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

Abstract

Snake pre-synaptic phospholipase A 2 neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the presynaptic 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 A 2 activity (PLA 2 ) hydrolysis of notexin, b-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 PLA 2 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 PLA 2 s appear to contribute little, if any, to the phospholipid hydrolysis measured here. Keywords: lysophospholipids, phospholipase A 2 activity, snake neurotoxins, toxicity. It is still impossible to analyse quantitatively the lipid products released by SPANs at the NMJ, their principal target in humans, for obvious technical reasons. However, it is well established that SPANs are highly toxic when injected into the CNS (Gandolfo et al. 1996;Kolko et al. 1999) and act on isolated brain-derived preparations (Rehm and Betz 1982;Nicholls et al. 1985;Rugolo et al. 1986). Therefore, data obtained with cultured CNS neurons were relevant and were as close to the in vivo situation as is currently experimentally possible. Methods are available to maintain many CNS neurons in primary cultures, but these cultures are mixtures of neurons and glial cells except for the granular neurons of the cerebellum (CGNs, cerebellar granule neurons), wherein cultures consist almost entirely of neurons (Lasher and Zaigon 1972;Levi et al. 1984). We have shown previously that SPANs are very active on these neurons (Rigoni et al. 2004). Using a pure neuronal culture is essential to achieve consistent and reliable MS analysis of changes in lipid compositions, as the presence of a large component of SPAN-resistant glial cells would dilute the changes induced by the toxins. Moreover, glial cells and neurons will have distinct and different compositions of membrane lipid, which will further complicate the MS analysis. CGN neurons are highly sensitive to SPANs and develop a well-defined bulging at axon and dendrite terminals within few minutes from toxin addition; such morphological alteration is accompanied by cytosolic calcium increase at nerve terminals and glutamate release from neurons (Rigoni et al. 2004(Rigoni et al. , 2007. These effects are mimicked by the additi...

Cellular Mechanisms of Action of Snake Phospholipase A2 Toxins

“…Once bound to the nerve terminal, snake PLA2 neurotoxins begin to hydrolyze phospholipids of the external layer, and this hydrolysis leads to a rapid accumulation of lysophospholipids and fatty acids. The extent and kinetics of phospholipid hydrolysis by the four toxins are similar, in contrast to the very different values of PLA2 activities obtained for the same toxins in in vitro PLA2 assays (Paoli et al 2009). This study helped to clarify the puzzling lack of correlation between the neurotoxicity in vivo and the PLA2 enzymatic activity measured in vitro by different methods (Montecucco and Rossetto 2000).…”

Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain

“…This rapid small ATP release has the features of a pool of vesicle-stored ATP that could be induced to exocytose by the Mt-I toxin. ATP is stored in vesicles in a variety of excitatory and nonexcitatory cells (36), and this mechanism would make PLA 2 snake myotoxins very similar to the snake presynaptic PLA 2 neurotoxins that induce exocytosis of synaptic vesicles from neurons via the action of lysophospholipids and fatty acids (37,38).…”

Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells

“…Tpx and β-Btx are representative of a large family of presynaptic snake neurotoxins endowed with phospholipase A2 activity (SPANs), which are important, although neglected, human pathogens (12)(13)(14)(15). We have contributed to the definition of their mechanism of action, which involves generation of lysophospholipids and fatty acids on the external layer of the plasma membrane (16,17). The mixture of these lipid products favors exocytosis of ready-to-release synaptic vesicles and mediates the rise of cytosolic Ca 2+ , presumably via transient lipid ion channels (16,18).…”