Journal of Molecular Neuroscience Copyright © 2001 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/01/17:303–310/$12.00

Tetanus Toxin Modulates Serotonin Transport in Rat-Brain Neuronal Cultures Patricia Pelliccioni, Carles Gil, Abderrahim Najib, Elisabet Sarri, Fernando Picatoste, and José Aguilera* Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès (Barcelona), Spain Received February 28, 2001; Accepted March 4, 2001

Abstract As has been previously described, tetanus toxin (TeTx) and its HC fragment inhibit the sodium-dependent 5-hydroxytryptamine (5-HT) uptake in rat-brain synaptosomes, probably through a kinase mechanism affecting the 5-HT transporter. Now, the inhibition of 5-HT uptake in neurons in primary culture by TeTx in a dosedependent manner is described in this work. This effect is also produced by the nontoxic C-terminal fragment of the TeTx heavy chain (Hc-fragment), indicating that 5-HT uptake inhibition is a consequence of the toxin binding to the plasmatic membrane and not to its catalytic activity. This conclusion is supported by the fact that the 5-HT accumulation was not inhibited by the light chain of TeTx or the toxoid, and was even potentiated by botulinum neurotoxin A. These results correlate with the activation of phosphoinositide-phospholipase C activity in the cultures used in this study, this activity only being enhanced by TeTx and by its Hc-fragment. On the other hand, the use of tyrosine phosphorylation modulators indicates that both Na3VO4 and basic fibroblast growth factor (bFGF) produce an enhancement of 5-HT uptake in this system, which is also sensitive to TeTx inhibition. On the other hand, genistein alone is able to reduce the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests that TeTx and genistein may share some events in their respective mechanisms of action. Furthermore, the incubation at different concentrations of 12-0-tetradecanoylphorbol 13-acetate (TPA) confirms the involvement of protein kinase C (PKC) in 5-HT transport modulation in rat-brain neuronal primary cultures. In summary, we shall demonstrate in this work that TeTx induces, through its Hc fragment, an inhibition of both basal and stimulated serotonin uptakes in primary neuronal cultures, in parallel to the activation of phosphoinositide-phospholipase C activity and PKC activation. Index Entries: 5-hydroxytryptamine (serotonin) uptake; phospholipids hydrolysis; protein kinase C; phospholipase C; tyrosine kinase receptors.

Introduction Tetanus toxin is a high-molecular-weight (150 kDa) polypeptide produced by the Gram-positive, spore-forming bacilli Clostridium tetani, which produces an illness characterized by muscular contractions and recurring spasms, which causes death in a high percentage of cases as a result of respiratory failure (Niemann, 1991). TeTx, as well as other clostridial neurotoxins, is synthesized as a single

polypeptide chain (scTeTx). Following limited proteolysis, an activation occurs in which bacterial or host proteases cleave the scTeTx in a hinge region, yielding two chains bound by an intermolecular disulfide bond (Weller et al., 1988; Krieglstein et al., 1990). Various techniques have been used to generate fragments from the toxins. The reduction of the disulfide bond gives rise to the L (light)-chain (50 kDa) fragment that is physiologically active as a metalloprotease, and also to the H (heavy)-chain

