The recent outbreaks of avian influenza (AI) worldwide have highlighted the difficulties in controlling this disease. Vaccination has become a recommended tool to support the eradication efforts and to limit the economic losses due to AI. A vaccination system based on the use of a vaccine containing a heterologous neuraminidase to the field virus has been shown to be efficacious in reducing the viral shedding and clinical symptoms and in differentiating vaccinated from infected animals (2). To further develop this so called differentiating infected from vaccinated animal vaccination system, two reassortant avian influenza viruses of the H7N5 subtype have been generated. The aim of this study was to generate a prototype strain with a rare N subtype to avoid interference with the anti-N discriminatory test.
The neuromuscular junction is exposed to different types of insults including mechanical traumas, toxins or autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain-Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, that activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regenerative program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron derived ATP acts as alarm messenger for Schwann cells, where it induces the activation of intracellular pathways including calcium signaling, cyclic AMP and CREB, which in turn produce signals that promote nerve regeneration. These results contribute to define the cross-talk taking place at the neuromuscular junction attacked by anti-gangliosides autoantibodies plus complement, functional to nerve regeneration, that are likely to be valid also for other peripheral neuropathies.
Snake presynaptic neurotoxins with phospholipase A2 activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca2+ from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A2 neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity. Snake presynaptic neurotoxins with phospholipase A2 activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca2+ from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A2 neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity. Two classes of neurotoxins can paralyze the neuromuscular junction through their enzymatic activity: (i) the clostridial neurotoxins, metalloproteases acting specifically on SNARE (soluble NSF attachment protein receptor) proteins to cause tetanus and botulism, and (ii) the SPANs (1Rossetto O. Morbiato L. Caccin P. Rigoni M. Montecucco C. J. Neurochem. 2006; 97: 1534-1545Crossref PubMed Scopus (94) Google Scholar). SPANs 3The abbreviations used are: SPANs, snake presynoptic phospholipase A2 neurotoxins; β-Btx, β-bungarotoxin; PTP, permeability transition pore; CGNs, cerebellar granular neurons; CRC, calcium retention capacity; CsA, cyclosporin A; mLysoPC, 1-myristoyllysophosphatidylcholine; Ntx, notexin; PLA2, phospholipase A2; OA, oleic acid; SCMNs, spinal cord motor neurons; Tpx, taipoxin; Tetx, textilotoxin. play a major role in envenomation and cause a botulism-like flaccid paralysis with autonomic symptoms (2Connolly S. Warrell D.A. Ann. Neurol. 1995; 38: 916-920Crossref PubMed Scopus (48) Google Scholar, 3Prasarnpun S. Walsh J. Awad S.S. Harris J.B. Brain. 2005; 128: 2987-2996Crossref PubMed Scopus (70) Google Scholar). The enzymatic activity and the neurospecificity make these toxins very effective; however, like botulinum neurotoxins, SPANs do not affect the cell body and axon of the motor neuron, allowing complete recovery in most patients (4Rossetto O. Montecucco C. Handb. Exp. Pharmacol. 2008; 184: 129-170Crossref Scopus (55) Google Scholar). Impairment of neuromuscular transmission by SPANs is traditionally measured in nerve-muscle preparations isolated from the mouse hemidiaphragm or from the chicken biventer cervicis. A simpler and more sensitive assay, based on SPAN-induced irreversible bulging of nerve terminals in culture, was recently described (5Rigoni M. Schiavo G. Weston A.E. Caccin P. Allegroni F. Pennuto M. Valtorta F. Montecucco C. Rossetto O. J. Cell Sci. 2004; 117: 3561-3570Crossref PubMed Scopus (61) Google Scholar). It was also shown that an early consequence of the action of SPANs is the hydrolysis of phosphatidylcholine into lysophosphatidylcholine and fatty acids and that their equimolar mixture mimics the swelling response of nerve terminals to the toxin itself (6Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (155) Google Scholar). The SPAN-induced nerve bulges accumulate Ca2+, and, this event is accompanied by mitochondrial rounding and depolarization (7Rigoni M. Pizzo P. Schiavo G. Weston A.E. Zatti G. Caccin P. Rossetto O. Pozzan T. Montecucco C. J. Biol. Chem. 2007; 282: 11238-11245Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The cytosolic [Ca2+] increase could also trigger the activity of many Ca2+-activated hydrolases of nucleic acids, proteins, and lipids, all factors that could account for the pronounced degeneration of nerve terminals poisoned by SPANs (8Cull-Candy S.G. Fohlman J. Gustavsson D. Lullmann-Rauch R. Thesleff S. Neuroscience. 1976; 1: 175-180Crossref PubMed Scopus (117) Google Scholar, 9Gopalakrishnakone P. Hawgood B.J. Toxicon. 1984; 22: 791-804Crossref PubMed Scopus (51) Google Scholar, 10Dixon R. Harris J.B. Am. J. Pathol. 1999; 154: 447-455Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Montecucco C. Gutiérrez J.M. Lomonte B. CMLS Cell. Mol. Life Sci. 2008; 65: 2897-2912Crossref PubMed Scopus (196) Google Scholar). Previous studies indicated that SPANs can gain access to the cell interior. Indeed, fluorescein-conjugated β-Btx was found to rapidly enter hippocampal neurons in culture and was suggested to associate at least in part with lysosomes (12Herkert M. Shakhman O. Schweins E. Becker C.M. Eur. J. Neurosci. 2001; 14: 821-828Crossref PubMed Scopus (54) Google Scholar). By antibody labeling, Tpx was found to localize inside chromaffin cells in culture (13Neco P. Rossetto O. Gil A. Montecucco C. Gutiérrez L.M. J. Neurochem. 2003; 83: 329-337Crossref Scopus (35) Google Scholar). Fluorophore-conjugated ammodytoxin A (a 14-kDa PLA2 neurotoxin isolated from the venom of Vipera ammodytes) was detected in the nucleus of hippocampal neurons (14Petrovic U. Sribar J. Paris A. Rupnik M. Krzan M. Vardjan N. Gubensek F. Zorec R. Kriźaj I. Biochem. Biophys. Res. Commun. 2004; 324: 981-985Crossref PubMed Scopus (37) Google Scholar) and in the cytosol of undifferentiated NSC34 cells (15Praźnikar Z.J. Kovaćić L. Rowan E.G. Romih R. Rumini P. Poletti A. Kriźaj I. Pungerćar J. Biochim. Biophys. Acta. 2008; 1783: 1129-1139Crossref PubMed Scopus (35) Google Scholar), a mouse neuroblastoma × spinal cord hybrid cell line (16Cashman N.R. Durham H.D. Blusztajn J.K. Oda K. Tabira T. Shaw I.T. Dahrouge S. Antel J.P. Dev. Dyn. 1992; 194: 209-221Crossref PubMed Scopus (582) Google Scholar). In addition, Tpx was reported to bind an endoplasmic reticulum-located protein in vitro (17Dodds D. Schlimgen A.K. Lu S.Y. Perin M.S. J. Neurochem. 1995; 64: 2339-2344Crossref PubMed Scopus (46) Google Scholar), and ammodytoxin A was found to bind a variety of cytosolic proteins (18Sribar J. Copic A. Paris A. Sherman N.E. Gubensek F. Fox J.W. Kriźaj I. J. Biol. Chem. 2001; 276: 12493-12496Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 19Sribar J. Sherman N.E. Prijatelj P. Faure G. Gubensek F. Fox J.W. Aitken A. Pungerćar J. Kriźaj I. Biochem. Biophys. Res. Commun. 