- Email: [email protected]
Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins
Toxicon 54 (2009) 138–144
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins Erik Tedesco a,1, Michela Rigoni a,1, Paola Caccin a, Eugene Grishin b, Ornella Rossetto a, Cesare Montecucco a, * a b
` di Padova and Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Padova, Italy Dipartimento di Scienze Biomediche Sperimentali, Universita Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2008 Received in revised form 12 March 2009 Accepted 23 March 2009 Available online 31 March 2009
Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca2þ which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of b-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca2þ influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca2þ overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Snake presynaptic PLA2 neurotoxins a-Latrotoxin Calcium imaging Primary neuronal cultures Neuromuscular junction
1. Introduction The venom of many snakes of four major families (Crotalidae, Elapidae, Hydrophiidae and Viperidae) contains phospholipase A2 neurotoxins which bind to the presynaptic membrane and hydrolyse phospholipids leading to a progressive accumulation of lysophospholipids (LysoPLs) and fatty acids (FAs) (Harris, 1985; Kini, 1997; Pungercar and Krizaj, 2007; Rossetto and Montecucco, 2008). In many Abbreviations: b-Btx, b-bungarotoxin; [Ca2þ], calcium concentration; CGNs, cerebellar granule neurons; EGTA, ethylene glycol tetraacetic acid; FA, fatty acid; a-Ltx, alpha-latrotoxin; LysoPL, lysophospholipid; NF, neurofilament; NMJ, neuromuscular junction; Ntx, notexin; PLA2, phospholipase A2; SCMNs, spinal cord motoneurons; SypI, synaptophysin I; SytI, synaptotagmin I; SPAN, snake PLA2 presynaptic neurotoxin; SV, synaptic vesicle; Tpx, taipoxin; Tetx, textilotoxin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-attached membrane protein 2. * Corresponding author. Tel.: þ39 0498276058; fax: þ39 0498276049. E-mail address: [email protected] (C. Montecucco). 1 These authors contributed equally to the present work. 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.03.025
cases, the neurotoxins play a major role in envenomation and cause a botulism-like flaccid paralysis with autonomic symptoms (Harris, 1985; Connolly et al., 1995; Prasarnpun et al., 2005). These neurotoxins have been abbreviated as SPANs (snake presynaptic PLA2 neurotoxins). The SPANintoxicated neuromuscular junction (NMJ) appears as much swollen as its anatomical constriction allows it to be. Nearly all synaptic vesicles (SVs) have disappeared and the plasma membrane is marked by the presence of U-shaped membrane indentations that suggests aborted endocytosis of synaptic vesicles. Mitochondria are rounded, swollen and vacuolated and there is degeneration of neurofilaments (Chen and Lee, 1970; Cull-Candy et al., 1976; Lee et al., 1984; Gopalakrishnakone and Hawgood, 1984; Dixon and Harris, 1999; Harris et al., 2000). We have recently found that the products of PLA2 hydrolysis of membrane phospholipids are sufficient to cause inhibition and degeneration of nerve terminals very similar to those caused by SPANs (Rigoni et al., 2005; Caccin et al., 2006). In addition, several studies have shown that
E. Tedesco et al. / Toxicon 54 (2009) 138–144
SPANs can enter directly into neurons in culture, though it is not yet known how they enter nor if a preliminary display of PLA2 activity is a pre-requisite for entry (Herkert et al., 2001; Neco et al., 2003; Petrovic et al., 2004; Praznikar et al., 2008; Rigoni et al., 2008); accordingly, these neurotoxins could as well attack internal cell membrane phospholipids. The effects of LysoPLs and FAs on cell membranes have been studied in different cells and in different contexts (Woodley et al., 1991; Leung et al., 1998; Wilson-Ashworth et al., 2004; Li et al., 2007). As discussed in detail elsewhere (Rossetto and Montecucco, 2008), the presence of LysoPLs on the outer layer of the nerve terminal plasma membrane and of FAs on both layers changes the membrane curvature in such a way as to increase the probability of fusion of the ready to release pool of synaptic vesicles (Zimmerberg and Chernomordik, 2005). This was shown to be indeed the case in hippocampal neurons in culture by using FM1-43 to follow specifically the recycling pool of vesicles (Bonanomi et al., 2005). In addition, in cultured neurons the plasma membrane becomes leaky and allows the entry of Ca2þ ions, driven by a large concentration gradient (millimolar outside and
b-Bungarotoxin (b-Btx) was obtained from SIGMA, taipoxin (Tpx) and textilotoxin (Tetx) were purchased from
139
Venom Supplies, notexin (Ntx) from Latoxan. a-Latrotoxin (a-Ltx) was purified from the venom of Latrodectus tredecimguttatus (Ushkaryov and Grishin, 1986). The neuroparalytic activity of the toxins was tested using the mouse phrenic nerve-hemidiaphragms preparation, as described previously (Rigoni et al., 2005). The enzymatic activity of SPANs was checked with a commercial kit based on the use of 1,2-dithio analogue of di-heptanoyl phosphatidylcholine as substrate (Cayman Chemicals). Polyclonal antibody against vesicle-associated membrane protein 2 (VAMP2) (Rossetto et al., 1996) was used at 1:300 dilution; monoclonal antibody against synaptophysin I (SypI) (DAKO) was used at a dilution of 1:10; monoclonal antibody against synaptotagmin I (SytI) luminal domain (Synaptic Systems) was used at a working dilution of 1:100; monoclonal antibody anti-NF (SIGMA) and polyclonal antibody antiVAChT (Synaptic Systems) were diluted 1:500. a-Bungarotoxin (Molecular Probes) was used at a working dilution of 1:200; Alexa555 and Alexa350 secondary antibodies (Molecular Probes) were used at a dilution of 1:200. 2.2. Primary neuronal cultures Rat cerebellar granule neurons (CGNs) were prepared from 6-day-old Wistar rats as previously described (Levi et al., 1984) and used 6–8 days after plating. Primary rat spinal cord motoneurons (SCMNs) were isolated from Sprague–Dawley (embryonic day 14) rat embryos and cultured following previously described protocols (Arce et al., 1999; Bohnert and Schiavo, 2005). Experiments were performed using SCMNs after 5–8 days of neuronal differentiation in vitro. 2.3. Immunofluorescence Neurons were seeded onto 24-well plates and incubated with each of the four SPANs (6 nM) for 60 min or with a-Ltx (0.1 nM) for 20 min at 37 C in serum-free medium, washed, fixed for 20 min at room temperature with 4% paraformaldeyde in PBS, quenched (0.38% glycine, 0.24% NH4Cl in PBS, 2 10 min) and permeabilized with 5% acetic acid in ethanol for 20 s at 20 C, in the case of CGNs, or with 0.1% Triton X-100 in PBS for 3 min, in the case of SCMNs. After saturation with 0.5% BSA in PBS, in the case of CGNs, and with 10% normal goat serum, 2% BSA, 0.2% gelatin in PBS, in the case of SCMNs, for 30 min at room temperature, samples were incubated with the primary antibodies (diluted in the saturation solution) for 60 min at room temperature, washed with 0.5% BSA in PBS and then incubated with the correspondent Alexa555-conjugated secondary antibodies for 1 h. To test the effect of the toxins on SV recycling, both control cells and cells treated with the toxins (60 min in the case of SPANs or 20 min in the case of a-Ltx) were incubated with a primary antibody directed against the luminal domain of synaptotagmin I (SytI-ecto Abs); incubation was carried out for 5 min at 37 C, then samples were extensively washed, fixed, quenched and incubated without permeabilization with Alexa555-conjugated anti-mouse secondary antibody. Coverslips were mounted in 90% glycerol in PBS containing 3% N-propylgallate and