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

Journal of Molecular Neuroscience

303

Volume 17, 2001

304 (100 kDa), which mediates binding to the target tissue and internalization. Limited proteolysis using papain generates the L-HN and the non-toxic HC fragments, with molecular weights of 100 and 50 kDa, respectively (Niemann, 1992). The main target of TeTx in vivo is the central nervous system (CNS), where it is believed to block synaptic release by several mechanisms, which probably work synergistically. The first mechanism identified emerged with the discovery of its action as a Zn2+-metalloprotease. The finding of the selective proteolytic cleavage of both the vesicle-associated membrane protein (synaptobrevin) and cellubrevin, a synaptobrevin homolog (Link et al., 1993; McMahon et al., 1993) proved this action. A second mechanism involves the activation by TeTx of Ca2+-dependent transglutaminase Type II (Facchiano et al., 1993) an enzyme that blocks neurosecretion even when the protease activity of TeTx is abolished (Ashton et al., 1995). Finally, the effects of TeTx may arise from the sustained translocation of PKC elicited by the toxin (Aguilera and Yavin, 1990), even before classical tetanus symptoms are evident (Aguilera et al., 1993). Rat-brain synaptosomes treated with TeTx, scTeTx, or some TeTx-fragments show inhibition of Na+-dependent high-affinity 5-HT uptake, which points out another mechanism altered by the neurotoxin that could be related to its poisoning effects (Inserte et al., 1999). Little is known about the mechanisms involved in this action of the toxin, although some descriptions point to some role of protein kinases. First, the evidence that the 5-HT uptake is modulated by tyrosine kinases in human platelets (Helmeste and Tang, 1995) and, second, by direct action of PKC over the SERT in HEK-293 cells (Ramamoorthy et al., 1998). Since TeTx elicits translocation to the membrane of PKC isoforms in rat-brain primary neurons (Gil et al., 1998) and in rat-brain synaptosomes, together with an enhancement of the tyrosine phosphorylation in trkA, PLCγ-1, and ERK-1/2 (Gil et al., 2000), these events could play an important role in the regulation of 5-HT transport by TeTx. In this work, we shall show that TeTx and the HC-TeTx fragment, which also contains ganglioside and neuronal cell-binding activities, substantially reduce both basal and tyrosine phosphorylation-stimulated 5-HT transport in cultured rat-brain neurons, as well as induce PLC activity. This suggests a mechanism of TeTx action over SERT in which some members of the signal transduction pathway, i.e., PLC, PKC and tyrosine kinases, could be involved.

Journal of Molecular Neuroscience

Pelliccioni et al.

Materials and Methods Clostridial Neurotoxins TeTx, Botulinum neurotoxin A(BoNT/A), HC-TeTx, and L-TeTx were kindly provided by Prof. Juan Blasi (Departament de Biologia Cel•lular i Anatomia Patològica, Universitat de Barcelona, Spain). The toxicity of TeTx and BoNT/A, determined according to Weller et al. (1998) was 6.7 × 106 and 2.0 × 107 mouse minimum lethal doses per milligram of protein, respectively. Tetanus toxoid was obtained by toxin incubation in 0.1% formaldehyde for 24 h at 37°C with extensive dialysis. Materials Tissue culture-grade myo-inositol, carbachol (carbamylcholine chloride), Tween 20, 5-hydroxytryptamine, genistein (4′,5,7-trihydroxyisoflavone), 12-0-tetradecanoylphorbol 13-acetate and bFGF were from Sigma Chemical (St. Louis, MO). Myo-[2-3H]inositol (55 Ci/mmol) was from NEN Research (Boston, MA). 5-[1,2- 3 H]Hydroxytryptamine ([ 3 H]5-HT) (34 Ci/mmol) was from Amersham International (Buckinghamshire, UK). Alomone Labs supplied NGF (Jerusalem, Israel). Paroxetine was a gift of SmithKline-Beecham. All other reagents were of the highest grade possible from standard commercial sources. Neuronal Primary Culture Primary cultures of neurons were routinely prepared according to Yavin et al. (1981) with minor modifications. Primary cultures of cerebral cortical neuron were prepared from 16-d-old fetal rat embryos. Cells were seeded on 24-well plates precoated with 25 µg/mL poly-L-lysine at a density of 0.7 × 106 viable cells/mL in Dulbecco’s modified Eagle’s basal medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics. After 2 h of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the medium was changed for Eagle basal medium enriched with 1% FCS, 19.4 mM glucose, 4 mM glutamine, 20 nM progesterone, 100 µM putrescine, 5 µg/mL transferrin, 10 µg/mLinsulin, 30 nM sodium selenite, and antibiotics. Cells grown in these conditions become an effectively pure population of cerebral neurons identified by their ability to interact with tetanus toxin (Yavin et al., 1981). All studies were performed on cells grown for at least 10 d in culture. Assay of [ 3H]5-HT Uptake Cells were washed twice with 500 µLKrebs-Ringer bicarbonate buffer (containing 125 mM NaCl, 3 mM Volume 17, 2001