2003; 302: 691-696Crossref PubMed Scopus (46) Google Scholar) and R25, an integral protein of mitochondria (20Sribar J. Copic A. Poljsak-Prijatelj M. Kuret J. Logonder U. Gubensek F. Kriźaj I. FEBS Lett. 2003; 553: 309-314Crossref PubMed Scopus (33) Google Scholar). As SPANs require Ca2+ for their hydrolytic activity, the biological relevance of these findings was considered to be questionable. However, we recently documented that SPANs do induce the accumulation of Ca2+ within nerve terminals (7Rigoni M. Pizzo P. Schiavo G. Weston A.E. Zatti G. Caccin P. Rossetto O. Pozzan T. Montecucco C. J. Biol. Chem. 2007; 282: 11238-11245Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and this finding reopened the possibility of a contribution of the entry of SPANs in the nerve terminal cytosol to the pathogenesis of envenomation. Here, we report that active fluorescent derivatives of Ntx, β-Btx, and Tpx enter nerve terminals and bind specifically to mitochondria, whose morphology changes from the elongated, spaghetti-like shape to a rounded one. Rounded mitochondria were detected inside the toxin-induced bulges of nerve terminals. To understand the mechanistic basis for the mitochondrial changes, we investigated the effect of these neurotoxins on isolated mitochondria and discovered that SPANs are inducers of the mitochondrial PTP, with a relative potency that matches their PLA2 activity. These findings have important consequences in defining the molecular events that lead to the pathogenesis of peripheral nerve paralysis caused by snake presynaptic PLA2 neurotoxins in general. Neurotoxins and Lipid Mixture Preparation—Ntx, Tpx, and Tetx were purchased from Venom Supplies; fluorescein isothiocyanate-conjugated β-Btx and β-Btx were from Sigma. Their purity was controlled by SDS-PAGE. 1-Myristoyllysophosphatidylcholine (mLysoPC; Sigma) and an oleic acid (OA; Sigma) mixture (mLysoPC + OA) were prepared as described previously (6Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (155) Google Scholar). Toxin Labeling and Assay—One hundred and fifty micrograms of purified toxin (Ntx, Tpx, and Tetx) were resuspended in 150 μl of 10 mm Hepes, 150 mm NaCl, pH 7.4; the pH of the reaction buffer was adjusted to 8.0 by adding sodium bicarbonate. Fifteen micrograms of Alexa568 dye (Molecular Probes) (from a stock solution of 10 μg/μl in Me2SO) were added to the toxin solution. The reaction was carried out in the dark at room temperature for 1 h under continuous stirring and was stopped by the addition of 15 μl of 1.5 m hydroxylamine, pH 8.5. Excess dye was removed by extensive dialysis against 10 mm Hepes, 150 mm NaCl, pH 7.4 (Slide-A-Lyzer dialysis cassette, 10-kDa cut-off, Pierce). The conjugate was collected; its absorbance spectrum was recorded; and ratios of 0.5 Alexa568/Ntx molecule, of 1.2 Alexa568/Tpx molecule, and 3.5 Alexa568/Tetx molecule were determined. The toxicity of Alexa568-conjugated toxins was assayed in the mouse nerve-hemidiaphragm preparation as before (6Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (155) Google Scholar). The fluorescent Ntx and Tpx derivatives, as well as the fluorescein isothiocyanate-conjugated β-Btx, were nearly as neurotoxic as their nonconjugated counterparts (supplemental Table S1). Alexa568-Tetx showed pronounced absorption onto the polylysine/polyornithine-laminin coating of the neuronal cultures and could not be used for neuron imaging. Chemical Modifications of Notexin—Acetylation of lysine residues with acetic anhydride (Sigma) was performed as described (21Means G.E. Feeney R.E. Chemical Modification of Proteins. Holden-Day, Inc., San Francisco1971: 214Google Scholar) with minor modifications. Briefly, 30 μg of Ntx were dissolved in 100 μl of a saturated solution of sodium acetate in 50 mm sodium borate buffer, pH 8.2, and then cooled in an ice-water bath. The solution was treated with a total amount of 15 μl of a 1:500 dilution of acetic anhydride, distributed over five additions during 1 h at 4 °C. Acetylated Ntx was then dialyzed against 150 mm NaCl, 10 mm Hepes, pH 7.4 (Slide-A-Lyzer dialysis cassette), and conjugated with Alexa568 as described above. Histidine modifications of Ntx with diethyl pyrocarbonate (Sigma) or p-bromophenacyl bromide (Sigma) were performed as described previously (22Papini E. Schiavo G. Sandoná D. Rappuoli R. Montecucco C. J. Biol. Chem. 1989; 264: 12385-12388Abstract Full Text PDF PubMed Google Scholar, 23Halpert J. Karlsson E. FEBS Lett. 1975; 61: 72-76Crossref Scopus (118) Google Scholar). In the case of modification with diethyl pyrocarbonate, the reaction was performed in 50 mm phosphate buffer, pH 7.8, at 25 °C (toxin concentration = 0.2 mg/ml) by adding aliquots of a freshly prepared solution of diethyl pyrocarbonate in anhydrous ethanol. The reaction was followed by monitoring the absorbances at 243 and 278 nm in a Perkin-Elmer Lambda 5 spectrophotometer (22Papini E. Schiavo G. Sandoná D. Rappuoli R. Montecucco C. J. Biol. Chem. 1989; 264: 12385-12388Abstract Full Text PDF PubMed Google Scholar) and was stopped by the addition of imidazole (5 mm final concentration). The modified toxin was then dialyzed against 50 mm phosphate buffer, pH 7.0. Notexin histidines were modified also with p-bromophenacyl bromide. Briefly, 100 μg of Ntx were resuspended in 100 μl of conjugation buffer (0.1 m sodium cacodylate-HCl, pH 6, 0.1 m NaCl). Incubation with p-bromophenacyl bromide was carried out at 30 °C at a molar reagent:protein ratio of 5:1 for 7 h and was followed by extensive dialysis against 10 mm Hepes, pH 7.4, 150 mm NaCl. Neurotoxicity, PLA2 activity, and effects on isolated brain mitochondria of modified toxins were tested (supplemental Table S2). Cell Culture Preparation—Rat CGNs were prepared from 6-day-old Wistar rats as described previously (24Levi G. Aloisi F. Ciotti M.T. Gallo V. Brain Res. 1984; 290: 77-86Crossref PubMed Scopus (389) Google Scholar) and used 6–8 days after plating. Primary rat SCMNs were isolated from Sprague-Dawley (embryonic day 14) rat embryos and cultured following previously described protocols (25Arce V. Garces A. de Bovis B. Filippi P. Henderson C.E. Pettmann B. de Lapeyriere O. J. Neurosci. Res. 1999; 55: 119-126Crossref PubMed Scopus (133) Google Scholar, 26Bohnert S. Schiavo G. J. Biol. Chem. 2005; 280: 42336-42344Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). SCMNs were used after 5–8 days of neuronal differentiation in vitro. Fluorescence Cell Imaging—SCMNs or CGNs were grown on 24-mm diameter coverslips and exposed to Alexa568-Tpx or Alexa568-Ntx or fluoresceinated β-Btx (25–50 nm) for different time periods at 37 °C in E4 medium (in the case of SCMNs) or Krebs-Ringer Hepes buffer (in the case of CGNs). E4 composition was 120 mm NaCl, 3 mm KCl, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 10 mm Hepes, pH 7.4. Krebs-Ringer Hepes buffer composition was 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 2 mm CaCl2, 1.2 mm KH2PO4, 6 mm glucose, and 25 mm Hepes, pH 7.4. After incubation, cells were extensively washed with the same buffers, and the coverslips were placed on the stage of an inverted epifluorescence microscope (Leica ADMIRE3) equipped with a Leica DC500 CCD camera, 63× oil immersion objective (NA 1.4). Images were acquired using Leica FW4000 software and analyzed with Leica Deblur and ImageJ v1.35 software. For colocalization studies, neurons were loaded with the mitochondrial dye nonyl acridine orange (5 nm, Molecular Probes) for 30 min at 37 °C and then washed and incubated with the fluorescent toxins. Images were