140
E. Tedesco et al. / Toxicon 54 (2009) 138–144
examined by confocal (Bio-Rad MRC1024ES) or epifluorescence (Leica DMIRE2) microscopy. 2.4. Calcium imaging Measurements of intracellular [Ca2þ] changes following SPANs or a-Ltx exposure were performed as described previously (Rigoni et al., 2007). 2.5. Immunohistochemistry All experiments were performed in accordance with Italian animal care guidelines, law no. 116/1992. Adult CD1 mice, weighting between 35 and 37 g, were anaesthetized by intraperitoneal injection of 15 mg/kg of a combination of tiletamine hypochloride and zolazepam hypochloride (Laboratories Virbac, Carros, France) and 3 mg/kg of xylazine hydrochloride (Laboratoires Calier, Barcelona, Spain). 50 ml of a-Ltx or SPANs (5 mg/kg and 3–6 mg/kg respectively, dissolved in physiological saline) were injected intramuscularly in the mouse tibialis anterior muscle of the left hind limb; an equal amount of physiological saline was injected in the contra-lateral hind limb as a control. After 24 h (SPANs) or 12 h (a-Ltx), mice were sacrificed and injected muscles of both hind limbs were dissected. Muscles were then processed in skinning solution (137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 24 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose and 5 mM EGTA, pH 7.4), washed and fixed for 30 min at room temperature with 4% paraformaldeyde in PBS. Samples were quenched with 50 mM NH4Cl in PBS for 10 min and incubated with saturation solution (0.1% Triton X-100, 3% BSA, 10% normal goat serum in PBS) for 60 min. Processed muscles were incubated with primary antibodies
in 5% BSA, 0.1% Triton X-100 in PBS for 3 h at room temperature, washed with PBS and then incubated with the correspondent secondary antibodies plus a-Btx conjugated with Alexa488 in 5% BSA in PBS for 90 min. Samples were mounted in Mowiol and examined by Nikon Eclipse 80i Video-Confocal microscope. 3. Results 3.1. SPANs and a-latrotoxin induce similar bulging on rat primary neurons SPANs induce a defined phenotype in different types of neurons in culture (Rigoni et al., 2004). Within few minutes from toxin addition, bulges appear as defined rounded swellings of the plasma membrane at neurons’ terminals and extensions (right panels of Fig. 1). The left panels of Fig. 1 show that a-Ltx induces, at very low concentration, a similar phenotype in both cerebellar granular neurons and in spinal cord motoneurons. Neuronal bulges induced by SPANs and a-Ltx were similar in size, appearance and distribution. These findings indicate that also the activity of a-Ltx can be tested with this sensitive and easy to perform cellular assay. 3.2. SPAN and a-latrotoxin induced bulges accumulate synaptic vesicle markers We have previously established that the neuronal bulges induced by SPANs are sites of accumulation of synaptic vesicles (SVs), whose presence could be shown by labelling with antibodies specific for SV proteins such as the vesicle-associated membrane protein 2 (VAMP2) or
Fig. 1. Morphological changes induced by a-Ltx and SPANs in primary neurons in culture. Spinal cord motoneurons (SCMNs, upper panels) and cerebellar granule neurons (CGNs, lower panels) were treated with a-Ltx 0.1 nM for 20 min (A) or with b-Btx 6 nM for 30 min (B). Both toxins induce closely similar membrane enlargements of neuronal processes (bulges). Very similar results were obtained with taipoxin, notexin and textilotoxin. Scale bar: 20 mm.
E. Tedesco et al. / Toxicon 54 (2009) 138–144
synaptophysin I (SypI) (Rigoni et al., 2004). Fig. 2 shows examples of such accumulation caused by SPANs (right panels) and that the action of a-Ltx (central panels) is very similar also in this respect, as previously found in hippocampal neurons (Pennuto et al., 2002). These pictures were obtained in permeabilised neurons and do not allow one to distinguish between an internal localization or a surface exposure of the antigens. Therefore the action of the toxins was also analysed in non-permeabilised cells (see next paragraph). 3.3. SPANs and a-latrotoxin induce exposure of synaptic vesicle luminal proteins in cultured neurons According to the electron microscopy analyses discussed in the introduction, SVs are largely reduced, or absent, in the cytosol after the action of a-Ltx or SPANs. The most likely possibility is that SVs were induced to fuse and that exocytosis was not followed by SV retrieval, owing to an impairment in the exo–endocytic balance. If this is the case, the luminal domains of the SV membrane proteins have to be permanently exposed on the surface of the neurons. Fig. 3 shows that, whilst the luminal part of the SV membrane marker synaptotagmin I is very little stained in control non-permeabilised neurons, it is intensively stained on the surface of bulges induced by both a-Ltx and SPANs. Such intense surface fluorescent signal indicates that SVs are incorporated in the plasma membrane at sites of
bulging following neurotoxins.
141
the
exposure
to
both
types
of
3.4. SPANs and a-latrotoxin induce entry of calcium into cultured neurons Previous studies have established that a very large Ca2þ influx is induced at nerve terminals by a-Ltx (Rosenthal and Meldolesi, 1989; Grishin, 1998; Ushkaryov et al., 2008) and that SPAN-induced bulges are sites of Ca2þ influx that can be monitored by Ca2þ imaging (Rigoni et al., 2007). It remained to show that the a-Ltx induced bulges are as well sites of Ca2þ entry. The left panel of Fig. 4 shows that this is indeed the case, but the time course of this Ca2þ influx is different from that observed in SPAN-treated neurons (middle and right panels). In cerebellar granular neurons exposed to 0.1 nM a-Ltx Ca2þ ions begin to enter about 700 s after toxin addition and this timing is very similar in all terminals that we have analysed. This is in agreement with the notion that the ions enter through a tetrameric channel made by the a-Ltx on the plasma membrane (Orlova et al., 2000), and indicates that several hundreds of seconds are needed to assemble a functional toxin channel on the plasma membrane. Fig. 4 shows that calcium imaging in a homogeneous population of neurons such as CGNs is very useful in the study of the kinetics of the action of this toxin in vivo. In contrast, SPANs induce a progressive increase of Ca2þ entry and this is in agreement with the
Fig. 2. Effect of a-Ltx and SPANs on the distribution of synaptic vesicle markers in primary neurons in culture. Cerebellar granule neurons were incubated with a-Ltx and b-Btx (0.1 nM for 20 min and 6 nM for 60 min respectively) and the distribution of the vesicular proteins VAMP2 and synaptophysin I was analysed by indirect immunofluorescence. In both cases the staining of the two proteins changes with respect to the control, showing an accumulation within the toxininduced membrane enlargements. Overlapping results were obtained with spinal cord motoneurons and with taipoxin, notexin and textilotoxin. Scale bar: 20 mm.
