Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.

Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.vaccine adjuvants | danger associated molecular pattern | DAMP | inflammation V accine adjuvants are used to enhance immune responses toward coadministered antigens, thereby improving vaccine potency, immunological memory, or cross-protection (1, 2). Experimental adjuvants range from simple molecules such as calcium phosphate (CaPi) to very complex mixtures such as incomplete Freund's adjuvant (IFA), made of a water-in-oil emulsion, or complete Freund's adjuvant, which also includes killed Mycobacteria (3). However, for human vaccines, adjuvants of highly defined properties that combine efficacy with complete safety are needed; to date, very few compounds have been licensed. Some of the safest and most efficient adjuvants licensed for human use, such as aluminum hydroxide (alum) and the oil-in-water squalene-based emulsion MF59, have been empirically identified, and their mechanism of action is still not fully understood (4, 5). A better understanding of their mechanism of action is needed to develop improved adjuvants that further enhance vaccine efficacy. This is particularly important for diseases for which protective vaccines do not exist (6).An examination of the chemical nature of four major vaccine adjuvants (alum, CaPi, IFA, and MF59) suggested they could interact with the phospholipid bilayer of cell membranes via hydrogen bonding or ionic interactions with the head groups of phospholipids/glycolipids and/or via hydrophobic...

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Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/ A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gammaaminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.

Botulinum neurotoxins produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD50 values in the 1-5 ng/kg range, and are solely responsible for the pathophysiology of botulism. These metalloproteinases enter peripheral cholinergic nerve terminals and cleave proteins of the neuroexocytosis apparatus, causing a persistent, but reversible, inhibition of neurotransmitter release. They are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation, causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals. Keywords: botulinum neurotoxins, dystonia, muscle paralysis, neuroexocytosis, pharmaco-cosmetics, snake neurotoxins. Toxigenic anaerobic spore-forming bacteria of the genus Clostridium produce seven different botulinum neurotoxins (designated BoNT/A to G). Ingestion of BoNT-poisoned food causes an intoxication known as botulism, whose symptoms are the result of a generalized sustained blockade of acetylcholine release (ACh) at somatic and autonomic nerve terminals. They include diplopia, ptosis, dysphagia and paralysis of facial muscles, which progressively descends to the trunk, eventually involving the respiratory and visceral muscles. Dysfunctions of the autonomic nervous system include reduced salivation and lacrimation, nausea, vomiting and abdominal pain. Most patients survive botulism, but complete recovery is slow and may require mechanical ventilation. The recovery time is longer after BoNT/A intoxication than after BoNT/B and BoNT/E intoxications ( Presynaptic snake neurotoxins endowed with PLA2 activity (SPANs) are major components of the venom of four families of venomous snakes (Crotalidae, Elapidae, Hydrophiidae and Viperidae). These neurotoxins play a major role in envenomation of the prey (Harris 1997) by causing a persistent blockade of neurotransmitter release from nerve terminals (Kini 1997;Montecucco and Rossetto 2000;Schiavo et al. 2000). This process is more complicated than botulism, as several venom components are biologically active. However, almost invariably, most of the neurological signs and symptoms are due to the action of the SPANs. In fact, independently on the anatomical site of biting, patients are reported to have ptosis, diplopia, ophthalmoplegia, difficulty in swallowing, respiratory paralysis, abdominal pain and autonomic symptoms (Warrell et al. 1983;Theakston et al. 1990;Connolly et al. 1995;Kularatne 2002;Prasarnpun et al. 2005). The progression of paralysis in isolated nerve-muscle preparations exposed to SPANs is ...

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.

Snake presynaptic neurotoxins with phospholipase A 2 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 Ca 2؉ 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 A 2 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 (1). SPANs 3 play a major role in envenomation and cause a botulism-like flaccid paralysis with autonomic symptoms (2, 3). 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 (4). 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 SPANinduced irreversible bulging of nerve terminals in culture, was recently described (5). 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 (6). The SPAN-induced nerve bulges accumulate Ca 2ϩ , and, this event is accompanied by mitochondrial rounding and depolarization (7). The cytosolic [Ca 2ϩ ] increase could also trigger the activity of many Ca 2ϩ -activated hydrolases of nucleic acids, proteins, and lipids, all factors that could account for the pronounced degeneration of nerve terminals poisoned by .Previous studies indicated that SPANs can gain access to the cell interior. Indeed, fluorescein-conjugated ␤-Btx was found to rapidly e...

The mechanisms of action of four snake presynaptic phospholipase A2 neurotoxins were investigated in cultured neurons isolated from various parts of the rat brain. Strikingly, physiological concentrations of notexin, β-bungarotoxin, taipoxin or textilotoxin induced a dose-dependent formation of discrete bulges at various sites of neuronal projections. Neuronal bulging was paralleled by the redistribution of the two synaptic vesicle markers synaptophysin I (SypI) and vesicle-attached membrane protein 2 (VAMP2) to the bulges, and by the exposure of the luminal domain of synaptotagmin on the cell surface. These neurotoxins induced glutamate release from cultured neurons similarly to the known evoked release of acetylcholine from neuromuscular junctions. In addition, partial fragmentation of F-actin and neurofilaments was observed in neurons, but not in astrocytes. These findings indicate that these snake presynaptic neurotoxins act with by same mechanism and that the observed phenotype results from the fusion of synaptic vesicles with the plasma membrane not balanced by an adequate membrane retrieval. These changes closely resemble those occurring at neuromuscular junctions of intoxicated animals and fully qualify these primary neuronal cultures as pertinent models for studying the molecular mode of action of these neurotoxins.