acquired at different times from toxin addition, and the fluorescent signals were superimposed. PLA2 Activity—The enzymatic activity of the four SPANs was measured with a commercial kit based on the use of the 1,2-dithio analogue of diheptanoylphosphatidylcholine as substrate (Cayman Chemicals). The hydrolysis of the thioester bond at the sn-2 position by PLA2 generates free thiols that interact with 5,5′-dithiobis(nitrobenzoic acid), leading to an increase in the absorbance at 405 nm. ΔA405 was measured with a Beckman SpectraCount. Rat Brain Mitochondrial Preparation—Two adult Wistar rat forebrains were used for each mitochondrial preparation. Rats were killed by cervical dislocation, and forebrains were immediately transferred to ice-cold isolation medium (250 mm sucrose, 10 mm Tris-HCl, pH 7.4, 0.1 mm EGTA). Dissected forebrains were chopped with scissors and homogenized with 5–7 strokes of a loose-fitting Wheaton pestle. The homogenate was centrifuged for 3 min at 2,000 × g in isolation medium + 0.5% bovine serum albumin to precipitate the nuclei, and the supernatant was centrifuged twice for 8 min at 12,000 × g. The resulting pellet was resuspended in isolation medium without bovine serum albumin and centrifuged for 8 min at 12,000 × g. The resulting pellet was finally resuspended in isolation buffer to a protein concentration of 50–60 mg/ml. Protein concentration was quantified with the biuret assay. Assessment of Permeability Transition in Isolated Mitochondria—Onset of the permeability transition was monitored as the fast Ca2+ release following accumulation of multiple 10 μm Ca2+ pulses at 1-min intervals (27Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Extra-mitochondrial Ca2+ concentration was monitored with the Ca2+ indicator Calcium Green-5N (excitation/emission, 505/535 nm, Invitrogen) with a PerkinElmer 650–40 fluorescence spectrometer. Mitochondria were resuspended to a final protein concentration of 1 mg/ml in 2 ml of the following medium: 120 mm KCl, 10 μm EGTA, 5 mm glutamate, 2.5 mm malate, 1 mm Tris phosphate, 10 mm Tris-HCl, pH 7.4, 1 μm Calcium Green-5N. A quartz cuvette with continuous stirring through a magnetic bar was employed to ensure rapid mixing. The number of 10 μm Ca2+ pulses retained by the mitochondrial suspension before PTP opening was counted and set to 100% mitochondrial CRC. Similar experiments were carried out in the presence of the indicated toxins at concentrations ranging between 0.5 and 50 nm. Where indicated, 0.8 μm CsA (Sigma) was added to inhibit the opening of the PTP. CRC experiments were performed within 3 h of mitochondria isolation. Snake Presynaptic PLA2 Neurotoxins Enter Nerve Terminals—To obtain results of rather general value, we have used here four different SPANs and two different primary neuronal cultures. Alexa568 fluorescent derivatives of three SPANs with different quaternary structure, Ntx (monomeric, 14 kDa), Tpx (trimeric, 42 kDa), and Tetx (pentameric, 70 kDa), were prepared and their toxicities were tested. In the case of β-Btx (heterodimeric, 21 kDa) we used a commercial fluoresceinated toxin. The fluorescent derivatives were nearly as active as the native toxins; however, Alexa568-Tetx was strongly absorbed by the culture plate coating and could not be used for fluorescence imaging (see "Experimental Procedures"). Because the end plates of motor neurons in vivo are not readily accessible to investigation, we have studied the entry of fluorescent toxins in primary cultures of SCMNs, which are closer to peripheral motor neurons (26Bohnert S. Schiavo G. J. Biol. Chem. 2005; 280: 42336-42344Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and in a very homogeneous population of CGNs. Fig. 1A shows that Alexa568-Ntx rapidly entered neuronal projections of SCMNs. Remarkably, fluorescent neurotoxin was not homogeneously distributed in the cytosol but rather localized to elongated, spaghetti-like structures that are clearly reminiscent of mitochondria. A similar staining pattern was found also in cerebellar granular neurons and with Alexa568-Tpx (Fig. 1B) and fluoresceinated β-Btx (data not shown), indicating that the mitochondrial-like staining is a rather general feature of SPANs. With time, SPANs induce bulging of neuronal projections (5Rigoni M. Schiavo G. Weston A.E. Caccin P. Allegroni F. Pennuto M. Valtorta F. Montecucco C. Rossetto O. J. Cell Sci. 2004; 117: 3561-3570Crossref PubMed Scopus (61) Google Scholar). Fig. 2 shows the staining of SCMNs with Alexa568-Tpx at 30 min; similar patterns were obtained with fluorescent Ntx and β-Btx (data not shown). The shape of the structures stained by the toxin changed during intoxication, and after 30 min, labeled organelles appeared as rounded bodies, which were always localized inside toxin-induced bulges. SPANs Bind Specifically to Mitochondria within Neurons— The identification of the intracellular organelles stained by these neurotoxins as mitochondria is supported by the findings of Fig. 3, which shows a close superimposition between the staining patterns of Alexa568-Tpx and the mitochondrial dye nonyl acridine orange in SCMNs. Similar findings were obtained in CGNs and with fluorescent β-Btx and Ntx (data not shown). This latter observation is only apparently different from that of Herkert et al. (12Herkert M. Shakhman O. Schweins E. Becker C.M. Eur. J. Neurosci. 2001; 14: 821-828Crossref PubMed Scopus (54) Google Scholar) in hippocampal neurons, which was interpreted as partial localization of fluoresceinated β-Btx to lysosomes. In fact, a close inspection of the figures shows that the spotty distribution found in the neuronal projections is compatible with a staining of mitochondria after 30 min of incubation with the neurotoxin (see below). These observations are consistent with the electron microscopy pictures of motor neurons and CGNs exposed to these neurotoxins (3Prasarnpun S. Walsh J. Awad S.S. Harris J.B. Brain. 2005; 128: 2987-2996Crossref PubMed Scopus (70) Google Scholar, 7Rigoni M. Pizzo P. Schiavo G. Weston A.E. Zatti G. Caccin P. Rossetto O. Pozzan T. Montecucco C. J. Biol. Chem. 2007; 282: 11238-11245Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8Cull-Candy S.G. Fohlman J. Gustavsson D. Lullmann-Rauch R. Thesleff S. Neuroscience. 1976; 1: 175-180Crossref PubMed Scopus (117) Google Scholar, 9Gopalakrishnakone P. Hawgood B.J. Toxicon. 1984; 22: 791-804Crossref PubMed Scopus (51) Google Scholar, 10Dixon R. Harris J.B. Am. J. Pathol. 1999; 154: 447-455Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Montecucco C. Gutiérrez J.M. Lomonte B. CMLS Cell. Mol. Life Sci. 2008; 65: 2897-2912Crossref PubMed Scopus (196) Google Scholar, 28Fohlman J. Eaker D. Dowdall M.J. Lúllmann-Rauch R. Sjódin T. Leander S. Eur. J. Biochem. 