and Watanabe, 2007). Myosin Va associates with synaptic and secretory vesicles by binding to synaptophysin/ VAMP-2 complexes in vesicular membranes (Prekeris and Terrian, 1997; Rose et al., 2003; Rudolf et al., 2003; Ivarsson et al., 2005; Varadi et al., 2005; Takamori et al., 2006; Desnos et al., 2007). Myosin Va dissociates from vesicular membranes in the presence of Ca2þ ions at micromolar levels (Prekeris and Terrian, 1997; Rose et al., 2003). Expression of a dominant-negative tail domain of myosin Va in secretory cells reduces secretory vesicle density in the subplasma membrane region and potently reduces their motility in the actin cortex (Rudolf et al., 2003; Varadi et al., 2005; Desnos et al., 2007). Myosin V head antibodies inhibit catecholamine release from adrenal chromaffin cells by interfering with secretory vesicle transport from the reserve to the releaseready compartment (Rose et al., 2003). Transfection of the dominant-negative tail domain of myosin Va in primary hippocampal neurons enhances the exocytosis of large dense core vesicles, and a similar increase in exocytosis is observed by depolymerization of F-actin using latrunculin B (Bittins et al., 2009). Thus, myosin Va is thought to play a dual role. It functions as a transport motor that actively moves secretory vesicles toward release sites, and also acts as a tethering

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or capture factor that prevents the exit of vesicles from the reserve compartment (Rudolf et al., 2011). OBA reduced the dissociation of myosin Va from synaptic vesicles, and suppressed fluctuating movements near the

plasma membrane and the long distance movement of secretory vesicles in PC12 cells. These results suggest that CaM is involved in Ca2þ-dependent dissociation of myosin Va from secretory vesicles, and that OBA may exert its inhibitory

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effect by preventing secretory vesicle detachment from Factin filaments by inhibiting myosin Va dissociation from secretory vesicles. We also found that myosin Va is associated with secretory vesicles in PC12 cells and cerebral cortex neurons, but not with those in cerebellar granule cells. These results may suggest that the lack of an inhibitory effect of OBA on cerebellar granule cells may reflect the absence of myosin Va in synaptic vesicles of these cells. An outstanding question is why myosin Va does not associate with synaptic vesicles in cerebellar granule cells despite the fact that it is abundantly expressed in the cerebellum, along with synaptophysin/VAMP-2 (Tilelli et al., 2003; Fig. 7). Cerebellar granule cells require a culture medium with high KCl (25 mM), conditions which depolarize the membrane and presumably induce a higher basal Ca2þ concentration than in cortical cultures, possibly chronically promoting the dissociation of myosin V from synaptic vesicles. Another possibility is that proteins that associate with myosin Va differ between cerebellar granule cells and PC12 cells and cerebral cortical neurons. The expression and intracellular distribution of myosin Va varies in different brain regions and neuronal cells (Tilelli et al., 2003). Indeed, contradictory results have been obtained on the role of myosin Va in neurotransmitter and hormonal release with different experimental systems. Depolarization-induced glutamate release from cerebrocortical synaptosomes is suppressed by 2,3-butanedione-2-monoxime, a potent inhibitor of myosin ATPase (Prekeris and Terrian, 1997), whereas expression of the dominant-negative tail domain of myosin Va enhances the exocytosis of large dense-core vesicles in cerebral cortical neurons (Bittins et al., 2009). Expression of the dominant-negative tail domain of myosin Va suppresses catecholamine release from PC12 cells, while knockdown of myosin Va expression by shRNA enhances exocytosis considerably in the same cells (Kögel et al., 2010). Syntaxin 1A, a synaptic t-SNARE protein essential for neurotransmitter release, binds to the neck region of myosin Va, and disruption of its association with myosin Va using an inhibitory peptide suppresses catecholamine release from adrenal chromaffin cells (Watanabe et al., 2005). Thus, it is possible that myosin Va function may differ depending on its expression as well as the expression of its modulator proteins. Additional studies will be needed to further understand the precise roles of myosin Va and CaM in neurotransmitter and hormone release in different cell types.

4.

Experimental procedures

4.1.

