β-Bungarotoxin induction of neurite outgrowth in NB41A3 cells

Toxicon 52 (2008) 354–360

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b-Bungarotoxin induction of neurite outgrowth in NB41A3 cells Pei-Fung Wu a, *, Long-Sen Chang b, Yu-Ling Kao c, Kuang-Tao Wang a a b c

Department of Kinesiology, Health and Leisure Studies, National Kaohsiung University, Kaohsiung, Taiwan Institute of Biomedical Sciences, National SunYat-Sen University, Kaohsiung, Taiwan Department of Life Science, National Kaohsiung University, Kaohsiung, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2008 Received in revised form 20 May 2008 Accepted 5 June 2008 Available online 21 June 2008

In this study, different concentrations of b-Bgt were used to treat cultured NB41A3 cells. Inverted phase contrast microscopy was then used 24 h after treatment to observe the outgrowth of neurite. We found a clear outgrowth of neurite at b-Bgt concentrations of 357 nM. However, using a cytotoxicity assay to study apoptosis, we found no significant difference in the rate of cell death in cell cultures treated with either 357 nM or 714 nM. Western blotting showed that after treatment with b-Bgt, there was a notable decrease in small G protein Cdc42 and a marked increase in RhoA protein. Flow cytometry revealed that b-Bgt did not alter the calcium influx in NB41A3 cells. The neurite outgrowth induced by b-Bgt was not affected by extracellular EGTA, suggesting that the internalization of b-Bgt from extracellular was independent of phospholipase. Taken together, our results suggest the b-Bgt-induced outgrowth of neurite from NB41A3 cells may be mediated by small G proteins. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Small G protein EGTA Internalization

1. Introduction

b-Bungarotoxin (b-Bgt), which is purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains (A chain and B chain) cross-linked by an interchain disulfide bond (Yang and Lee, 1963; Kondo et al., 1978a,b). The A chains are structurally homologous to PLA2, and the B chains are sequentially homologous with trypsin inhibitor, toxin I and dendrotoxin (Kelly and Brown, 1974; Kondo et al., 1982a,b). The B chain may block certain voltage-gated potassium channels without involving the A chain (Benishin, 1990). Moreover, according to synaptosome-binding studies on an isolated B chain, the B chains may not be exclusively related to the specific binding of b-Bgt with its target (Wu et al., 1998). Both A and B chains have been reported to be necessary for b-Bgt to facilitate spontaneous synaptic current frequency with a Xenopus nerve–muscle culture (Liou et al., 2004, 2006). To date the mechanism by which presynaptic neurotoxins, which * Corresponding author. Tel.: þ886 7 5919212; fax: þ886 7 5919264. E-mail address: [email protected] (P.-F. Wu). 0041-0101/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.06.009

have intrinsic phospholipase A2 (PLA2) activity, block the release of neurotransmitters from nerve terminals remains unknown Recent studies using cell cultures to investigate how b-Bgt blocks neurotransmitter release have been inconclusive. While one such study has suggested that b-Bgt activates NMDA receptor and causes neuronal cell death (Tseng and Lin-Shiau, 2003a), another has suggested that b-Bgt cannot activate NMDA receptor (Chen, 2005). Therefore, the role of b-Bgt remains unresolved. Several bacterial protein toxins have been found to target eukaryotic cells by modulating the functions involved in Rho GTPase signaling processes and regulation of actin cytoskeleton (Lerm et al., 2000). These bacterial toxins include Clostridium toxin A, B, C2, C3, Escherichia coli cytotoxic necrotizing factors, Yersinia toxin, Samonella toxin and Pseudomonas toxin (Aktories and Schmidt, 2003). Crotoxin, the main neurotoxin component of Crotalus durissus terrificus snake venom, has been reported to induce the reorganization of actin and inhibit tyrosine phosphorylation and the activity of small GTPases in rat macrophages (Sampaio et al., 2006). Therefore, this study investigated whether b-Bgt was carried out to interact with small GTPases like

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bacterial toxins and Crotoxin, and induce the outgrowth of neurite in NB41A3 cells. To do this, we first determined by microscopy whether there was outgrowth of neurites in NB41A3 cells treated with different concentrations of b-Bgt, and used Western blotting and densitometry to detect and measure changes in expression of Cdc42 and RhoA proteins.