1979; 94: 531-540Crossref PubMed Scopus (46) Google Scholar), whose mitochondria show rounding and alteration of cristae indicative of their loss of function. The action of SPANs is known to be very specific for the presynaptic nerve terminals in vivo. Also in our cultures SPANs staining appears to be very specific for mitochondria within neurons, as shown by lack of toxin staining in non-neuronal cells (Fig. 4). These findings prompted us to investigate the effects of SPANs in mitochondria isolated from rat brain. SPANs Open the Mitochondrial Permeability Transition Pore—A common cause of mitochondrial swelling and depolarization in situ is the opening of the mitochondrial PTP, an inner membrane high-conductance channel that can be desensitized by CsA (29Bernardi P. Krauskopf A. Basso E. Petronilli V. Blachly-Dyson E. Di Lisa F. Forte M.A. FEBS J. 2006; 273: 2077-2099Crossref PubMed Scopus (558) Google Scholar). The propensity of the PTP to open in a population of mitochondria can be monitored with a sensitive technique based on the CRC, i.e. the amount of Ca2+ that can be taken up by mitochondria in the presence of inorganic phosphate before onset of PTP opening (27Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Untreated, control mitochondria accumulated 10 pulses of 10 μm Ca2+ before onset of the permeability transition, which is readily detected by a precipitous release of the previously accumulated Ca2+ (Fig. 5A). Addition of as little as 1 nm Ntx dramatically decreased the threshold for PTP opening, which was observed after accumulation of three pulses of Ca2+ (Fig. 5B). It should be noted that prior to PTP opening, the rate of Ca2+ uptake in Ntx-treated mitochondria was indistinguishable from that of controls, indicating that, in the absence of added Ca2+, Ntx does not affect energy coupling. As expected for a PTP-dependent event, treatment with CsA increased the CRC both in the absence (Fig. 5C) and presence (Fig. 5D) of Ntx. We then investigated the effects of the four SPANs on the CRC and their relative potency. Ntx was the most effective, β-Btx and Tpx displayed an intermediate PTP sensitizing activity, whereas Tetx was nearly ineffective (Fig. 6A). This order of potency correlates well with the PLA2 activity of the four SPANs measured by an in vitro assay (Ntx, 371 μmol/min/mg; β-Btx, 218 μmol/min/mg; Tpx, 100 μmol/min/mg; Tetx, 10 μmol/min/mg; see "Experimental Procedures"). To test the hypothesis that the enzymatic activity is indeed responsible for facilitation of PTP opening by SPANs, we determined the direct effect of the products of the PLA2 activity, mLysoPC, and OA. These were added to rat brain mitochondria either individually or in the 1:1 molar mixture that is produced by SPANs (Fig. 6B). Consistent with our hypothesis, the equimolar mixture of mLysoPC + OA (1 μm) facilitated PTP opening, whereas mLysoPC alone was less effective. OA had a strong effect, in line with previous observations, demonstrating that fatty acids (i.e. arachidonic and palmitic) are effective inducers of the PTP in isolated mitochondria and intact cells (30Scorrano L. Penzo D. Petronilli V. Pagano F. Bernardi P. J. Biol. Chem. 2001; 276: 12035-12040Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Together with our previous findings that fatty acid alone has a minor inhibitory effect on the transmission of the nerve impulse to the muscle (6Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (155) Google Scholar), the present result indicates that very little fatty acid is able to partition from the plasma membrane into the mitochondria of the nerve terminals after its release by the PLA2 activity of these neurotoxins. Further evidence that the PLA2 enzymatic activity of the SPANs is instrumental in inducing the mitochondrial change in permeability was obtained in experiments performed with chemically inactivated Ntx. The toxin was acetylated, and this derivative retained 2.9 ± 2.5% of the PLA2 activity of the unmodified toxin (n = 3). Acetylated Ntx did not significantly inhibit neurotransmission of the mouse hemidiaphragm preparation, did not stain or induce any bulging of neurons in culture, and failed to induce opening of the mitochondrial PTP. Notexin was also chemically modified with the histidine-specific reagents diethyl pyrocarbonate and p-bromophenacyl bromide following established procedures (22Papini E. Schiavo G. Sandoná D. Rappuoli R. Montecucco C. J. Biol. Chem. 1989; 264: 12385-12388Abstract Full Text PDF PubMed Google Scholar, 23Halpert J. Karlsson E. FEBS Lett. 1975; 61: 72-76Crossref Scopus (118) Google Scholar), which led to partial loss of PLA2 activity (supplemental Table S2). Importantly, the percentage of loss of enzymatic activity correlated well with the percentages of loss of neurotoxicity and of capability of opening the mitochondrial PTP. These data strongly support the proposal that the PLA2 activity of SPANs is involved in their effect on the mitochondrial PTP. Fig. 7 reports the relative protective effect of the PTP inhibitor CsA in the absence or presence of the four SPANs. This parameter has similar values whether or not a SPAN was present, whichever SPAN is considered. This indicates that SPANs do not directly permeabilize the mitochondrial membrane, with ensuing unspecific Ca2+ leak. On the other hand, the effect of SPANs appears to be rather specific for the PTP channel, as CsA inhibited the effect of the toxins to a similar extent, with relative values close to those of the controls. The main findings of the present study are (i) presynaptic snake neurotoxins of different size (from 14 to 42 kDa) endowed with PLA2 activity enter neurons within a short time of addition; (ii) they bind specifically to mitochondria and induce a shape change within regions of nerve terminals that undergo swelling to form round bulges of the plasma membrane; and (iii) these neurotoxins induce opening of the mitochondrial PTP, which leads to release of Ca2+, with an order of potency that matches their PLA2 enzymatic activities. The entry of SPANs inside cells was reported before. β-Btx and ammodytoxin A were detected within hippocampal neurons (12Herkert M. Shakhman O. Schweins E. Becker C.M. Eur. J. Neurosci. 2001; 14: 821-828Crossref PubMed Scopus (54) Google Scholar, 14Petrovic U. Sribar J. Paris A. Rupnik M. Krzan M. Vardjan N. Gubensek F. Zorec R. Kriźaj I. Biochem. Biophys. Res. Commun. 2004; 324: 981-985Crossref PubMed Scopus (37) Google Scholar), ammodytoxin A was recently found also in NSC34 cells (15Praźnikar Z.J. Kovaćić L. Rowan E.G. Romih R. Rumini P. Poletti A. Kriźaj I. Pungerćar J. Biochim. Biophys. Acta. 2008; 1783: 1129-1139Crossref PubMed Scopus (35) Google Scholar), and Tpx staining by antibody labeling was reported within chromaffin cells (13Neco P. Rossetto O. Gil A. Montecucco C. Gutiérrez L.M. J. Neurochem. 2003; 83: 329-337Crossref Scopus (35) Google
Abstract Snake envenoming is a major, but neglected, tropical disease. Among venomous snakes, those inducing neurotoxicity such as kraits ( Bungarus genus) cause a potentially lethal peripheral neuroparalysis with respiratory deficit in a large number of people each year. In order to prevent the development of a deadly respiratory paralysis, hospitalization with pulmonary ventilation and use of antivenoms are the primary therapies currently employed. However, hospitals are frequently out of reach for envenomated patients and there is a general consensus that additional, non-expensive treatments, deliverable even long after the snake bite, are needed. Traumatic or toxic degenerations of peripheral motor neurons cause a neuroparalysis that activates a pro-regenerative intercellular signaling program taking place at the neuromuscular junction (NMJ). We recently reported that the intercellular signaling axis melatonin-melatonin receptor 1 (MT1) plays a major role in the recovery of function of the NMJs after degeneration of motor axon terminals caused by massive Ca 2+ influx. Here we show that the small chemical MT1 agonists: Ramelteon and Agomelatine, already licensed for the treatment of insomnia and depression, respectively, are strong promoters of the neuroregeneration after paralysis induced by krait venoms