142
E. Tedesco et al. / Toxicon 54 (2009) 138–144
Fig. 3. Effect of a-Ltx and SPANs on synaptic vesicle recycling of cultured neurons. Cerebellar granule neurons were exposed to a-Ltx and Tpx (0.1 nM for 20 min and 6 nM for 60 min respectively) and then incubated for 5 min at 37 C before fixation and permeabilization with a monoclonal antibody that specifically recognizes a luminal epitope of the vesicular protein synaptotagmin I and then processed for indirect immunofluorescence. In both cases the fluorescent signal is brighter than the control and accumulates within the membrane enlargements, indicating that these bulges are sites of unbalanced exo–endocytosis. Similar results were obtained in SCMNs and with b-bungarotoxin, notexin and textilotoxin. Scale bar: 20 mm.
proposal that this influx is mediated by transient lipidic pores made by lysophospholipids and fatty acids in the plasma membrane (Rigoni et al., 2007; Rossetto and Montecucco, 2008). The end result is, however, in both cases the same, i.e. toxin-induced bulges accumulate Ca2þ and this calcium overload may set in motion a series of degenerative events.
an evident loss not only of neurofilaments but also of the specific nervous terminal marker VAChT. One may conclude that also in this respect the two types of toxins share a similar pharmacological/pathophysiological endpoint. As expected from presynaptic neurospecific toxins, there is no evident modification of the postsynaptic component of the NMJ (except for notexin, which is well known to cause also myotoxic effects).
3.5. Degeneration of presynaptic nerve terminals at NMJ after SPAN or a-Ltx exposure
4. Discussion
An end result of the chain of events started by the Ca2þ influx caused by SPANs and a-Ltx is the degeneration of nervous terminal (Chen and Lee, 1970; Ceccarelli et al., 1972; Cull-Candy et al., 1976; Ceccarelli and Hurlbut, 1980; Duchen et al., 1981; Lee et al., 1984; Gopalakrishnakone and Hawgood, 1984; Dixon and Harris, 1999; Harris et al., 2000). Fig. 5 compares the staining of neurofilaments and vesicular acetylcholine transporter (VAChT), which are essential components of nerve terminals, in control and toxininjected mice muscles. With both SPANs and a-Ltx there is
The main purpose of the present work was the comparison of the action of snake presynaptic PLA2 neurotoxins with that of the black widow spider a-latrotoxin. Apart from that such a side-by-side comparison was not performed before, the rationale for such a work is based on the fact that the molecular pathogenesis of the degeneration of nerve terminals induced by the snake presynaptic PLA2 neurotoxins is not yet defined (Pungercar and Krizaj, 2007; Rigoni et al., 2008), whilst that caused by a-Ltx is well established to be due mainly to the large influx of
Fig. 4. Intracellular [Ca2þ] increase induced by a-Ltx and SPANs in neurons in culture. The traces give the time course of changes in 340/380 fluorescence ratios of Fura2-loaded CGNs, showing the intracellular [Ca2þ] rises within different bulges induced by a-Ltx (0.1 nM), Tpx and Tetx (6 nM), added at time 0; cell bodies remain largely unaffected (black line in the left panel, red trace in the middle panel and green trace in the right panel). Closely similar results were obtained in SCMNs and with the other two snake neurotoxins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
E. Tedesco et al. / Toxicon 54 (2009) 138–144
143
Fig. 5. Effect of a-Ltx and SPANs on mouse neuromuscular junction. Mice hind limbs were injected with a-Ltx and Tpx and processed for indirect immunohistochemistry after 12 and 24 h respectively. Both neurotoxins cause degradation of neurofilaments (blue) and disappearance of vesicular acetylcholine transporter staining (red). No morphological modifications of the postsynaptic element of the NMJ stained with a-Btx (green) were observed. Scale bars: 10 mm. Similar results were obtained with the three other SPANs (with the exception of Ntx that caused also muscle damage). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Ca2þ which induces mitochondrial collapse and activation of many degradative enzymes (Ushkaryov et al., 2008). The present work documents that the two types of presynaptic neurotoxins cause similar alterations in neurons in culture and in vivo. Both of them cause bulging of neurons in culture and these bulges expose on their surface the luminal domain of proteins of the synaptic vesicle membrane. Bulges accumulate calcium although with different kinetics, which are accounted for by the different biochemical activities of the two types of toxins, i.e. PLA2 activity for SPANs and ion channel activity for a-Ltx. A high cytosolic [Ca2þ] is known to trigger a series of degradative events and, as a marker, we followed here the degradation of neurofilaments and the disappearance of VAChT. Here, we found that neurofilaments and VAChT disappear at the motoneuron endings of mice injected with the two types of neurotoxins, similarly to what previously found with b-Btx (Prasarnpun et al., 2004). Taken together, all these similarities suggest that a major role in the degeneration of the nerve terminals caused by SPANs is the calcium overloading that follows the change in membrane composition caused by the PLA2 activity of SPANs. The fact that Ca2þ ions appear to enter into nerve terminals soon after SPANs addition and that they accumulate within bulges are indications that the toxin hydrolytic activity may be, at least in the beginning of the process, concentrated close to their binding sites and that the concentration of the phospholipids hydrolysis products may reach significant values such as to change the plasma membrane permeability. The precise localization of the binding sites of SPANs within the nerve termini remains to be determined and should throw light on this ill-defined, but fundamental, aspect of the process.
Acknowledgments The present work was supported by Telethon (grant no. GGP06133) and by Fondazione Cariparo Progetto ‘‘Physiopathology of the synapse: neurotransmitters, neurotoxins and novel therapies’’ to CM and by CMB program of RAS to EG.