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TBST (25 mM Tris pH 7.5, 135 mM NaCl, and 0.15% Tween 20) for 1 h and then incubated with anti-Rho A, antiCdc42 and anti-Actin overnight, respectively. Blots were then washed with TBST and incubated with secondary antibody conjugated with horseradish peroxidase for 50 min. The immunoreactive proteins were detected using an ECL detection system (Amersham Biosciences) according to the manufacturer’s directions.

2. Materials and methods NB41A3 cell line was purchased from ATCC. b-Bungarus multicinctus, 40 ,6-diamidino-2-phenylindole (DAPI) and chemicals were purchased from Sigma Chemical Co. (USA). Anti-Cdc42, anti-RhoA and anti-actin were purchased from Santa Cruz Biotechnology (USA). Anti-a-tubulin and second antibodies were purchased from Invitrogen. 2.1. Cell culture The cells cultured in this study were mouse neuroblastoma NB41A3 cells (ATCC CCL 147). According to the supplier, in cultures, these cells appear as mature neurons and secrete the enzymes choline acetylase and tyrosine hydroxylase for the synthesis of the neurotransmitters in a manner similar to the motor neuron. NB41A3 cells were maintained in Ham’s F10 medium (containing L-glutamine) supplemented with 2.5% FBS and 15% horse serum (Gibco). To induce the outgrowth of neurite, NB41A3 cells were cultured in Ham’s F10 medium containing various concentrations of b-Bgt for 24, 48 and 72 h, respectively.

2.4. Flow cytometry Calcium signal was measured by flow cytometry following procedures used by Wheatly et al. (2001) with some modifications. The NB41A3 cells were treated with 357 nM of b-Bgt for 0.5, 1, 3 and 5 h, respectively. The cells were harvested and washed with PBS. After washing with PBS, Fluo-4 (Invitrogen) was added to the cell suspension to a final concentration of 4 mM and incubated at 37  for 1 h while gently being mixed. The cells were counted using Coulter Epics XL flow cytometry. 2.5. Data analysis The number of cells that were condensed and the number of neurites extending from the cell bodies were counted and compared with untreated control cells. All data reported in this study represent the mean  SEM of measurements done in triplicate. A p value <0.05 (paired t-test) was considered significant. All statistical operations were performed on Sigmaplot Version 10.0.

2.2. Immunofluorescence staining 3. Results After treatment of b-Bgt, cells were washed with PBS and fixed with 4% formaldehyde in PBS for 5 min at room temperature and blocked with blocking buffer (5% BSA and 0.5% Triton X-100 in PBS) at 37  for 45 min. After washing with PBS twice, the cells were probed with rabbit anti-b-Bgt serum (1:500) and mouse anti-atubulin antibody (1:1000). The goat anti-rabbit and rabbit anti-mouse antibodies were used to react with primary antibodies, respectively. The cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI, 2 mg/ ml). Immunofluorescent cell images were obtained using an Olympus LSM Fluovies 500 confocal laser scanning microscope (Olympus). 2.3. SDS-PAGE and Western blot analysis NB41A3 cells were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1% NP-40 and 0.25% sodium deoxycholate) with protease inhibitors (Complete, Boehringer Mannheim). Samples were kept on ice for 5 min and centrifuged at 10,000  g for 15 min to remove insoluble material. The protein concentration of the supernatants was measured using a BCA kit (Pierce Biotechnology, Inc., USA). For each sample, 100 mg of protein was separated on 15% polyacrylamide gel and then transferred onto PVDF membrane in a semi-dry transfer apparatus (BioRad). Membranes were blocked in 5% non-fat dry milk in