in mice, which is also Ca 2+ mediated. The venom from a Bungarus species representative of the large class of neurotoxic snakes (including taipans, coral snakes, some Alpine vipers in addition to other kraits) was chosen. The functional recovery of the NMJ was demonstrated using electrophysiological, imaging and lung ventilation detection methods. According to the present results, we propose that Ramelteon and Agomelatine should be tested in human patients bitten by neurotoxic snakes acting presynaptically to promote their recovery of health. Noticeably, these drugs are commercially available, safe, non-expensive, have a long bench life and can be administered long after a snakebite even in places far away from health facilities. Synopsis Snakebite envenomings cause important tropical human diseases that often include a lethal muscle paralysis. Current treatments consist in hospitalization and antivenoms, which are not always quickly accessible to victims. In fact, these snakebites take place mainly in rural and low income countries. In this work, researchers discovered, in mice, a novel function of melatonin and of its type 1 receptor in promoting functional recovery after snake-induced peripheral neuroparalysis with nerve terminal degeneration. In particular, researchers found that drugs approved for the treatment of insomnia (Ramelteon) and depression (Agomelatine), activate melatonin receptor and promote the functional recovery after a krait venom induced paralysis. These drugs are on sell in pharmacies, are safe and stable, and are ready to be tried for promoting the recovery from peripheral neuroparalysis in human victims bitten by neurotoxic snakes, even without hospitalization.
Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC + FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC + FA. Here, we show that LysoPC + FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC + FA lipid mixture as a novel and effective agonist of synaptic vesicle release. Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC + FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC + FA. Here, we show that LysoPC + FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC + FA lipid mixture as a novel and effective agonist of synaptic vesicle release. Toxins in general, and neurotoxins in particular, are invaluable tools in the molecular analysis of specific cellular processes, from the activation of G protein-coupled receptors to the characterization of the events controlling regulated exocytosis (1Rappuoli R. Montecucco C. Protein Toxins and Their Use in Cell Biology. Oxford University Press, Oxford1997Google Scholar). Much attention has been recently dedicated to a class of neurotoxins with phospholipase A2 (PLA2) 2The abbreviations used are: PLA2, phospholipase A2; CGNs, cerebellar granule neurons; EM, electron microscopy; FA, fatty acid(s); LysoPC, lysophosphatidylcholine; mLysoPC, 1-myristoyl-lysophosphatidylcholine; OA, oleic acid; SCMN, spinal cord motor neuron; SPAN, snake presynaptic phospholipase A2 neurotoxin; SV, synaptic vesicle(s); TMRE, tetramethylrhodamine methyl ester; VDCC, voltage-gated Ca2+ channel. 2The abbreviations used are: PLA2, phospholipase A2; CGNs, cerebellar granule neurons; EM, electron microscopy; FA, fatty acid(s); LysoPC, lysophosphatidylcholine; mLysoPC, 1-myristoyl-lysophosphatidylcholine; OA, oleic acid; SCMN, spinal cord motor neuron; SPAN, snake presynaptic phospholipase A2 neurotoxin; SV, synaptic vesicle(s); TMRE, tetramethylrhodamine methyl ester; VDCC, voltage-gated Ca2+ channel. activity that are produced by different families of poisonous snakes (SPANs), whose precise biochemical and cellular mode of action has long remained elusive (2Kini R.M. Venom Phospholipase A2 Enzymes: Structure, Function, and Mechanism. John Wiley & Sons, Inc., Chichester, UK1997Google Scholar). A hallmark of their action in vivo and in vitro is the induction of enlargement of nerve terminals with large depletion of their content of synaptic vesicles (SV) (3Chen I.L. Lee C.Y. Virchows Arch. B Cell Pathol. 1970; 6: 318-325PubMed Google Scholar, 4Cull-Candy S.G. Fohlman J. Gustavsson D. Lullmann-Rauch R. Thesleff S. Neuroscience. 1976; 1: 175-180Crossref PubMed Scopus (115) Google Scholar, 5Gopalakrishnakone P. Hawgood B.J. Toxicon. 1984; 22: 791-804Crossref PubMed Scopus (50) Google Scholar, 6Dixon R.W. Harris J.B. Am. J. Pathol. 1999; 154: 447-455Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Montecucco C. Rossetto O. Trends Biochem. Sci. 2000; 25: 266-270Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).We have recently shown that SPANs hydrolyze phospholipids of cultured neurons with generation of lysophosphatidylcholine (LysoPC) and fatty acids (FA) (8Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (153) Google Scholar). This leads to a massive release of SV, with their incorporation into the presynaptic plasma membrane and consequent surface exposure of SV luminal epitopes (8Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (153) Google Scholar, 9Rigoni M. Schiavo G. Weston A.E. Caccin P. Allegrini F. Pennuto M. Valtorta F. Montecucco C. Rossetto O. J. Cell Sci. 2004; 117: 3561-3570Crossref PubMed Scopus (61) Google Scholar, 10Bonanomi D. Pennuto M. Rigoni M. Rossetto O. Montecucco C. Valtorta F. Mol. Pharmacol. 2005; 67: 1901-1908Crossref PubMed Scopus (29) Google Scholar). These and other experiments performed with models of SNARE-mediated membrane fusion provided evidence for the involvement of hemifusion lipid intermediates in exocytosis (11Chernomordik L.V. Kozlov M.M. Annu. Rev. Biochem. 2003; 72: 175-207Crossref PubMed Scopus (588) Google Scholar, 12Chernomordik L.V. Kozlov M.M. Cell. 2005; 123: 375-382Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 13Giraudo C.G. Hu C. You D. Slovic A.M. Mosharov E.V. Sulzer D. Melia T.J. Rothman J.E. J. Cell Biol. 2005; 170: 249-260Crossref PubMed Scopus (112) Google Scholar, 14Xu Y. Zhang F. Su Z. McNew J.A. Shin Y.K. Struct. Mol. Biol. 2005; 12: 417-422Crossref PubMed Scopus (193) Google Scholar, 15Reese C. Mayer A. J. Cell Biol. 2005; 171: 981-990Crossref PubMed Scopus (53) Google Scholar, 16Zimmerberg J. Chernomordik L.V. Science. 2005; 310: 1626-1627Crossref PubMed Scopus (32) Google Scholar, 17Corda D. Colanzi A. Luini A. Trends Cell Biol. 2006; 16: 167-173Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The presence of LysoPC on the external leaflet of the presynaptic plasma membrane and of FA on both sides, caused by SPANs or by the addition of LysoPC and FA mixture (LysoPC + FA), promotes the formation of the hemifusion intermediate and its transition to an open pore. At the same time, this change in lipid composition of the membrane inhibits the reverse process (i.e. the fission and retrieval of SV). A balanced SV exocytosis-endocytosis cycle is at the basis of synaptic transmission at nerve terminals (18Murthy V.N. De Camilli P. Annu. Rev. Neurosci. 2003; 26: 701-728Crossref PubMed Scopus (272) Google Scholar, 19Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1852) Google Scholar, 20Ryan T.A. Curr. Opin. Cell Biol. 2006; 18: 416-421Crossref PubMed Scopus (43) Google Scholar). SPANs promote exocytosis and inhibit endocytosis and, therefore, disrupt this finely tuned balance, causing the fusion of SV and formation of nerve terminal bulges decorated with the luminal domain(s) of SV proteins on their surface (9Rigoni M. Schiavo G. Weston A.E. Caccin P. Allegrini F. Pennuto M. Valtorta F. Montecucco C. Rossetto O. J. Cell Sci. 2004; 117: 3561-3570Crossref PubMed Scopus (61) Google Scholar, 10Bonanomi D. Pennuto M. Rigoni M. Rossetto O. Montecucco C. Valtorta F. Mol. Pharmacol. 