Conflicts of interest The authors declare that there are no conflicts of interest. References Arce, V., Garces, A., de Bovis, B., Filippi, P., Henderson, C., Pettmann, B., deLapeyrie`re, O., 1999. Cardiotrophin-1 requires LIFRbeta to promote survival of mouse motoneurons purified by a novel technique. J. Neurosci. Res. 55, 119–126. Bernardi, P., Rasola, A., 2007. Calcium and cell death: the mitochondrial connection. Subcell. Biochem. 45, 481–506. Bertipaglia, I., Carafoli, E., 2007. Calpains and human disease. Subcell. Biochem. 45, 29–53. Bohnert, S., Schiavo, G., 2005. Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation. J. Biol. Chem. 280, 42336–42344. Bonanomi, D., Pennuto, M., Rigoni, M., Rossetto, O., Montecucco, C., Valtorta, F., 2005. Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2. Mol. Pharmacol. 67, 1901–1908. Caccin, P., Rigoni, M., Risceglie, A., Rossetto, O., Montecucco, C., 2006. Reversible skeletal neuromuscular paralysis induced by different lysophospholipids. FEBS Lett. 580, 6317–6321. Ceccarelli, B., Hurlbut, W.P., Mauro, A., 1972. Depletion of vesicle from frog neuromuscular junction by prolonged tetanic stimulation. J. Cell Biol. 50, 30–38. Ceccarelli, B., Hurlbut, W.P., 1980. Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60, 396–441. Chen, I.L., Lee, C.Y., 1970. Ultrastructural changes in the motor nerve terminals caused by beta-bungarotoxin. Virchows Arch. B Cell. Pathol. 6, 318–325. Connolly, S., Trevett, A.J., Nwokolo, N.C., Lalloo, D.G., Naraqi, S., Mantle, D., Schofield, I.S., Fawcett, P.R., Harris, J.B., Warrell, D.A., 1995. Neuromuscular effects of Papuan Taipan snake venom. Ann. Neurol. 38, 916–920. Cull-Candy, S.G., Fohlman, J., Gustavsson, D., Lu¨llmann-Rauch, R., Thesleff, S., 1976. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1, 175–180. Dixon, R.W., Harris, J.B., 1999. Nerve terminal damage by beta-bungarotoxin: its clinical significance. Am. J. Pathol. 154, 447–455. Duchen, L.W., Gomez, S., Queiroz, L.S., 1981. The neuromuscular junction of the mouse after black widow spider venom. J. Physiol. 316, 279–291. Gopalakrishnakone, P., Hawgood, B.J., 1984. Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon 22, 791–804. Grishin, E.V., 1998. Black widow spider toxins: the present and the future. Toxicon 36, 1693–1701. Harris, J.B., 1985. Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol. Ther. 31, 79–102.
144
E. Tedesco et al. / Toxicon 54 (2009) 138–144
Harris, J.B., Grubb, B.D., Maltin, C.A., Dixon, R., 2000. The neurotoxicity of the venom phospholipases A2, notexin and taipoxin. Exp. Neurol. 161, 517–526. Herkert, M., Shakhman, O., Schweins, E., Becker, C.M., 2001. Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization. Eur. J. Neurosci. 14, 821–828. Kini, R.M., 1997. Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism. Chichester, UK. Lee, C.Y., Tsai, M.C., Chen, Y.M., Ritonja, A., Gubensek, F., 1984. Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom. Arch. Int. Pharmacodyn. Ther. 268, 313–324. Leung, Y.M., Xion, Y., Ou, Y.J., Kwan, C.Y., 1998. Perturbation by lysophosphatidylcholine of membrane permeability in cultured vascular smooth muscle and endothelial cells. Life Sci. 63, 965–973. Levi, G., Aloisi, F., Ciotti, M.T., Gallo, V., 1984. Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures. Brain Res. 290, 77–86. Li, X.H., Long, D.X., Li, W., Wu, Y.J., 2007. Different mechanisms of lysophosphatidylcholine-induced Ca2þ mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells. Neurosci. Lett. 424, 22–26. Montecucco, C., Gutie´rrez, J.M., Lomonte, B., 2008. Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell. Mol. Life Sci. [Epub ahead of print]. Neco, P., Rossetto, O., Gil, A., Montecucco, C., Gutie´rrez, L.M., 2003. Taipoxin induces F-actin fragmentation and enhances release of catecholamines in bovine chromaffin cells. J. Neurochem. 85, 329–337. Orlova, E.V., Rahman, M.A., Gowen, B., Volynski, K.E., Ashton, A.C., Manser, C., van Heel, M., Ushkaryov, Y.A., 2000. Structure of a-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores. Nat. Struct. Biol. 7, 48–53. Pennuto, M., Dunlap, D., Contestabile, A., Benfenati, F., Valtorta, F., 2002. Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses. Mol. Biol. Cell 13, 2706–2717. Petrovic, U., Sribar, J., Paris, A., Rupnik, M., Krzan, M., Vardjan, N., Gubensek, F., Zorec, R., Krizaj, I., 2004. Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell. Biochem. Biophys. Res. Commun. 324, 981–985. Prasarnpun, S., Walsh, J., Harris, J.B., 2004. Beta-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction. Neuropharmacology 47, 304–314. Prasarnpun, S., Walsh, J., Awad, S.S., Harris, J.B., 2005. Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis. Brain 128, 2987–2996.
Praznikar, Z.J., Kovacic, L., Rowan, E.G., Romih, R., Rusmini, P., Poletti, A., Krizaj, I., Pungercar, J., 2008. A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol. Biochim. Biophys. Acta 1783, 1129–1139. Pungercar, J., Krizaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892. Rigoni, M., Schiavo, G., Weston, A.E., Caccin, P., Allegrini, F., Pennuto, M., Valtorta, F., Montecucco, C., Rossetto, O., 2004. Snake presynaptic neurotoxins with phospholipase A2 activity induce punctuate swellings of neurites and exocytosis of synaptic vesicles. J. Cell Sci. 117, 561–570. Rigoni, M., Caccin, P., Gschmeissner, S., Koster, G., Postle, A.D., Rossetto, O., Schiavo, G., Montecucco, C., 2005. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid–fatty acid mixtures. Science 310, 1678–1680. Rigoni, M., Pizzo, P., Schiavo, G., Weston, A.E., Zatti, G., Caccin, P., Rossetto, O., Pozzan, T., Montecucco, C., 2007. Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products. J. Biol. Chem. 282, 11238–11245. Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., Montecucco, C., 2008. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria and open their transition pores. J. Biol. Chem. 283, 34013–34020. Rosenthal, L., Meldolesi, J., 1989. Alpha-latrotoxin and related toxins. Pharmacol. Ther. 42, 115–134. Rossetto, O., Gorza, L., Schiavo, G., Schiavo, N., Scheller, R.H., Montecucco, C., 1996. VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in non-neuronal tissues. J. Cell Biol. 132, 167–179. Rossetto, O., Montecucco, C., 2008. Presynaptic neurotoxins with enzymatic activities. Handb. Exp. Pharmacol. 184, 129–170. Ushkaryov, Y.A., Grishin, E.V., 1986. Black widow spider neurotoxin and its interaction with rat brain receptor. Bioorg. Chem. (Russ.) 12, 71–80. Ushkaryov, Y.A., Rohou, A., Sugita, S., 2008. alpha-Latrotoxin and its receptors. Handb. Exp. Pharmacol. 184, 171–206. Woodley, S.L., Ikenouchi, H., Barry, W.H., 1991. Lysophosphatidylcholine increases cytosolic calcium in ventricular myocytes by direct action on the sarcolemma. J. Mol. Cell. Cardiol. 23, 671–680. Wilson-Ashworth, H.A., Judd, A.M., Law, R.M., Freestone, B.D., Taylor, S., Mizukawa, M.K., Cromar, K.R., Sudweeks, S., Bell, J.D., 2004. Formation of transient non-protein calcium pores by lysophospholipids in S49 lymphoma cells. J. Membr. Biol. 200, 25–33. Zimmerberg, J., Chernomordik, L.V., 2005. Neuroscience. Synaptic membranes bend to the will of a neurotoxin. Science 310, 1626–1627.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins Erik Tedesco a,1, Michela Rigoni a,1, Paola Caccin a, Eugene Grishin b, Ornella Rossetto a, Cesare Montecucco a, * a b