Reagents

9

Reagents were obtained from the following manufacturers: trypsin, Becton, Dickinson and Company; CaM, digitonin, and W-7, Merck; DY-9760e, Daiichi Sankyo; B27 supplement, fetal calf serum (FCS), horse serum, Lipofectamine 2000, minimum essential medium (MEM), neurobasal medium, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody, and HRPconjugated rabbit anti-mouse IgG antibody, Life Technologies; penicillin and streptomycin, Meiji Seika Pharma; pre-colostral newborn calf serum, Mitsubishi Chemical; ionomycin, papain, and protease inhibitor cocktails, Nacalai Tesque; dimethyl pimelimidate, Pierce; bovine serum albumin (BSA), cytosine arabinofuranoside, deoxyribonuclease (DNase) I, Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), and ophiobolin A, Sigma-Aldrich; and Dynabeads M-270 epoxy, Veritas. All other chemicals used were reagent grade.

4.2.

Antibodies

Monoclonal anti-botulinum toxin antibody (B102), a kind gift from Dr. Shunji Kozaki of Osaka Prefecture University, was used as the control mouse IgG antibody. Monoclonal anti-synap tophysin antibody (171B5) (Obata et al., 1986), monoclonal anti-synaptotagmin I antibody (1D12) (Takahashi et al., 1991), monoclonal anti-syntaxin antibody (10H5) (Yoshida et al., 1992), and polyclonal antibodies to SNAP-25 and VAMP-2 (Ando et al., 2005) were prepared as described previously. The recombinant myosin-Va globular tail [residues 1444–1853], derived from mouse cDNA, was produced using the pGEX-6P-1 vector (Pharmacia Bioscience) as a glutathione-S-transferase (GST)-fusion protein. After purification with PreScission protease (Pharmacia Bioscience) cleavage, rabbits were immunized with 0.46 mg of the globular tail protein to obtain a polyclonal antibody against myosin V. Polyclonal anti-phosphorylated CREB antibody was purchased from Cell Signaling Technology.

4.3.

Cell culture

PC12-C3, a subclone of PC12 (Nishiki et al., 1997), was used in the present study. Cells were plated at a density of 1.2  106 per dish onto either polyethyleneimine-coated 35 mm dishes

Fig. 9 – Suppression of secretory vesicle movement by OBA in PC12 cells. Large dense-core vesicles in PC12 cells were visualized by expressing hGH-EGFP by transient transfection, and observed by total internal reflection fluorescence microscopy. PC12 cells were preincubated in low-Kþ solution in the presence or absence of either 3 μM OBA or 3 μM KN-62, a Ca2þ/CaM-dependent protein kinase II inhibitor, for 30 min at 37 1C. (A) Optical images were taken at 10 s intervals in the presence or absence of either 3 μM OBA or 3 μM KN-62, and were represented in pseudocolors (red and green). Merged images are shown on the right. Bars: 4 μm. (B) The percentage of immobile vesicles was quantified in the presence of either OBA (black bar) or KN-62 (gray bar), or in the absence of inhibitor (open bar). Values are presented as mean7S.E. Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (2, 42) ¼ 19.4, po0.00005. Control (n ¼ 22), OBA (n ¼18), and KN-62 (n ¼5), nnnpo0.00005. (C) A series of optical images were taken at 2 s intervals as indicated. Some vesicles moved a long distance (arrowheads). Bars: 2 μm. (D) The number of vesicles that moved more than 4 μm in 30 s was counted in 3 μM OBA-treated (closed bar) or untreated cells (open bar). Values are presented as mean7S.E. (n ¼9). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s honestly significant difference (HSD) post hoc tests. F (1, 17)¼ 31.9, po0.00005. nnnpo0.00005.