2005; 67: 1901-1908Crossref PubMed Scopus (29) Google Scholar). EM analysis reveals that nerve terminals are almost completely depleted of SV, with disappearance of both the "ready-releasable" pool and of the much larger "reserve" SV pool (3Chen I.L. Lee C.Y. Virchows Arch. B Cell Pathol. 1970; 6: 318-325PubMed Google Scholar, 4Cull-Candy S.G. Fohlman J. Gustavsson D. Lullmann-Rauch R. Thesleff S. Neuroscience. 1976; 1: 175-180Crossref PubMed Scopus (115) Google Scholar, 5Gopalakrishnakone P. Hawgood B.J. Toxicon. 1984; 22: 791-804Crossref PubMed Scopus (50) Google Scholar, 6Dixon R.W. Harris J.B. Am. J. Pathol. 1999; 154: 447-455Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Montecucco C. Rossetto O. Trends Biochem. Sci. 2000; 25: 266-270Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 8Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (153) Google Scholar). Whereas depletion of the already docked SV was expected (16Zimmerberg J. Chernomordik L.V. Science. 2005; 310: 1626-1627Crossref PubMed Scopus (32) Google Scholar), more surprising is the depletion of the "reserve" SV pool (21Rizzoli S.O. Betz W. Nat. Rev. Neurosci. 2005; 6: 57-69Crossref PubMed Scopus (637) Google Scholar), since the membrane changes induced by LysoPC + FA are predicted to act predominantly on SV bound to the presynaptic membrane or which can rapidly enter in contact with its cytosolic leaflet. These SV have been defined as rapidly releasable vesicles, to be distinguished from the reserve pool of vesicles whose release is caused by the rise of the nerve terminal cytosolic Ca2+ concentration, which follows an extensive stimulation (21Rizzoli S.O. Betz W. Nat. Rev. Neurosci. 2005; 6: 57-69Crossref PubMed Scopus (637) Google Scholar, 22Ceccarelli B. Hurlbut W.P. Mauro A. J. Cell Biol. 1972; 54: 30-38Crossref PubMed Scopus (219) Google Scholar, 23Ushkaryov Y.A. Volynski K.E. Ashton A.C. Toxicon. 2004; 43: 527-542Crossref PubMed Scopus (108) Google Scholar).Here we have investigated the mechanism by which SPANs and the LysoPC + FA lipid mixture cause a massive SV release. Using primary cultures of different types of neurons, we found that the synaptic bulging induced by SPANs and LysoPC + FA is followed by a sustained increase in [Ca2+]i. At the same time, the mitochondrial membrane potential collapses. Upon SPAN or lipid mixture incubation, mitochondria change shape and appear to accumulate within bulges characterized by high calcium. Based on these findings, we present here a general model of nerve terminal blockade induced by SPANs or by the LysoPC + FA mixture that explains the release of both the recycling and the reserve pools of SV.EXPERIMENTAL PROCEDURESCell Cultures—Rat cerebellar granular neurons (CGNs) were prepared from 6-day-old Wistar rats as previously described (9Rigoni M. Schiavo G. Weston A.E. Caccin P. Allegrini F. Pennuto M. Valtorta F. Montecucco C. Rossetto O. J. Cell Sci. 2004; 117: 3561-3570Crossref PubMed Scopus (61) Google Scholar, 24Levi G. Aloisi F. Ciotti M.T. Gallo V. Brain Res. 1984; 290: 77-86Crossref PubMed Scopus (389) Google Scholar) and used 6-8 days after plating. Primary rat spinal motor neurons (SCMNs) were isolated from Sprague-Dawley (embryonic day 14) rat embryos and cultured following previously described protocols (25Arce V. Garces A. de Bovis B. Filippi P. Henderson C.E. Pettmann B. de Lapeyriere O. J. Neurosci. Res. 1999; 55: 119-126Crossref PubMed Scopus (133) Google Scholar, 26Bohnert S. Schiavo G. J. Biol. Chem. 2005; 280: 42336-42344Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). All experiments were performed using SCMNs differentiated for 5-8 days in vitro.Fura2-AM Loading and Image Acquisition—Neurons grown on 24 mm coverslips were incubated in complete medium with 3 μm Fura-2/AM and 0.02% pluronic (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C and then washed. To prevent Fura-2 leakage and sequestration, 250 μm sulfinpyrazone (Sigma) was present throughout the loading procedure and [Ca2+]i measurements. After loading, cells were bathed in Krebs-Ringer buffer (KRH, in the case of CGNs) or E4 medium (Extra-4, in the case of SCMNs). KRH composition was as follows: 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 2 mm CaCl2, 1.2 mm KH2PO4, 6 mm glucose, and 25 mm HEPES, pH 7.4. E4 composition was as follows: 120 mm NaCl, 3 mm KCl, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 10 mm HEPES, pH 7.4. Coverslips were mounted on a thermostated chamber (Medical System Corp.), placed on the stage of an inverted epifluorescence microscope (Axiovert 100 TV; Zeiss), equipped for single cell fluorescence measurements and imaging analysis (27Giacomello M. Barbiero L. Zatti G. Squitti R. Binetti G. Pozzan T. Fasolato C. Ghidoni R. Pizzo P. Neurobiol. Dis. 2005; 18: 638-648Crossref PubMed Scopus (65) Google Scholar). Samples were alternatively illuminated at 340 and 380 nm (every 15 s for 20-30 min after SPAN addition and every 10 s for 15-20 min after lipid exposure) through a ×40 oil immersion objective (numerical aperture 1.30; Zeiss), exposure times of 100 ms. Data were analyzed with MATLAB™ (The Math-Works, Natick, MA) and ImageJ version 1.35.Lipid Mixture Preparation, Neurotoxins, and Inhibitors—1-Myristoyl-lysophosphatidylcholine (mLysoPC; Sigma) and oleic acid (OA; Sigma) mixture (mLysoPC + OA) was prepared as previously described (8Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (153) Google Scholar). Notexin, taipoxin, and textilotoxin were purchased from Venom Supplies; β-bungarotoxin was from Sigma. Their purity was controlled by SDS-PAGE, and their PLA2 activity was measured with the Cayman secretory PLA2 assay kit. ω-Conotoxin MVIIC was from Latoxan, and nimodipine was from Tocris. Ionomycin and monensin were purchased from Calbiochem.Mitochondrial Imaging—SCMNs or CGNs were loaded with tetramethylrhodamine methyl ester (TMRE) (10 nm; Molecular Probes) in medium supplemented with the multidrug resistance pump inhibitor CsH (1.6 μm final concentration) (28Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar) for 30 min at 37 °C. Fluorescence images were acquired with a Leica AD MIRE3 inverted microscope, equipped with a Leica DC500 CCD camera, ×63 oil immersion objective (NA 1.4), using an exposure time of 50 ms. Data were collected using Leica FW4000 software and analyzed with Leica Deblur and ImageJ version 1.35. The average fluorescence of isolated mitochondria exposed to SPANs or the lipid mixture was recorded as a function of time. Intensity variations were expressed as a percentage of the initial value. Data represent the average of at least five regions of interest. For morphological staining of mitochondria, CGNs were incubated with 300 nm Mitotracker Red (Molecular Probes) for 15 min at 37 °C and then washed; images were acquired as above.Measurement of Cellular ATP—CGNs (300,000/13-mm coverslip) were transfected at 5 days in vitro with cytosolic luciferase with a standard Lipofectamine™2000 procedure, using 1.5 μg of DNA. Measurements of cell luminescence were performed 24 h after transfection in cells pretreated or not with 25 nm taipoxin or the mLysoPC + OA lipid mixture (25 μm for 15 min; n = 4 for each condition) as described (29Jouaville L.S. Pinton P. Bastianutto C. Rutter G. Rizzuto