` di Padova and Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Padova, Italy Dipartimento di Scienze Biomediche Sperimentali, Universita Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2008 Received in revised form 12 March 2009 Accepted 23 March 2009 Available online 31 March 2009
Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca2þ which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of b-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca2þ influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca2þ overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Snake presynaptic PLA2 neurotoxins a-Latrotoxin Calcium imaging Primary neuronal cultures Neuromuscular junction
1. Introduction The venom of many snakes of four major families (Crotalidae, Elapidae, Hydrophiidae and Viperidae) contains phospholipase A2 neurotoxins which bind to the presynaptic membrane and hydrolyse phospholipids leading to a progressive accumulation of lysophospholipids (LysoPLs) and fatty acids (FAs) (Harris, 1985; Kini, 1997; Pungercar and Krizaj, 2007; Rossetto and Montecucco, 2008). In many Abbreviations: b-Btx, b-bungarotoxin; [Ca2þ], calcium concentration; CGNs, cerebellar granule neurons; EGTA, ethylene glycol tetraacetic acid; FA, fatty acid; a-Ltx, alpha-latrotoxin; LysoPL, lysophospholipid; NF, neurofilament; NMJ, neuromuscular junction; Ntx, notexin; PLA2, phospholipase A2; SCMNs, spinal cord motoneurons; SypI, synaptophysin I; SytI, synaptotagmin I; SPAN, snake PLA2 presynaptic neurotoxin; SV, synaptic vesicle; Tpx, taipoxin; Tetx, textilotoxin; VAChT, vesicular acetylcholine transporter; VAMP2, vesicle-attached membrane protein 2. * Corresponding author. Tel.: þ39 0498276058; fax: þ39 0498276049. E-mail address: [email protected] (C. Montecucco). 1 These authors contributed equally to the present work. 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.03.025
cases, the neurotoxins play a major role in envenomation and cause a botulism-like flaccid paralysis with autonomic symptoms (Harris, 1985; Connolly et al., 1995; Prasarnpun et al., 2005). These neurotoxins have been abbreviated as SPANs (snake presynaptic PLA2 neurotoxins). The SPANintoxicated neuromuscular junction (NMJ) appears as much swollen as its anatomical constriction allows it to be. Nearly all synaptic vesicles (SVs) have disappeared and the plasma membrane is marked by the presence of U-shaped membrane indentations that suggests aborted endocytosis of synaptic vesicles. Mitochondria are rounded, swollen and vacuolated and there is degeneration of neurofilaments (Chen and Lee, 1970; Cull-Candy et al., 1976; Lee et al., 1984; Gopalakrishnakone and Hawgood, 1984; Dixon and Harris, 1999; Harris et al., 2000). We have recently found that the products of PLA2 hydrolysis of membrane phospholipids are sufficient to cause inhibition and degeneration of nerve terminals very similar to those caused by SPANs (Rigoni et al., 2005; Caccin et al., 2006). In addition, several studies have shown that
E. Tedesco et al. / Toxicon 54 (2009) 138–144
SPANs can enter directly into neurons in culture, though it is not yet known how they enter nor if a preliminary display of PLA2 activity is a pre-requisite for entry (Herkert et al., 2001; Neco et al., 2003; Petrovic et al., 2004; Praznikar et al., 2008; Rigoni et al., 2008); accordingly, these neurotoxins could as well attack internal cell membrane phospholipids. The effects of LysoPLs and FAs on cell membranes have been studied in different cells and in different contexts (Woodley et al., 1991; Leung et al., 1998; Wilson-Ashworth et al., 2004; Li et al., 2007). As discussed in detail elsewhere (Rossetto and Montecucco, 2008), the presence of LysoPLs on the outer layer of the nerve terminal plasma membrane and of FAs on both layers changes the membrane curvature in such a way as to increase the probability of fusion of the ready to release pool of synaptic vesicles (Zimmerberg and Chernomordik, 2005). This was shown to be indeed the case in hippocampal neurons in culture by using FM1-43 to follow specifically the recycling pool of vesicles (Bonanomi et al., 2005). In addition, in cultured neurons the plasma membrane becomes leaky and allows the entry of Ca2þ ions, driven by a large concentration gradient (millimolar outside and
b-Bungarotoxin (b-Btx) was obtained from SIGMA, taipoxin (Tpx) and textilotoxin (Tetx) were purchased from
139
Venom Supplies, notexin (Ntx) from Latoxan. a-Latrotoxin (a-Ltx) was purified from the venom of Latrodectus tredecimguttatus (Ushkaryov and Grishin, 1986). The neuroparalytic activity of the toxins was tested using the mouse phrenic nerve-hemidiaphragms preparation, as described previously (Rigoni et al., 2005). The enzymatic activity of SPANs was checked with a commercial kit based on the use of 1,2-dithio analogue of di-heptanoyl phosphatidylcholine as substrate (Cayman Chemicals). Polyclonal antibody against vesicle-associated membrane protein 2 (VAMP2) (Rossetto et al., 1996) was used at 1:300 dilution; monoclonal antibody against synaptophysin I (SypI) (DAKO) was used at a dilution of 1:10; monoclonal antibody against synaptotagmin I (SytI) luminal domain (Synaptic Systems) was used at a working dilution of 1:100; monoclonal antibody anti-NF (SIGMA) and polyclonal antibody antiVAChT (Synaptic Systems) were diluted 1:500. a-Bungarotoxin (Molecular Probes) was used at a working dilution of 1:200; Alexa555 and Alexa350 secondary antibodies (Molecular Probes) were used at a dilution of 1:200. 2.2. Primary neuronal cultures Rat cerebellar granule neurons (CGNs) were prepared from 6-day-old Wistar rats as previously described (Levi et al., 1984) and used 6–8 days after plating. Primary rat spinal cord motoneurons (SCMNs) were isolated from Sprague–Dawley (embryonic day 14) rat embryos and cultured following previously described protocols (Arce et al., 1999; Bohnert and Schiavo, 2005). Experiments were performed using SCMNs after 5–8 days of neuronal differentiation in vitro. 2.3. Immunofluorescence Neurons were seeded onto 24-well plates and incubated with each of the four SPANs (6 nM) for 60 min or with a-Ltx (0.1 nM) for 20 min at 37 C in serum-free medium, washed, fixed for 20 min at room temperature with 4% paraformaldeyde in PBS, quenched (0.38% glycine, 0.24% NH4Cl in PBS, 2 10 min) and permeabilized with 5% acetic acid in ethanol for 20 s at 20 C, in the case of CGNs, or with 0.1% Triton X-100 in PBS for 3 min, in the case of SCMNs. After saturation with 0.5% BSA in PBS, in the case of CGNs, and with 10% normal goat serum, 2% BSA, 0.2% gelatin in PBS, in the case of SCMNs, for 30 min at room temperature, samples were incubated with the primary antibodies (diluted in the saturation solution) for 60 min at room temperature, washed with 0.5% BSA in PBS and then incubated with the correspondent Alexa555-conjugated secondary antibodies for 1 h. To test the effect of the toxins on SV recycling, both control cells and cells treated with the toxins (60 min in the case of SPANs or 20 min in the case of a-Ltx) were incubated with a primary antibody directed against the luminal domain of synaptotagmin I (SytI-ecto Abs); incubation was carried out for 5 min at 37 C, then samples were extensively washed, fixed, quenched and incubated without permeabilization with Alexa555-conjugated anti-mouse secondary antibody. Coverslips were mounted in 90% glycerol in PBS containing 3% N-propylgallate and