3.1. b-Bungarotoxin induced neurite outgrowth Fig. 1 shows the neurite outgrowth of NB41A3 induced by b-Bgt at concentrations of 0, 14, 71, 357 and 714 nM, for 24, 48 and 72 h. Condensed cell bodies were clearly observed in cultures treated with b-Bgt at concentrations of 357 and 714 nM for 24, 48 days 72 h (Fig. 1). Neurite outgrowth, defined as cells having more than five neurites extending from their cell bodies, was highest in cultures following 24 h exposure to 357 and 714 nM b-Bgt. At 48 h and 72 h, cell death may have hindered the outgrowth of neurite, though MTT assay showed no significant change in cell viability. Although cell bodies had shrunk after treatment with b-Bgt, there was no significant change in cell viability of the treated cells compared with the controls (data not shown). These results showed that b-Bgt clearly induced neurite outgrowth in NB41A3 while causing no cell death. Secretory phospholipase A2 activity (PLA2) has been found to induce neurite outgrowth in PC12 cells (Nakashima et al., 2003), and EGTA has been found to abolish PLA2 (Ballou and Cheung, 1983). To determine whether there was an association between neurite outgrowth and b-Bgt PLA2 activity, EGTA was added to the culture medium before the cells were treated with b-Bgt. b-Bgt-induced neurite outgrowth was not affected by extracellular EGTA and, therefore, it could be considered at b-Bgt internalized from extracellular is independent of PLA2 activity.

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Fig. 1. Cell morphology of NB41A3 cells observed by inverted phase-contrast microscopy. A1, B1,C1,D1 and E1 show NB41A3 cells maintained in Ham’s F-10 supplemented with 15% horse serum, 2.5% fetal bovine serum containing 0, 14, 71, 357 and 714 nM b-Bgt, respectively for 24 h. A2, B2,C2,D2 and E2 show NB41A3 cells maintained in Ham’s F-10 supplemented with 15% horse serum, 2.5% fetal bovine serum containing 0, 14, 71, 357 and 714 nM b-Bgt, respectively, for 48 h. A3, B3, C3, D3 and E3 show NB41A3 cells maintained in Ham’s F-10 supplemented with 15% horse serum, 2.5% fetal bovine serum containing 0, 14, 71, 357 and 714 nM of b-Bgt, respectively, for 72 h. The arrow shows the cell body was condensed. The data represent the mean  SEM based on measurements done in triplicate. *p < 0.05 and **p < 0.01 compared with the control.

3.2. b-Bungarotoxin was internalized into NB41A3 cells It has been demonstrated previously (Herkert et al., 2001) that b-Bgt was internalized into cultured hippocampal cells, suggesting a cytoplasmic site of action. In order to test whether b-Bgt is similarly internalized into NB41A3 cells we used confocal microscopy to detect the intracellular presence of b-Bgt. As can be seen in Fig. 2, b-Bgt was found at 24 h in the cytoplasma and the nucleus. b-Bgt

also colocalized with a-tubulin in NB41A3 cells. Cdc42 has been reported to regulate the microtubule organization center (MTOC) and cytoskeleton (Palazzo et al., 2001; Gundersen et al., 2005; Lee et al., 2005). Moreover, neurite outgrowth occurs as result of RhoGTPases-related cytoskeletal movement (Palazzo et al., 2001; Etienne-Manneville and Hall, 2001, 2003; Tzima et al., 2003). We used Western blotting to follow the changes in levels of small G protein, RhoA and Cdc42. One hundred micrograms of cell lysate

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were collected and subjected to immunoblot analysis at 24 h treatment with b-Bgt. We found marked reductions inCdc42 protein (Fig. 3A) and marked increases in RhoA protein (Fig. 3B), suggesting that b-Bgt-induced outgrowth of neurite from NB41A3 cells may be mediated by small G proteins. 3.3. b-Bungarotoxin cannot cause the calcium influx in NB41A3 cells Because PLA2 has been found to induce increases in intracellular Ca2þ (Evans et al., 1989), we used flow cytometry to measure intracellular calcium in NB41A3 cells once b-Bgt had been internalized. As shown in Fig. 4, b-Bgt did not alter the concentration of cytosolic calcium in these cells, which would explain why there was neurite outgrowth in the presence of extracellular EGTA. 4. Discussion For the last 40 years, studies have focused on the physiological and pharmacological properties of b-Bgt (Rowan,