R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13087-13812Crossref Scopus (636) Google Scholar). Cells were constantly perfused with a modified Krebs-Ringer buffer (mKRB: 125 mm NaCl, 5 mm KCl, 1 mm Na3PO4, 1 mm MgCl2, 1mm CaCl2, 20 mm HEPES, 5.5 mm glucose, pH 7.4, at 37 °C) with 20 μm luciferin. Complete equilibration in the chamber with the new medium was obtained in 5 s. Under these conditions, the light output of a coverslip of transfected cells was in the range of 5,000-10,000 counts/s versus a background lower than 100 counts/s.In addition, the cellular ATP content was determined in CGNs, plated in a Petri dish (2 × 106 cells), at 7 days in vitro by a luciferin luciferase assay (ATP Bioluminescent Assay kit CLS II; Roche Applied Science) with an LKB Wallac 1250 luminometer. After treatment (mKRB, 25 μm lipid mixture for 15 min or 25 nm taipoxin for 60 min), cells were diluted in Tyrode buffer medium (145 mm NaCl, 5 mm KCl, 1 mm MgCl2, 0.5 mm NaH2PO4, 5 mm glucose, 15 mm Hepes, pH 7.7), and ATP was extracted by adding 0.3 m perchloroacetic acid. Samples were kept on ice for 5 min and centrifuged at 18,000 × g for 10 min. The pH of the supernatant was adjusted to 7.7 ± 0.1 with a buffer solution of KHCO3 (1 m) and KOH (1 m); the suspension was centrifuged at 18,000 × g for 5 min, and the supernatant was used for the analysis. The ATP content of each sample was estimated using the internal standard method in which 25 pmol of ATP were added twice to the assay. Analysis was repeated on triplicates for each condition. In both procedures for ATP content assay, the number of cells analyzed in each condition was equal, as determined by protein concentration measurement.Electron Microscopy—CGNs were plated onto 13-mm polylysin-coated Thermanox coverslips (Nunc) and, after 6 days in culture, exposed to either SPANs (6 nm for 1 h) or mLysoPC + OA (25 μm for 15 min). Samples were then fixed for 1 h at room temperature in 2.5% glutaraldeyde (EM grade; Applichem) in phosphate buffer. Cells were then washed repeatedly with phosphate buffer and subjected to secondary fixation with 1% osmium tetroxide for 30 min, followed by extensive washes. Samples were then dehydrated with ascending grades of ethanol, araldite-embebbed for 2 days, and stained with aqueous uranyl acetate and lead citrate. Sections were imaged in a Jeol 1010 electron microscope equipped with a 2,000 × 2,000-pixel digital camera (GATAN).RESULTSCytosolic [Ca2+]i Increases in Nerve Terminals Exposed to Snake Presynaptic PLA2 Neurotoxins—A rise in intrasynaptic Ca2+, such as that caused by prolonged nerve stimulation, is known to trigger SV mobilization from the reserve pool (21Rizzoli S.O. Betz W. Nat. Rev. Neurosci. 2005; 6: 57-69Crossref PubMed Scopus (637) Google Scholar, 22Ceccarelli B. Hurlbut W.P. Mauro A. J. Cell Biol. 1972; 54: 30-38Crossref PubMed Scopus (219) Google Scholar, 23Ushkaryov Y.A. Volynski K.E. Ashton A.C. Toxicon. 2004; 43: 527-542Crossref PubMed Scopus (108) Google Scholar). We thus hypothesized that the extensive SPAN-induced release of SV, leading to a complete impairment of the nerve terminal, could indeed be caused by a rise in [Ca2+]i. To test this possibility, we have used primary cultures of rat cerebellar granular cells and spinal cord motor neurons. Cells were loaded with the intracellular calcium indicator Fura-2 and then treated with either SPANs or LysoPC + FA mixture. Fig. 1A shows three video images of Fura-2 loaded CGNs taken at different time points after application of 25 nm taipoxin, a SPAN isolated from the venom of Oxyuranus scutellatus scutellatus. The images are pseudocolor-coded and report the ratio of the probe emission intensity at 510 nm following alternative excitation at 340 and 380 nm. The 340/380 ratio is a function of the intracellular Ca2+ concentration ([Ca2+]i), whereby an increase in [Ca2+]i is encoded as a shift of the pseudocolors from blue to red (video provided as supplemental Movie 1). A significant increase of the 340/380 ratio was observed in the bulges already at 10 min after toxin application, and the rise continued with time. In contrast, [Ca2+]i was unchanged in cell bodies. This selected localization of the Ca2+ rise is likely to result from the specific binding, and therefore action, of the toxin at nerve terminals. The larger surface to volume ratio of nerve terminals compared with the cell body is also to be considered (see below). Fig. 1C shows a quantitative analysis of the 340/380 fluorescence ratio changes measured at the level of different bulges and a cell body (Fig. 1B) as a function of time. Individual bulges differ significantly in their rate of [Ca2+]i rise, but the overall trend is similar. Very similar results were obtained in SCMNs and with structurally different snake PLA2 neurotoxins (i.e. notexin, β-bungarotoxin, and textilotoxin (not shown)), indicating that the results obtained here are not restricted to a single neuronal subtype or to the SPAN used. Incubation with control medium did not alter the level and distribution of intracellular Ca2+ (Fig. S1 and supplemental Movie 2).Lysophosphatidylcholine/Fatty Acid Mixture Causes an Increase in [Ca2+]i in Cultured Neurons—A similar result was obtained when CGNs were treated with the LysoPC + FA lipid mixture (Fig. 2A). Again, [Ca2+]i increased with time within the bulges induced by the lipid mixture, but in this case a rise was also detected at the level of cell bodies (black trace). The complete time course of the increase in [Ca2+]i in different bulges and in a cell body is shown in Fig. 2C. The ability of the lipid mixture to increase [Ca2+]i both at the level of bulges and cell bodies, compared with the restricted action of SPANs on neurites, was expected on the basis of the fact that LysoPC and FA can partition into the plasma membrane at any site, whereas SPANs bind specifically to the presynaptic membrane of nerve terminals (2Kini R.M. Venom Phospholipase A2 Enzymes: Structure, Function, and Mechanism. John Wiley & Sons, Inc., Chichester, UK1997Google Scholar, 30Rossetto O. Morbiato L. Caccin P. Rigoni M. Montecucco C. J. Neurochem. 2006; 97: 1534-1545Crossref PubMed Scopus (91) Google Scholar). On the other hand, the larger [Ca2+]i increase in the presynaptic regions compared with the cell body could also be influenced by the different surface/volume ratio of the two compartments. Any modification of the homeostatic mechanisms controlling Ca2+ at the plasma membrane is expected to cause substantial variations in the tiny cytosolic rim of the presynaptic membrane, whereas the same modification should take longer to affect the bulk [Ca2+]i in the cell body.FIGURE 2Increase of [Ca2+]i induced by an equimolar mixture of lysophosphatidylcholine and oleic acid (mLysoPC + OA) in cultured neurons. A, three pseudocolor images from a movie of CGNs loaded with Fura2-AM and treated with 25 μm mLysoPC + OA for 15 min. At variance from SPAN-treated neurons, [Ca2+]i appears to increase also in the cell body, although its increase is lower than within nerve bulges. Scale bar, 10 μm. B, fluorescent images from the experiment in A at t = 0 and at t = 15 min. C, analysis of the 340/380 fluorescence ratio of the selected boxed region as a function of time. Each trace shows the ratio changes of individual regions of interest. The phenomenon was highly reproducible in many experiments performed with different neuron preparations, and the one shown here is very representative of the ensemble.View Large Image Figure ViewerDownload Hi-res image Download (PPT)supplemental Fig. S2 shows that the component of the LysoPC + FA lipid mixture most effective in raising the intracellular Ca2+ concentration is LysoPC and that FA, when added alone, had no effect. However, the two molecules clearly synergize in order to produce the effect shown by the mixture in Fig. 2C and in Supplemental Fig. 2A. This finding parallels the neuroparalytic effects that the lipid mixture and the two lipids alone have on the neuromuscular junction of the hemidiaphragm preparation (8Rigoni M. Caccin P. Gschmeissner S. Koster G. Postle A.D. Rossetto O. Schiavo G. Montecucco C. Science. 