140
E. Tedesco et al. / Toxicon 54 (2009) 138–144
examined by confocal (Bio-Rad MRC1024ES) or epifluorescence (Leica DMIRE2) microscopy. 2.4. Calcium imaging Measurements of intracellular [Ca2þ] changes following SPANs or a-Ltx exposure were performed as described previously (Rigoni et al., 2007). 2.5. Immunohistochemistry All experiments were performed in accordance with Italian animal care guidelines, law no. 116/1992. Adult CD1 mice, weighting between 35 and 37 g, were anaesthetized by intraperitoneal injection of 15 mg/kg of a combination of tiletamine hypochloride and zolazepam hypochloride (Laboratories Virbac, Carros, France) and 3 mg/kg of xylazine hydrochloride (Laboratoires Calier, Barcelona, Spain). 50 ml of a-Ltx or SPANs (5 mg/kg and 3–6 mg/kg respectively, dissolved in physiological saline) were injected intramuscularly in the mouse tibialis anterior muscle of the left hind limb; an equal amount of physiological saline was injected in the contra-lateral hind limb as a control. After 24 h (SPANs) or 12 h (a-Ltx), mice were sacrificed and injected muscles of both hind limbs were dissected. Muscles were then processed in skinning solution (137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 24 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose and 5 mM EGTA, pH 7.4), washed and fixed for 30 min at room temperature with 4% paraformaldeyde in PBS. Samples were quenched with 50 mM NH4Cl in PBS for 10 min and incubated with saturation solution (0.1% Triton X-100, 3% BSA, 10% normal goat serum in PBS) for 60 min. Processed muscles were incubated with primary antibodies
in 5% BSA, 0.1% Triton X-100 in PBS for 3 h at room temperature, washed with PBS and then incubated with the correspondent secondary antibodies plus a-Btx conjugated with Alexa488 in 5% BSA in PBS for 90 min. Samples were mounted in Mowiol and examined by Nikon Eclipse 80i Video-Confocal microscope. 3. Results 3.1. SPANs and a-latrotoxin induce similar bulging on rat primary neurons SPANs induce a defined phenotype in different types of neurons in culture (Rigoni et al., 2004). Within few minutes from toxin addition, bulges appear as defined rounded swellings of the plasma membrane at neurons’ terminals and extensions (right panels of Fig. 1). The left panels of Fig. 1 show that a-Ltx induces, at very low concentration, a similar phenotype in both cerebellar granular neurons and in spinal cord motoneurons. Neuronal bulges induced by SPANs and a-Ltx were similar in size, appearance and distribution. These findings indicate that also the activity of a-Ltx can be tested with this sensitive and easy to perform cellular assay. 3.2. SPAN and a-latrotoxin induced bulges accumulate synaptic vesicle markers We have previously established that the neuronal bulges induced by SPANs are sites of accumulation of synaptic vesicles (SVs), whose presence could be shown by labelling with antibodies specific for SV proteins such as the vesicle-associated membrane protein 2 (VAMP2) or
Fig. 1. Morphological changes induced by a-Ltx and SPANs in primary neurons in culture. Spinal cord motoneurons (SCMNs, upper panels) and cerebellar granule neurons (CGNs, lower panels) were treated with a-Ltx 0.1 nM for 20 min (A) or with b-Btx 6 nM for 30 min (B). Both toxins induce closely similar membrane enlargements of neuronal processes (bulges). Very similar results were obtained with taipoxin, notexin and textilotoxin. Scale bar: 20 mm.
E. Tedesco et al. / Toxicon 54 (2009) 138–144
synaptophysin I (SypI) (Rigoni et al., 2004). Fig. 2 shows examples of such accumulation caused by SPANs (right panels) and that the action of a-Ltx (central panels) is very similar also in this respect, as previously found in hippocampal neurons (Pennuto et al., 2002). These pictures were obtained in permeabilised neurons and do not allow one to distinguish between an internal localization or a surface exposure of the antigens. Therefore the action of the toxins was also analysed in non-permeabilised cells (see next paragraph). 3.3. SPANs and a-latrotoxin induce exposure of synaptic vesicle luminal proteins in cultured neurons According to the electron microscopy analyses discussed in the introduction, SVs are largely reduced, or absent, in the cytosol after the action of a-Ltx or SPANs. The most likely possibility is that SVs were induced to fuse and that exocytosis was not followed by SV retrieval, owing to an impairment in the exo–endocytic balance. If this is the case, the luminal domains of the SV membrane proteins have to be permanently exposed on the surface of the neurons. Fig. 3 shows that, whilst the luminal part of the SV membrane marker synaptotagmin I is very little stained in control non-permeabilised neurons, it is intensively stained on the surface of bulges induced by both a-Ltx and SPANs. Such intense surface fluorescent signal indicates that SVs are incorporated in the plasma membrane at sites of
bulging following neurotoxins.
141
the
exposure
to
both
types
of
3.4. SPANs and a-latrotoxin induce entry of calcium into cultured neurons Previous studies have established that a very large Ca2þ influx is induced at nerve terminals by a-Ltx (Rosenthal and Meldolesi, 1989; Grishin, 1998; Ushkaryov et al., 2008) and that SPAN-induced bulges are sites of Ca2þ influx that can be monitored by Ca2þ imaging (Rigoni et al., 2007). It remained to show that the a-Ltx induced bulges are as well sites of Ca2þ entry. The left panel of Fig. 4 shows that this is indeed the case, but the time course of this Ca2þ influx is different from that observed in SPAN-treated neurons (middle and right panels). In cerebellar granular neurons exposed to 0.1 nM a-Ltx Ca2þ ions begin to enter about 700 s after toxin addition and this timing is very similar in all terminals that we have analysed. This is in agreement with the notion that the ions enter through a tetrameric channel made by the a-Ltx on the plasma membrane (Orlova et al., 2000), and indicates that several hundreds of seconds are needed to assemble a functional toxin channel on the plasma membrane. Fig. 4 shows that calcium imaging in a homogeneous population of neurons such as CGNs is very useful in the study of the kinetics of the action of this toxin in vivo. In contrast, SPANs induce a progressive increase of Ca2þ entry and this is in agreement with the
Fig. 2. Effect of a-Ltx and SPANs on the distribution of synaptic vesicle markers in primary neurons in culture. Cerebellar granule neurons were incubated with a-Ltx and b-Btx (0.1 nM for 20 min and 6 nM for 60 min respectively) and the distribution of the vesicular proteins VAMP2 and synaptophysin I was analysed by indirect immunofluorescence. In both cases the staining of the two proteins changes with respect to the control, showing an accumulation within the toxininduced membrane enlargements. Overlapping results were obtained with spinal cord motoneurons and with taipoxin, notexin and textilotoxin. Scale bar: 20 mm.