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(Falcon) or glass-bottom culture dishes (MatTek), and at a density of 4.2  105 per well onto four-well culture plates (Nalge Nunc) in DMEM containing 5% (v/v) pre-colostral newborn calf serum and 5% (v/v) heat-inactivated horse serum. Cells were maintained in a 10% CO2-humidified atmosphere at 37 1C. Cerebral cortices were dissected from fetal Wistar rats at E17, and dissociated in 10 units/ml papain, 1.14 mM L-cystein, 27.8 mM glucose, 0.002% (w/v) BSA, and 280 units/ml DNase I for 10 min at 37 1C. After collection by centrifugation, papaintreated tissue was triturated in 10 ml of culture medium (MEM containing 10% (v/v) heat-inactivated FCS, 27.8 mM glucose, 6 mM NaHCO3, 50 units/ml penicillin, and 100 μg/ml streptomycin) using a 10 ml plastic pipette with a 200 μl plastic micropipette tip. Cells were then washed with culture medium, filtered through two layers of lens paper to remove cell aggregates, and plated onto polyethyleneimine-coated four-well culture plates at a density of 1.0  106 per well in culture medium. Neurons were maintained in a 5% CO2humidified atmosphere at 37 1C for 2 days, and the culture medium was changed to neurobasal medium containing 2% (v/v) B27 supplement, 1 μM cytosine arabinofuranoside, 50 units/ml penicillin, and 100 μg/ml streptomycin, and changed every 3 days. Neurons were cultured for 12–14 days before use. Cerebella were isolated from 7- to 9-day-old Wistar rats of either sex, and dissociated in 1% (w/v) trypsin, 12.5 mM glucose, and 700 units/ml DNase I for 15 min at 37 1C. After collecting by centrifugation, trypsinized tissue was triturated in 10 ml DMEM containing 10% (v/v) heat-inactivated FCS, 50 units/ml penicillin, and 100 μg/ml streptomycin, using a 10 ml plastic pipette with a 200 μl plastic micropipette tip. Cells were then washed with culture medium (90% (v/v) DMEM, 10% (v/v) heat-inactivated FCS, 25 mM KCl, 50 units/ ml penicillin, and 100 μg/ml streptomycin), filtered through two layers of lens paper to remove cell aggregates, and plated onto polyethyleneimine-coated four-well culture plates at a density of 1.0  106 per well in culture medium. Cells were maintained at 37 1C in a humidified 10% CO2 atmosphere. On the second day in vitro, 1 μM cytosine arabinofuranoside was added to the cultures to inhibit the proliferation of nonneuronal cells. Cells were cultured for 14–15 days before use.

4.4.

DA and ACh release assay

PC12 cells were plated onto polyethyleneimine-coated 35 mm dishes at a density of 1.2  106 per dish and cultured for 2 days. After several washes with low-Kþ solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, 15 mM HEPES-NaOH, pH 7.4), cells were preincubated under various conditions at 37 1C as indicated in the Figure Legends, and the medium was replaced subsequently with low-Kþ solutions with or without 1 μM ionomycin. Extracellular solutions were filtered through 0.2 μm Ultrafree-MC (Millipore) filters and stored at 80 1C until use. Released DA in low-Kþ solutions was assessed by HPLC (Jasco Corp) with a reverse-phase column (TSKgel ODS80TM, Tosoh) and an electrochemical detector (Eicom), as described previously (Nishiki et al., 1997). Released ACh in low-Kþ solutions was determined by HPLC (Jasco Corp) with a

reverse-phase column (Eicompak AC-GEL, Eicom), an immobilized acetylcholine esterase and choline oxidase column (AC-Enzympak, Eicom), and an electrochemical detector, as described previously (Nishiki et al., 1997). The amount of DA and ACh released into solution was expressed as a percentage of the total amount of DA and ACh in the cells.

4.5.

DA release assay using permeabilized PC12 cells

PC12 cells were plated onto polyethyleneimine-coated fourwell culture plates at a density of 4.2  105 per well and cultured for 2 days. After several washes with low-Kþ solution, cells were preincubated under various conditions at 37 1C as indicated in the Figure Legends. Cells were rinsed twice with Ca2þ-free Lock’s solution (156 mM NaCl, 5.6 mM KCl, 0.2 mM EGTA, 3.6 mM NaHCO3, 5.6 mM glucose, 5 mM HEPES-NaOH, pH 6.8), and permeabilized by incubation for 5 min with 8 μM digitonin, 5 mM ATP, and 5 mM MgSO4 in KGEP solution (140 mM potassium glutamate, 5 mM glucose, 5 mM EGTA, 20 mM piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES)-NaOH, pH 6.8). After permeabilization, the medium was replaced with KGEP solution containing 100 μM CaCl2 for 5 min, as described previously (Nishiki et al., 1997). Collected extracellular solutions were filtered through 0.2 μm Ultrafree-MC (Millipore) filters and stored at 80 1C until use. Released DA in KGEP solutions was determined by HPLC, as described above. The amount of DA released into solution was expressed as a percentage of the total amount of DA in the cells.

4.6.