Tetanus Toxin Modulates Serotonin Transport KCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 22 mM NaHCO3, 1 mM NaH2PO4, and 10 mM Glucose, it was gassed with a mixture of 95% O2 and 5% CO2 for 20 min and adjusted to pH 7.4) and resuspended in 950 µL of the buffer containing the toxin (BoNT/A, TeTx, or its fragments), or without toxin as a control. After a preincubation period of 30 min at 37°C in a shaking water-bath, uptake was started by the addition of 50 µL [3H]5-HT. For saturation experiments, the final tritiated amine concentration range was from 5 nM to 160 nM, and for inhibition experiments a concentration of 80 nM was used. After incubation of 5 min at 37°C, samples were washed twice with icecold serotonin (1 mM). 250 µL of NaOH (0.5 M) was added to each well and left for 30 min at room temperature. After that, the cells were collected and transferred to a microtube, incubated for 30 min at 50°C. 100 µL of the homogenate were then taken for counting total radioactivity in the cells. They then were placed in vials with 5 mLBiodegradable Counting Scintillant (Amersham Int.; Buckinghamshire, UK) and counted in a Wallac-1409 liquid scintillation counter, with an efficiency of 45%. The nonspecific transport was measured by cell incubation with the same [3H]5-HT concentrations at 37°C in the presence of paroxetine (50 µM), fenfluramine (50 µM) and imipramine (50 µM). The specific uptake was defined as the difference between uptake carried out at 37°C minus uptake carried out at 37°C plus serotonin-transport inhibitors. Uptake was calculated as fmol of [3H]5-HT per minute and mg of protein. Protein was determined according to Bradford (1976) using immunoglobulin G as a standard (BioRad; Hercules, CA).

Determination of PLC Activity PLC was assayed by measuring the accumulation of 3H-inositol phosphates ( 3H-InsPs) from myo[2-3H]inositol-prelabeled cells. Cultured neurons were labeled with myo-[2-3H]inositol (2 µCi/mL). After 24 h, labeled cells were washed twice, for 15 min each, with 500 µL of Krebs-Henseleit buffer (in mM: NaCl 116, KCl 4.7, MgSO4 1.2, NaHCO3 25, CaCl2 1.3, KH2PO4 1.2, and glucose 11, pH 7.4, equilibrated with O2/CO2 (95⬊5 v/v). The incubation was performed in the presence or absence of stimuli for 30 min at 37°C in the presence of 10 mM LiCl. The reactions were stopped by adding ice-cold methanol (800 µL), and cells were scraped and transferred to test tubes. Chloroform (900 µL) was added and two phases were generated by adding water (250 µL) and 0.25 M HCl (500 µL). Samples of the aqueous phases (1 mL) were neutralized with 1.5 M Journal of Molecular Neuroscience

305 NH4OH, diluted with 5 mL of water, and loaded onto columns with 0.5 mL of Dowex 1 × 8 resin (100–200 mesh, formate form) as described previously (Claro et al., 1993). After washing the columns with 4 × 4 mL of water and 2 × 4 mL of 60 mM sodium formate/5 mM borax to remove [3H]inositol and [3H]glycerophosphorylinositol, 3H-inositol phosphates were eluted together with 2 × 3 mL of 1.0 M ammonium formate/0.1 M formic acid. After the addition of 15 mL of a scintillation cocktail (Flo-Scint IV from Packard BioScience Co.), the vials were cooled to form a clear homogenous phase and were counted at a 30% efficiency rate. Accumulation of 3H-InsPs was calculated as the percentage of lipid-labeling in the same sample. To do so, [3H]inositol labeling of lipids was monitored by counting 0.2-mL aliquots of the organic phases.