142
E. Tedesco et al. / Toxicon 54 (2009) 138–144
Fig. 3. Effect of a-Ltx and SPANs on synaptic vesicle recycling of cultured neurons. Cerebellar granule neurons were exposed to a-Ltx and Tpx (0.1 nM for 20 min and 6 nM for 60 min respectively) and then incubated for 5 min at 37 C before fixation and permeabilization with a monoclonal antibody that specifically recognizes a luminal epitope of the vesicular protein synaptotagmin I and then processed for indirect immunofluorescence. In both cases the fluorescent signal is brighter than the control and accumulates within the membrane enlargements, indicating that these bulges are sites of unbalanced exo–endocytosis. Similar results were obtained in SCMNs and with b-bungarotoxin, notexin and textilotoxin. Scale bar: 20 mm.
proposal that this influx is mediated by transient lipidic pores made by lysophospholipids and fatty acids in the plasma membrane (Rigoni et al., 2007; Rossetto and Montecucco, 2008). The end result is, however, in both cases the same, i.e. toxin-induced bulges accumulate Ca2þ and this calcium overload may set in motion a series of degenerative events.
an evident loss not only of neurofilaments but also of the specific nervous terminal marker VAChT. One may conclude that also in this respect the two types of toxins share a similar pharmacological/pathophysiological endpoint. As expected from presynaptic neurospecific toxins, there is no evident modification of the postsynaptic component of the NMJ (except for notexin, which is well known to cause also myotoxic effects).
3.5. Degeneration of presynaptic nerve terminals at NMJ after SPAN or a-Ltx exposure
4. Discussion
An end result of the chain of events started by the Ca2þ influx caused by SPANs and a-Ltx is the degeneration of nervous terminal (Chen and Lee, 1970; Ceccarelli et al., 1972; Cull-Candy et al., 1976; Ceccarelli and Hurlbut, 1980; Duchen et al., 1981; Lee et al., 1984; Gopalakrishnakone and Hawgood, 1984; Dixon and Harris, 1999; Harris et al., 2000). Fig. 5 compares the staining of neurofilaments and vesicular acetylcholine transporter (VAChT), which are essential components of nerve terminals, in control and toxininjected mice muscles. With both SPANs and a-Ltx there is
The main purpose of the present work was the comparison of the action of snake presynaptic PLA2 neurotoxins with that of the black widow spider a-latrotoxin. Apart from that such a side-by-side comparison was not performed before, the rationale for such a work is based on the fact that the molecular pathogenesis of the degeneration of nerve terminals induced by the snake presynaptic PLA2 neurotoxins is not yet defined (Pungercar and Krizaj, 2007; Rigoni et al., 2008), whilst that caused by a-Ltx is well established to be due mainly to the large influx of
Fig. 4. Intracellular [Ca2þ] increase induced by a-Ltx and SPANs in neurons in culture. The traces give the time course of changes in 340/380 fluorescence ratios of Fura2-loaded CGNs, showing the intracellular [Ca2þ] rises within different bulges induced by a-Ltx (0.1 nM), Tpx and Tetx (6 nM), added at time 0; cell bodies remain largely unaffected (black line in the left panel, red trace in the middle panel and green trace in the right panel). Closely similar results were obtained in SCMNs and with the other two snake neurotoxins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
E. Tedesco et al. / Toxicon 54 (2009) 138–144
143
Fig. 5. Effect of a-Ltx and SPANs on mouse neuromuscular junction. Mice hind limbs were injected with a-Ltx and Tpx and processed for indirect immunohistochemistry after 12 and 24 h respectively. Both neurotoxins cause degradation of neurofilaments (blue) and disappearance of vesicular acetylcholine transporter staining (red). No morphological modifications of the postsynaptic element of the NMJ stained with a-Btx (green) were observed. Scale bars: 10 mm. Similar results were obtained with the three other SPANs (with the exception of Ntx that caused also muscle damage). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Ca2þ which induces mitochondrial collapse and activation of many degradative enzymes (Ushkaryov et al., 2008). The present work documents that the two types of presynaptic neurotoxins cause similar alterations in neurons in culture and in vivo. Both of them cause bulging of neurons in culture and these bulges expose on their surface the luminal domain of proteins of the synaptic vesicle membrane. Bulges accumulate calcium although with different kinetics, which are accounted for by the different biochemical activities of the two types of toxins, i.e. PLA2 activity for SPANs and ion channel activity for a-Ltx. A high cytosolic [Ca2þ] is known to trigger a series of degradative events and, as a marker, we followed here the degradation of neurofilaments and the disappearance of VAChT. Here, we found that neurofilaments and VAChT disappear at the motoneuron endings of mice injected with the two types of neurotoxins, similarly to what previously found with b-Btx (Prasarnpun et al., 2004). Taken together, all these similarities suggest that a major role in the degeneration of the nerve terminals caused by SPANs is the calcium overloading that follows the change in membrane composition caused by the PLA2 activity of SPANs. The fact that Ca2þ ions appear to enter into nerve terminals soon after SPANs addition and that they accumulate within bulges are indications that the toxin hydrolytic activity may be, at least in the beginning of the process, concentrated close to their binding sites and that the concentration of the phospholipids hydrolysis products may reach significant values such as to change the plasma membrane permeability. The precise localization of the binding sites of SPANs within the nerve termini remains to be determined and should throw light on this ill-defined, but fundamental, aspect of the process.
Acknowledgments The present work was supported by Telethon (grant no. GGP06133) and by Fondazione Cariparo Progetto ‘‘Physiopathology of the synapse: neurotransmitters, neurotoxins and novel therapies’’ to CM and by CMB program of RAS to EG.
