Michela Rigoni

Equivalent Effects of Snake PLA2 Neurotoxins and Lysophospholipid-Fatty Acid Mixtures

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.

Presynaptic enzymatic neurotoxins.

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.

Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles

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.

Snake Phospholipase A2 Neurotoxins Enter Neurons, Bind Specifically to Mitochondria, and Open Their Transition Pores

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.

Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products

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.

Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins.

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 Ca(2+) 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 beta-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 Ca(2+) 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 Ca(2+) overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.

VAMP/synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes

Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N‐terminal 50 kDa light chain (L) domain, which is a zinc‐dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter‐containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.

Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion‐pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine > lysophosphatidylethanolamine > lysophosphatidic acid > lysophosphatidylserine > lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.

Mass spectrometry analysis of the phospholipase A2 activity of snake pre‐synaptic neurotoxins in cultured neurons

Snake pre‐synaptic phospholipase A2 neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre‐synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time–course of the phospholipase A2 activity (PLA2) hydrolysis of notexin, β‐bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA2 hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA2s appear to contribute little, if any, to the phospholipid hydrolysis measured here.