Data Analysis Data obtained are presented as means with standard error of mean (mean ± SEM) of indicated samples. Differences between groups were assessed using the one-way analysis of variance (ANOVA) test and one-way Dunnett’s test for multiple comparisons. p < 0.05 was considered statistically significant.

Results Saturation of 5-HT Uptake in Neuronal Cultures As shown in Fig. 1, untreated neuronal cultures showed saturable specific [3H]5-HT uptake with kinetic parameters, Vmax and KM, of 564.9 ± 39 fmol per min and mg of protein, and 28.1 ± 5.6 nM, respectively (n = 3). The nonspecific uptake, measured at substrate concentration (80 nM) not far from the K M value, was in the range 20–30% of the total [3H] 5-HT uptake. The 5-HT uptake determined corresponds to an effectively pure population of cerebral neurons, as is described in the Materials and Methods section. The presence of SERT protein was confirmed by immunodetection in Western blot (result not shown). Inhibition of 5-HT Uptake by TeTx and by Hc-TeTx TeTx inhibited the specific uptake of [3H]5-HT (80 nM) in a concentration-dependent manner (82 ± 9% of control at 1 nM of TeTx, 75 ± 5% of control at 5 nM and 40 ± 7% of control at 10 nM), whereas the same concentrations of another clostridial neurotoxin, BoNT/A, produced low, but significant, increases in 5-HT accumulation (132 ± 8%, 162 ± 12% Volume 17, 2001

306

Fig 1. [3H]5-HT uptake evaluated by saturation curve. Na+-dependent 3H5-HT uptake into rat-brain primary neurons preincubated for 30 min with and without the neurotoxin was evaluated for Krebs-Ringer’s buffer. The transport activity was measured at different concentrations [3H]5-HT (5–160 nM) for 5 min at 37°C for each treatment. The nonspecific transport was measured after cell incubation at the same [3H]5-HT concentrations at 37°C in the presence of paroxetine (50 µM), fenfluramine (50 µM), and imipramine (50 µM). Uptake was calculated as fmol of [3H]5-hydroxytryptamine per minute and mg of protein. Data are mean ± SD (bars) values of triplicate determinations from representative experiments. The straight line at the bottom of the figure corresponds to the nonspecific [3H] 5-HT uptake, measured in presence of uptake inhibitors.

and 144 ± 11% of control, respectively) (Fig. 2A). Among the TeTx fragments tested, HC-TeTx (10 nM) reduced [3H] 5-HT uptake to 53 ± 6% of control (n = 3), but both L-TeTx (10 nM) and the tetanus toxoid (10 nM) were ineffective (108 ± 13% and 99 ± 3% of the control [3H] 5-HT uptake, respectively) (Fig. 2B).

TeTx-Induced Inhibition of 5-HT Uptake Enhanced by Tyrosine Phosphorylation Modulators In order to further explore the TeTx effect on [3H] 5-HT transport in our system, experiments with sodium vanadate (100 µM), a nonspecific tyrosine phosphatase inhibitor, and agonists of TyrKRs, such as nerve growth factor (NGF) and bFGF, were performed. Sodium vanadate (Fig. 3) and bFGF (Fig. 4) induced an enhancement of [3H] 5-HT transport (178 ± 29% and 169 ± 6%, respectively), which was also inhibited by TeTx. Interestingly, the tyrosine kinase Journal of Molecular Neuroscience

Pelliccioni et al.