Conflicts of interest The authors declare that there are no conflicts of interest. References Arce, V., Garces, A., de Bovis, B., Filippi, P., Henderson, C., Pettmann, B., deLapeyrie`re, O., 1999. Cardiotrophin-1 requires LIFRbeta to promote survival of mouse motoneurons purified by a novel technique. J. Neurosci. Res. 55, 119–126. Bernardi, P., Rasola, A., 2007. Calcium and cell death: the mitochondrial connection. Subcell. Biochem. 45, 481–506. Bertipaglia, I., Carafoli, E., 2007. Calpains and human disease. Subcell. Biochem. 45, 29–53. Bohnert, S., Schiavo, G., 2005. Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation. J. Biol. Chem. 280, 42336–42344. Bonanomi, D., Pennuto, M., Rigoni, M., Rossetto, O., Montecucco, C., Valtorta, F., 2005. Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2. Mol. Pharmacol. 67, 1901–1908. Caccin, P., Rigoni, M., Risceglie, A., Rossetto, O., Montecucco, C., 2006. Reversible skeletal neuromuscular paralysis induced by different lysophospholipids. FEBS Lett. 580, 6317–6321. Ceccarelli, B., Hurlbut, W.P., Mauro, A., 1972. Depletion of vesicle from frog neuromuscular junction by prolonged tetanic stimulation. J. Cell Biol. 50, 30–38. Ceccarelli, B., Hurlbut, W.P., 1980. Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60, 396–441. Chen, I.L., Lee, C.Y., 1970. Ultrastructural changes in the motor nerve terminals caused by beta-bungarotoxin. Virchows Arch. B Cell. Pathol. 6, 318–325. Connolly, S., Trevett, A.J., Nwokolo, N.C., Lalloo, D.G., Naraqi, S., Mantle, D., Schofield, I.S., Fawcett, P.R., Harris, J.B., Warrell, D.A., 1995. Neuromuscular effects of Papuan Taipan snake venom. Ann. Neurol. 38, 916–920. Cull-Candy, S.G., Fohlman, J., Gustavsson, D., Lu¨llmann-Rauch, R., Thesleff, S., 1976. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1, 175–180. Dixon, R.W., Harris, J.B., 1999. Nerve terminal damage by beta-bungarotoxin: its clinical significance. Am. J. Pathol. 154, 447–455. Duchen, L.W., Gomez, S., Queiroz, L.S., 1981. The neuromuscular junction of the mouse after black widow spider venom. J. Physiol. 316, 279–291. Gopalakrishnakone, P., Hawgood, B.J., 1984. Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon 22, 791–804. Grishin, E.V., 1998. Black widow spider toxins: the present and the future. Toxicon 36, 1693–1701. Harris, J.B., 1985. Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol. Ther. 31, 79–102.
144
E. Tedesco et al. / Toxicon 54 (2009) 138–144
Harris, J.B., Grubb, B.D., Maltin, C.A., Dixon, R., 2000. The neurotoxicity of the venom phospholipases A2, notexin and taipoxin. Exp. Neurol. 161, 517–526. Herkert, M., Shakhman, O., Schweins, E., Becker, C.M., 2001. Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization. Eur. J. Neurosci. 14, 821–828. Kini, R.M., 1997. Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism. Chichester, UK. Lee, C.Y., Tsai, M.C., Chen, Y.M., Ritonja, A., Gubensek, F., 1984. Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom. Arch. Int. Pharmacodyn. Ther. 268, 313–324. Leung, Y.M., Xion, Y., Ou, Y.J., Kwan, C.Y., 1998. Perturbation by lysophosphatidylcholine of membrane permeability in cultured vascular smooth muscle and endothelial cells. Life Sci. 63, 965–973. Levi, G., Aloisi, F., Ciotti, M.T., Gallo, V., 1984. Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures. Brain Res. 290, 77–86. Li, X.H., Long, D.X., Li, W., Wu, Y.J., 2007. Different mechanisms of lysophosphatidylcholine-induced Ca2þ mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells. Neurosci. Lett. 424, 22–26. Montecucco, C., Gutie´rrez, J.M., Lomonte, B., 2008. Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell. Mol. Life Sci. [Epub ahead of print]. Neco, P., Rossetto, O., Gil, A., Montecucco, C., Gutie´rrez, L.M., 2003. Taipoxin induces F-actin fragmentation and enhances release of catecholamines in bovine chromaffin cells. J. Neurochem. 85, 329–337. Orlova, E.V., Rahman, M.A., Gowen, B., Volynski, K.E., Ashton, A.C., Manser, C., van Heel, M., Ushkaryov, Y.A., 2000. Structure of a-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores. Nat. Struct. Biol. 7, 48–53. Pennuto, M., Dunlap, D., Contestabile, A., Benfenati, F., Valtorta, F., 2002. Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses. Mol. Biol. Cell 13, 2706–2717. Petrovic, U., Sribar, J., Paris, A., Rupnik, M., Krzan, M., Vardjan, N., Gubensek, F., Zorec, R., Krizaj, I., 2004. Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell. Biochem. Biophys. Res. Commun. 324, 981–985. Prasarnpun, S., Walsh, J., Harris, J.B., 2004. Beta-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction. Neuropharmacology 47, 304–314. Prasarnpun, S., Walsh, J., Awad, S.S., Harris, J.B., 2005. Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis. Brain 128, 2987–2996.
Praznikar, Z.J., Kovacic, L., Rowan, E.G., Romih, R., Rusmini, P., Poletti, A., Krizaj, I., Pungercar, J., 2008. A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol. Biochim. Biophys. Acta 1783, 1129–1139. Pungercar, J., Krizaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892. Rigoni, M., Schiavo, G., Weston, A.E., Caccin, P., Allegrini, F., Pennuto, M., Valtorta, F., Montecucco, C., Rossetto, O., 2004. Snake presynaptic neurotoxins with phospholipase A2 activity induce punctuate swellings of neurites and exocytosis of synaptic vesicles. J. Cell Sci. 117, 561–570. Rigoni, M., Caccin, P., Gschmeissner, S., Koster, G., Postle, A.D., Rossetto, O., Schiavo, G., Montecucco, C., 2005. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid–fatty acid mixtures. Science 310, 1678–1680. Rigoni, M., Pizzo, P., Schiavo, G., Weston, A.E., Zatti, G., Caccin, P., Rossetto, O., Pozzan, T., Montecucco, C., 2007. Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products. J. Biol. Chem. 282, 11238–11245. Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., Montecucco, C., 2008. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria and open their transition pores. J. Biol. Chem. 283, 34013–34020. Rosenthal, L., Meldolesi, J., 1989. Alpha-latrotoxin and related toxins. Pharmacol. Ther. 42, 115–134. Rossetto, O., Gorza, L., Schiavo, G., Schiavo, N., Scheller, R.H., Montecucco, C., 1996. VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in non-neuronal tissues. J. Cell Biol. 132, 167–179. Rossetto, O., Montecucco, C., 2008. Presynaptic neurotoxins with enzymatic activities. Handb. Exp. Pharmacol. 184, 129–170. Ushkaryov, Y.A., Grishin, E.V., 1986. Black widow spider neurotoxin and its interaction with rat brain receptor. Bioorg. Chem. (Russ.) 12, 71–80. Ushkaryov, Y.A., Rohou, A., Sugita, S., 2008. alpha-Latrotoxin and its receptors. Handb. Exp. Pharmacol. 184, 171–206. Woodley, S.L., Ikenouchi, H., Barry, W.H., 1991. Lysophosphatidylcholine increases cytosolic calcium in ventricular myocytes by direct action on the sarcolemma. J. Mol. Cell. Cardiol. 23, 671–680. Wilson-Ashworth, H.A., Judd, A.M., Law, R.M., Freestone, B.D., Taylor, S., Mizukawa, M.K., Cromar, K.R., Sudweeks, S., Bell, J.D., 2004. Formation of transient non-protein calcium pores by lysophospholipids in S49 lymphoma cells. J. Membr. Biol. 200, 25–33. Zimmerberg, J., Chernomordik, L.V., 2005. Neuroscience. Synaptic membranes bend to the will of a neurotoxin. Science 310, 1626–1627.