2005; 310: 1678-1680Crossref PubMed Scopus (153) Google Scholar).Mechanisms of [Ca2+]i Increase inside Nerve Terminals Poisoned with Lysophosphatidylcholine/Fatty Acid Mixtures—These findings rise the question as to whether the Ca2+ increase triggered by the toxins or by the lipid mixture derives from Ca2+ influx from the extracellular medium or depends on Ca2+ mobilization from intracellular stores, or both. To address this question, the experiments shown in Fig. 2 were repeated in CGNs incubated with medium without CaCl2 and supplemented with 150 μm EGTA (Ca2+-free buffer) (Fig. 3B). SPANs could not be used in these tests as Ca2+ is strictly required for their phospholipase activity (2Kini R.M. Venom Phospholipase A2 Enzymes: Structure, Function, and Mechanism. John Wiley & Sons, Inc., Chichester, UK1997Google Scholar). Under these conditions, the lipid mixture caused a very small Ca2+ rise and at very late time points (Fig. 3, compare A and B). This Ca2+ increase is small and must derive from intracellular stores. In order to distinguish in our primary neuronal culture the different Ca2+-containing compartments on the basis of their luminal pH, we used a simple procedure (31Fasolato C. Zottini M. Clementi E. Zacchetti D. Meldolesi J. Pozzan T. J. Biol. Chem. 1991; 266: 20159-20167Abstract Full Text PDF PubMed Google Scholar). Cells were first treated with ionomycin in Ca2+-free buffer. This Ca2+ ionophore exchanges Ca2+ for 2H+ and can very effectively release the cation but only from organelles with neutral/alkaline pH lumena (primarily ER, mitochondria) (32Fasolato C. Pozzan T. J. Biol. Chem. 1989; 264: 19630-19636Abstract Full Text PDF PubMed Google Scholar). A very small and transient rise in [Ca2+]i was observed in control cells under these conditions (Fig. 3C). The acidic pH of other organelles (Golgi, secretory vesicles, and lysosomes) was then collapsed by the addition of monensin, and this induced a much larger increase in [Ca2+]i, indicating that most Ca2+ stores in these cells have an acidic luminal pH (Fig. 3C). Monensin was ineffective without ionomycin (not shown). The experiments presented in Fig. 3, D and E, demonstrate that the residual late rise of [Ca2+]i shown in Fig. 3B derives mainly from intracellular acidic compartments. Indeed, in the experiments presented in Fig. 3D, neurons were first preincubated with 1 μm ionomycin in Ca2+-free buffer and then exposed to 25 μm mLysoPC + OA. Under these conditions, the lipid mixture still elicited a late Ca2+ rise, indicating that it acted on stores insensitive to ionomycin. A complete suppression of the Ca2+ rise by mLysoPC + OA was achieved by the concomitant pretreatment with ionomycin and monensin (Fig. 3E).FIGURE 3Origin of [Ca2+]i increase within synaptic bulges induced by mLysoPC + OA. CGNs were exposed to 25 μm mLysoPC + OA for 15 min in the presence (A) or absence (B) of extracellular Ca2+ (Ca2+-free buffer: KRH without CaCl2 + EGTA 150 μm). C, control neurons were sequentially exposed to ionomycin and monensin (1 and 5 μm, respectively) in Ca2+-free buffer to determine the increase in [Ca2+]i contributed by the emptying of internal Ca2+ stores. D, cells were pretreated with 1 μm ionomycin (final concentration) in Ca2+-free buffer and then incubated with 25 μm mLysoPC + OA for 15 min in the same medium, whereas in E, cells were pretreated with monensin (5 μm, final concentration) in addition to ionomycin in Ca2+-free buffer and then exposed to the lipid mixture. Lipid mixture was added at t = 0. F, the result of the same experiment performed in C on cells pretreated with 25 μm mLysoPC + OA for 15 min in Ca2+-free buffer. These traces refer to experiments performed on the same cell culture and are representative of three different sets of experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Last, but not least, it should be noted that neurons exposed to the lipid mixture in Ca2+-free buffer showed a higher response to ionomycin with respect to control neurons, and most bulges did not respond at all to monensin (Fig. 3, compare F with C). This latter observation suggests that, following exposure to the lipid mixture, there is an intracellular partition of fatty acids, which are known to act similarly to monensin and to quench transmembrane proton gradients (33Wojtczak L. Schonfeld P. Biochim. Biophys. Acta. 1993; 1183: 41-57Crossref PubMed Scopus (314) Google Scholar). Intracellular acidic compartments, that under normal conditions cannot be emptied by ionomycin, become thus available to its action. Indeed, the same result was obtained when neurons were pretreated in Ca2+-free buffer with the protonophore carbonylcyanide p-trifluoromethoxyphenyl hydrazone, a well known quencher of transmembrane pH gradients (not shown).In order to investigate the nature of Ca2+ influx through the plasma membrane, neurons were incubated with the P/Q and N-type voltage-gated Ca2+ channel (VDCC) inhibitor ω-conotoxin MVIIC and with the L-channel inhibitor nimodipine. This treatment did not prevent the rise of [Ca2+]i caused by the lipid mixture, although a very small reduction in the rate of the Ca2+ increase was observed in some neurons treated with these VDCC inhibitors (Fig. 4, compare A and B). Using isolated neuromuscular junction preparations, whose VDCCs are well characterized and known to be effectively inhibited by ω-conotoxin MVIIC (34Wright C.E. Angus J.A. Br. J. Pharmacol. 1996; 119: 49-56Crossref PubMed Scopus (75) Google Scholar), we did not observe any change in the paralysis time induced by SPANs (not shown). Control experiments performed with neurons treated with these channel inhibitors and depolarized with 55 mm KCl showed about 50% reduction in the calcium influx (Fig. 4, compare C and D). Taken together, these results indicate that these synaptic VDCCs may contribute to the rise of [Ca2+]i induced by SPANs in primary cultures of neurons but are clearly dispensable. One possible explanation is that LysoPC + OA induces in cultured neurons a nonspecific increase in the membrane permeability for small molecules, as it was shown to occur for cultured vascular smooth muscle and endothelial cells (35Leung Y.M. Xion Y. Ou Y.J. Kwan C.Y. Life Sci. 1998; 63: 965-973Crossref PubMed Scopus
Abstract The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL 12α, also abbreviated as stromal‐derived factor‐1 ( SDF ‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL 12α acts via binding to the neuronal CXCR 4 receptor. A CXCL 12α‐neutralizing antibody or a specific CXCR 4 inhibitor strongly delays recovery from motor neuron degeneration in vivo . Recombinant CXCL 12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro . These findings indicate that the CXCL 12α‐ CXCR 4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.
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