Fig 2. Effect of neurotoxins on Na+-dependent serotonin transport in rat-brain neurons. The cells were preincubated for 30 min with or without the corresponding neurotoxin or fragment at the indicated dose. Control uptake = 718 ± 91 fmol/min/mg protein. (A) Dose-dependent effect of tetanus toxin and botulinim neurotoxin A. One-way ANOVA tests were performed for TeTx (p < 0.001) and for BoNT/A (p < 0.01). (B) Effect of TeTx (10 nM), HC-TeTx (10 nM), L-TeTx (10 nM), and Toxoid (10 nM). Each point represents the mean ± SEM (n = 3–4). To compare each treatment with its respective control group, the one-way Dunnett’s test was performed, **p < 0.01, ***p < 0.001, and n.s. = not significant.

inhibitor genistein, at 160 µM, elicited an inhibition of basal [3H] 5-HT transport (61 ± 3% of control) to a similar extent as that of TeTx (10 nM), but both effects were not additive.

Inhibition of 5-HT Uptake by Direct Stimulation of PKC Since SERT has been described to be modulated by PKC, the direct activation of this kinase by concenVolume 17, 2001

Tetanus Toxin Modulates Serotonin Transport

Fig. 3. Effect of tetanus toxin, genistein, and sodium vanadate (Na3VO4) on Na+-dependent serotonin transport in rat-brain neurons. The cells were preincubated for 30 min with and without TeTx (10 nM), Na3VO4 (100 µM), or genistein (160 µM). Shaded bars represent incubation of tyrosine phosphorylation modulator (Na3VO4 or genistein) and no subsequent addition of toxin. Each point represents the mean ± SEM (n = 3–4). The one-way ANOVA test was p < 0.001, and the one-way Dunnett’s test was performed, **p < 0.01 and n.s., not significant.

307

Fig. 5. Effect of phorbol ester TPA on Na+-dependent serotonin transport in rat-brain neurons. The cells were preincubated for 30 min with and without the corresponding concentration of TPA for 5 min at 37°C. Each point represents the mean SEM (n = 3–4). The one-way ANOVA test was p < 0.001 and the one-way Dunnett’s test was performed, **p < 0.01, ***p < 0.001.

trations (0.1, 1, and 5.0 µM) of the TPA, a phorbol ester typically used as an activator of classical and novel PKC isoforms, has been tested. The results obtained showed a reduction of the [3H] 5-HT uptake rates (70, 32, and 49% of control, respectively) (Fig. 5).

Activation of Phospholipase C Activity by TeTx and by Hc-TeTx Since PKC activation may result from phosphoinositide phospholipase C (PLC) stimulation, the action of TeTx on this effector system was studied and this effect with those triggered by activation of TyrKRs and muscarinic-cholinergic receptors was compared. As shown in Fig. 6A, TeTx (10 nM) substantially enhanced the PLC-derived accumulation of [3H]inositol phosphates in [3H]inositol-prelabelled neurons (281 ± 41% of basal). This effect was somewhat lower than the stimulation induced by 1 mM of the cholinFig. 4. Effect of tetanus toxin and growth factors on Na+dependent serotonin transport in rat-brain neurons. The ergic agonist carbachol (441 ± 38%) but higher than cells were preincubated for 30 min in control conditions, those produced by 200 nM bFGF or 50 nM NGF (128 in the presence of the neurotoxin (10 nM) or of growth ± 5% and 166 ± 14%, respectively). Of the two TeTx factors (NGF [50 nM], bFGF [200 nM]), and with both fragments, only HC-TeTx produced an increase of toxin and growth factors, when indicated. Each point phosphoinositide hydrolysis (161 ± 16% of basal), and represents the mean ± SEM (n = 3–4). The one-way ANOVA the tetanus toxoid failed to activate PLC (Fig. 6B). test was p < 0.001 and the one-way Dunnett’s test was performed, *p < 0.05, **p < 0.01, ***p < 0.001, and n.s., not significant.