Glu release assay

Neurons were plated on polyethyleneimine-coated four-well culture plates at a density of 1.0  106 per well. After washing with low-Kþ solution, neurons were preincubated under various conditions at 37 1C as indicated in the Figure Legends. Medium was subsequently replaced with the low-Kþ solution or low-Kþ solution containing 10 μM ionomycin. Collected extracellular solutions were filtered through 0.45 μm filters (Cosmonice Filter W, Nacalai Tesque) and stored at 80 1C until use. The amount of Glu was determined by reversephase high performance liquid chromatography on a Crestpak C18S column (4.6  150 mm) (JASCO), using pre-column derivation with ο-phthalaldehyde and fluorescence detection (FP-920-S, JASCO).

4.7. cells

Immunoisolation of synaptic vesicles from cultured

PC12 cells were homogenized with a glass homogenizer (Dounce Grinder Tight, Wheaton) in PBS containing protease inhibitor cocktails with 300 strokes on ice, and centrifuged at 7700 g for 15 min. The resulting supernatant was saved for use. Cerebral cortical neurons and cerebellar granule cells were treated with 5 mM HEPES (pH 7.4 adjusted with NaOH) containing protease inhibitor cocktails for 5 min on ice. After hypotonic shock, cell lysates were mixed with an equal volume of 0.64 M sucrose, 5 mM HEPES (pH 7.4 adjusted with NaOH), and protease inhibitor cocktails, and centrifuged at 17,400 g for 30 min to obtain the supernatant. Cell lysates

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were mixed with antibody-conjugated magnetic beads, followed by incubation for 24 h at 4 1C with continuous rotation. Beads were washed five times with PBS containing protease inhibitor cocktails, denatured at 100 1C for 3 min in sodium dodecyl sulphate (SDS) sample buffer (70 mM SDS, 3.3% (w/v) glycerol, 0.03 mM bromophenol blue, 125 mM Tris–HCl, pH 6.8), and stored at 80 1C until use.

4.8.

Immunoblotting

SDS-polyacrylamide gel electrophoresis (PAGE) was performed with a 12.5% (w/v) acrylamide gel or 2–15% (w/v) gradient gel (Daiichi Kagaku). Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon-p, Millipore) with a semi-dry transblotting apparatus. Membranes were blocked in 10% (w/v) non-fat milk in TBST (0.05% (w/v) Tween 20, 150 mM NaCl, 25 mM Tris–HCl, pH 7.5) and incubated with primary antibodies in TBST containing 10% (w/v) non-fat milk for 1 h at room temperature. After washing in TBST, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies in TBST containing 10% (w/v) non-fat milk. After washing in TBST, immunoreactive bands were visualized with the Super Signal chemiluminescent detection kit (Pierce) in a linear range with a luminescence image analyzer connected to an electronically cooled CCD camera system (LAS-1000, Fuji Photo Film).

4.9. Total internal reflection fluorescence microscopy and image analysis A DNA fragment corresponding to the human growth hormone (hGH) gene was isolated from the pXGH5 vector and subcloned into the pEGFP vector (Promega). PC12 cells were plated onto 35 mm glass-bottom culture dishes at a density of 1.2  106 cell per dish. After 20–24 h, the cells were transfected with the vector using Lipofectamines 2000 (Invitrogen) in the presence of serum according to manufacturer’s instructions. For live imaging of PC12 cells transfected with hGH-EGFP, we performed total internal reflection fluorescence microscopy (TIRFM or evanescent field microscopy), as described previously (Stout and Axelrod, 1989; Aoyagi et al., 2005). Digital images were captured on a CCD camera (CCD 300RC, Dage-MTI), with a pixel size of 63.5 nm. hGH-EGFP-labeled vesicles were identified and tracked through a time-sequence stack of images with StreamPix 3.4.0 digital video recording software (Norpix). Frames were acquired as streams at 30 Hz with an exposure time of 30 s. For stability of vesicle movement analysis, overlap was determined by assigning red and green colors to the images taken 10 s apart. Stable movement was defined for cases in which the red and green dots had merged more than half-way. For long distance migrating vesicle analysis, migrating vesicles were scored as positive when the hGH-EGFP-labeled granule signal moved more than 4 μm over 30 s. The percentage of long distance migrating vesicles among all vesicles was determined for single cells.

4.10.

Statistics

Data are presented as mean7SE. Groups were analyzed by oneway or two-way ANOVA tests followed by Tukey–Kramer’s

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honestly significant difference (HSD) post hoc tests. Statistical significance is indicated as npo0.05, nnpo0.01, and nnnpo0.001.

Acknowledgments This work was supported by Grants-in Aid 21300141 and 23650193 for Scientific Research on Priority Areas (B) from the Japan Society for the Promotion of Science.

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