2001). In a functional study of b-Bgt A and B chains, Chang and Yang (1993) were able to successfully separate the two, but found neither of them to exhibit phospholipase enzyme activity nor did they find them to have the lethality of the native toxin. Recent studies have suggested the recombinant B chain might block the current of the potassium channel in the vascular smooth-muscle cells in rats (Wu et al., 1998). Interestingly, a neuronal calcium sensor protein, KChIP3, has been found to bind with the B chain (Lin et al., 2006). In other words, while much is known about the physiological and pharmacological properties of b-Bgt as well as its effects, the mechanism underlying the effect of b-Bgt on the presynaptic motor nerve terminal remain largely unknown. The presynaptic neurotoxin b-Bgt has been reported to cause triphasic muscular changes in amphibians (Abe et al., 1976; Caratsch et al., 1981). b-Bgt-induced phase I changes have been found to be phospholipase-independent, and phase II and III phospholipase-dependent (Caratsch et al., 1985). In this study, b-Bgt induction of neurite outgrowth was not affected by extracellular EGTA at all, suggesting that the internalization of b-Bgt from extracellular was independent of phospholipase.

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Fig. 2. Cell morphology of NB41A3 cells as shown by confocal microscopy. The blue fluorescent staining by DAPI shows the nucleus of NB41A3 cells. Red fluorescence indicates the position of the a-tubulin and the green fluorescence shows the effect of b-Bgt. (a) and (d) shows that there was no b-Bgt treatment. (b) and (e) show cells containing 71 nM b-Bgt for 24 h. (c) and (f) show cells containing 357 nM b-Bgt for 24 h. (a–c) show cells observed by the 100 amplified field and (d–f) show cells observed by the 400 amplified field. The arrow shows the site of b-Bgt.

Tseng and Lin-Shiau, 2003a suggest that NMDA receptor and L-type Ca2þ channels might be involved in b-Bgtinduced neurotoxity in cultured CGNs. However, Chen (2005) found that nifedipine (a Ca2þ channel blocker), AP-4 and suramin (an NMDA receptor antagonist) did not alter the cytotoxicity of b-Bgt on cultured CGNs. Herkert et al. (2001) proposed that b-Bgt induced neuronal apoptosis is not mediated by caspase activity, whereas Chen (2005) found an increase in caspase 3 activity in b-Bgttreated CGNs. We did not find b-Bgt to have a cytotoxic effect on NB41A3 cells. Neither did we find any appreciable cell death even at the 714 nM b-Bgt treatment. This lack of cytotoxicity may be because there was no flux in Ca2þ after treatment with b-Bgt. Although Tseng and Lin-Shiau

(2003b) and Shakhman et al. (2003) both indicate reactive oxygen species (ROS) induced by b-Bgt is dependent upon Ca2þ, how it interacts with ROS is still unclear. In conclusion, this study found the outgrowth of neurite to be induced by b-Bgt. Accumulated evidence has shown that neurite outgrowth is closely associated with small G proteins and the outgrowth of neurite triggered by retinoic acid or lysophosphatidic acid in cultured neuronal cells contributes to neuronal plasticity in the development of the nervous system (Kozma et al., 1997; Manning et al., 2000; Singh et al., 2001; Ueda et al., 2001; Cimmino et al., 2007). Because b-Bgt induced the outgrowth of neurite of NB41A3 cells without significant cytotoxicity, this model may be used in the study of neurogenesis.

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Fig. 3. Effect of Rho GTPase on neurite outgrowth. (A–C) show the total protein lysate harvested from NB41A3 cells and against anti-Cdc42, anti-RhoA and antiactin (loading control), respectively. Lane 1 depicts the 100 mg protein lysate of NB41A3 cell. Lanes 2 and 3 depict the 100 mg protein lysates containing 71 nM and 357 nM of b-Bgt for 24 h, respectively. The data are represented as the mean  SEM based of measurements done in triplicate. *p < 0.05 compared with the control (lane 1).

Fig. 4. Flow cytometry was used to detect the calcium signal of NB41A3 cells (white area). The black area shows the calcium influx of NB41A3 cells after treatment with b-Bgt. The total cell count was 105.

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