Discussion Na +-dependent, high-affinity serotonin transporters are responsible for efficient synaptic clear-

Journal of Molecular Neuroscience

Volume 17, 2001

308

Fig. 6. Phosphoinositide hydrolysis in rat-brain neurons. Cells prelabeled with [3H]inositol were incubated in KrebsHenseleit buffer containing 10 mM LiCl. (A) Basal (no further additions), carbachol (1 mM) (Cch), TeTx (10 nM), bFGF (200 nM), or NGF (50 nM) for 30 min. (B) Basal, and with 10 nM of each respective toxin. The radioactivity detected in the Basal corresponded to 8.7 ± 1.3% of the total radioactivity of 3H-lipids. Data are expressed as means ± SEM (n = 5–7). The one-way ANOVA test in panels A and B were p < 0.001. The one-way Dunnett’s test was performed, *p < 0.05, **p < 0.01, and n.s., not significant.

ance of extracellular 5-HT. In the present work, the effects of two clostridial neurotoxins, TeTx and BoNT/A, have been studied on 5-HT transport in primary cultures of rat-brain neurons. Previous to any experiment, we determined that these cells show saturable [3H] 5-HT uptake, with similar kinetic parameters to those found in the case of adult rat-brain synaptosomal preparations (Inserte et al., 1999). As in synaptosomes, [3H] 5-HT uptake in cultured neurons was inhibited by the antidepressant drugs fluoxetine, paroxetine, and imipramine (Demchyshyn

Journal of Molecular Neuroscience

Pelliccioni et al. et al., 1994; Helmeste and Tang, 1995), which were used to define the nonspecific uptake. This approach was chosen instead of measuring [3H] 5-HT uptake at 0°C since cells lost their adherence to culture plaques in this condition. The present study demonstrates that the two clostridial neurotoxins assayed have opposite effects on 5-HT transport in cultured neurons. The inhibition of the neurotransmitter uptake by TeTx and the significant 5-HT accumulation elicited by BoNT/A, both observed in this work, are in agreement with previous data showing that in rat-brain synaptosomes 5-HT uptake is inhibited by TeTx, but not by BoNT/A, in a time-, and concentration-dependent manner (Najib et al., 1999). The different actions of the two structurally related toxins could be due to their lesser similarity in the C-terminal domain of the heavy chain, which is the domain that binds to the plasma membrane (Niemann, 1991) since, as is shown here, the HC-TeTx fragment, but not the L-TeTx chain, mimics the effect of the whole TeTx molecule on 5-HT transport. Our data also indicate that the inhibitory action of TeTx on 5-HT transport is independent of the metalloprotease activity, which, in both toxins, is located in the L-chains. On the other hand, the serotonin accumulation elicited by BoNT/Acould be a consequence of its metalloprotease activity (Link et al., 1992; Schiavo et al., 1992a; Schiavo et al., 1992b). Evidence of 5-HT transport modulation by tyrosine kinases has been reported in human platelets, where two structurally distinct inhibitors of these activities, genistein and methyl 2,5-dihydroxycinnamate, induce a rapid and dramatic decrease of 5-HT uptake (Helmeste and Tang, 1995). It is herein shown that genistein reduced the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests an event shared by TeTx and genistein in their respective mechanisms of action over 5-HT uptake. In addition, the tyrosine phosphatase inhibitor sodium vanadate as well as bFGF enhanced 5-HT uptake, which was blocked by TeTx, approx at a 50% rate. The different effects of the growth factors might result from a different relative density of their respective receptors in the cultured neurons. In this context, a high expression of bFGF receptors in the CNS has been reported (Lindholm et al., 1994). Activators of PKC, such as phorbol esters, have been shown in a number of systems to produce transporter phosphorylation and redistribution (Qian et al., 1997) and 5-HT uptake inhibiton (Myers et al., 1989; Anderson and Horne, 1992). In addition, activation and translocation of PKC by TeTx in rat-brain

Volume 17, 2001

Tetanus Toxin Modulates Serotonin Transport primary neurons have been previously shown (Gil et al., 1998), as well as PKC activation by TPA in ratbrain synaptosomes, resulting in a decreased Vmax value of the 5-HT uptake (Najib et al., 1999). The possibility that the actions of TeTx on 5-HT transport could be mediated by PKC-mediated SERT phosphorylation has been recently demonstrated in ratbrain synaptosomes (Najib et al., 2000). In addition to PKC activation, TeTx also activates ERK-1/2 (Gil et al., 2000), an event also related to PKC activity. The maximal inhibition exerted by TPA in this work is observed at 1 µM, whereas at 5 µM a partial reversion of the inhibition is detected. This event is possibly related with the PKC downregulation subsequent to activation of the kinase, when cells are treated with high TPA concentrations. The activation of PKC by TeTx in cultured neurons may result from the PLC-mediated generation of the second messenger diacylglycerol, since the toxin and its HC-TeTx fragment were able to stimulate the PLCmediated hydrolysis of phosphoinositides, as seen in Fig. 6. The extent of the effect of the toxin was lower than that elicited by a maximally acting concentration of the cholinergic agonist carbachol, and higher than the activation triggered by the growth factors bFGF and NGF. Considering that both TeTx and growth factors activate PLC but have opposite effects on 5-HT uptake, it appears that 5-HT transport in neurons is not directly controlled by PLC stimulation. Focusing on the PLC isoforms’ activation by TeTx, the toxin-induced tyrosine phosphorylation of PLCγ-1 in rat-brain synaptosomes has been described (Gil et al., 2000). Moreover, the membrane translocation of PLCγ-1 after incubation of synaptosomes with HC-TeTx has been observed (submitted), reinforcing both of these results on PLCγ-1 activation in the putative participation of TyrKRs in the TeTx action. In conclusion, our studies show that the HC domain of tetanus toxin might modulate some signaling pathways involving tyrosine kinase, PLC, and/or PKC activation to regulate the transport of 5-HT. Further work is needed to uncover the relationship between PLC, PKC, and tyrosine kinase modulation by TeTx and the involvement of these systems in the regulation of 5-HT transport, either by affecting SERT activity or decreasing the surface SERT protein.

Acknowledgment This work was supported in part by grant PB970169 from the Ministerio de Educación y Cultura,

Journal of Molecular Neuroscience

309 Dirección General de Enseñanza Superior e Investigación Científica. We would also like to thank Mr. Chuck Simmons for the revision of this manuscript.

References Aguilera J., Padrós-Giralt C., Habig W. H., and Yavin E. (1993) GT1b ganglioside prevents tetanus toxin-induced protein kinase-C activation and down-regulation in the neonatal brain in vivo. J. Neurochem. 60, 709–713. Aguilera J. and Yavin E. (1990) In vivo translocation and down-regulation of protein kinase C following intraventricular administration of tetanus toxin. J. Neurochem. 54, 339–342. Anderson G. M. and Horne W. C. (1992) Activators of protein kinase-C decrease serotonin transport in human platelets. Biochim. Biophys. Acta 1137, 331–337. Ashton A. C., Li X., Doussau F., Weller U., Dougan G., Poulain B., and Dolly O. (1995) Tetanus toxin inhibits neuroexocytosis even when its Zn2+-dependent protease activity is removed. J. Biol. Chem. 270, 31,386–31,390. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Claro E., Fain J. N., and Picatoste F. (1993) Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices. J. Neurochem. 60, 2078–2086. Demchyshyn L. L., Pristupa Z. B., Sugamori K. S., Barker E. L., Blakely R. D., Wolfgang W. J., et al. (1994) Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 91, 5158–5162. Facchiano F., Benfenati F., Valtorta F., and Luini A. (1993) Covalent modification of synapsin I by a tetanus toxinactivated transglutaminase. J. Biol. Chem. 268, 4588–4591. Gil C., Ruiz-Meana M., Álava M., Yavin E., and Aguilera J. (1998) Tetanus Toxin Enhances Protein Kinase C activity translocation and increases polyphosphoinositide hydrolysis in rat cerebral cortex preparations. J. Neurochem. 70, 1636–1643. Gil C., Chaib I., Pelliccioni P., and Aguilera J. (2000) Activation of signal transduction pathways involving trkA, PLCγ-1, PKC isoforms and ERK-1/2 by tetanus toxin. FEBS Lett. 481, 177–182. Helmeste D. M. and Tang S. W. (1995) Tyrosine kinase inhibitors regulate serotonin uptake in platelets. Eur. J. Pharmacol. 280, R5–R7. Inserte J., Najib A., Pelliccioni P., Gil C., and Aguilera J. (1999) Inhibition by tetanus toxin of the sodiumdependent, high-affinity [ 3H]5-hydroxytryptamine uptake in rat synaptosomes. Biochem. Pharmacol. 57, 111–120. Krieglstein K., Weller U., Henschen A., and Habermann E. (1990) Arrangement of disulfide bridges and posi-

Volume 17, 2001

310 tions of sulfhydryl groups in tetanus toxin. Eur. J. Biochem. 188, 39–45. Lindholm D., Hartikka J., Berghazui M. D., Castren E., Tzimagiorgis G., Hughes R. A., and Thoene H. (1994) Fibroblast growth factor-5 promotes differentiation of cultured rat septal cholinergic and raphe serotonergic neurons: comparison with the effects of neurotrophins. Eur. J. Neurosci. 6, 244–252. Link E., Edelmann E., Chou J. H., Binz T., Yamasaki S., Eisel U., et al. (1992) Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis. Biochem. Bioph. Res. Comm. 189, 1017–1023. Link E., McMahon H., Vonmollard G. F., Yamasaki S., Niemann H., Sudhof T. C., and Jahn R. (1993) Cleavage of cellubrevin by tetanus toxin does not affect fusion of early endosomes. J. Biol. Chem. 268, 18,423–18,426. McMahon H. T., Ushkaryov Y. A., Edelman L., Link E., Binz T., Niemann H., et al. (1993) Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature 364, 346–349. Myers C. L., Lazo J. S., and Pitt B. R. (1989) Translocation of protein kinase-C is associated with inhibition of 5-HT uptake by cultured endothelial cells. Am. J. Physiol. 257, L253–L258. Najib A., Pelliccioni P., Gil C., and Aguilera J. (1999) Clostridium neurotoxins influence serotonin uptake and release differently in rat-brain synaptosomes. J. Neurochem. 72, 1991–1998. Najib A., Pelliccioni P., Gil C., and Aguilera J. (2000) Serotonin transporter phosphorylation modulated by tetanus toxin. FEBS Lett. 486, 136–142. Niemann H. (1992) Clostridial neurotoxins: proposal of a common nomenclature. Toxicon 30, 223–225.

Journal of Molecular Neuroscience

Pelliccioni et al. Niemann H. (1991) Molecular biology of clostridial neurotoxins, in Sourcebook of Bacterial Toxins (Alouf J. E. and Freer J. E., eds.), Academic Press, New York, pp. 303–348. Qian Y. Galli A., Ramamoorthy S., Risso S., DeFelice L. J., and Blakely R. D. (1997) Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J. Neurosci. 17, 45–57. Ramamoorthy S., Giovanetti E., Qian Y., and Blakely R. D. (1998) Phosphorylation and regulation of antidepressant-sensitive serotonin transporters. J. Biol. Chem. 273, 2458–2466. Schiavo G., Benfenati F., Poulain B., Rossetto O., Polverino de Laureto P., DasGupta B. R., and Montecucco C. (1992a) Tetanus toxin and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832–834. Schiavo G., Poulain B., Rossetto O., Benfenati F., Tauc L., and Montecucco C. (1992b) Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc. EMBO J. 11, 3577–3583. Weller U., Mauler F., and Habermann E. (1988) Tetanus toxin: Biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis. Naunyn-Schmiedeberg’s Arch. Pharmacol. 338, 99–106. Yavin E., Yavin Z., Habig W. H., Handegree M. C., and Kohn L. D. (1981) Tetanus toxin association with developing neuronal cell cultures. Kinetic parameters and evidence for ganglioside-mediated internalization. J. Biol. Chem. 256, 7014–7022.

Volume 17, 2001