Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

Petrovič, Uroš; Šribar, Jernej; Pariš, Alenka; Rupnik, Marjan Slak; Kržan, Mojca; Vardjan, Nina; Gubenšek, Franc; Zorec, Robert; Križaj, Igor

doi:10.1016/j.bbrc.2004.09.144

Cited by 37 publications

(26 citation statements)

References 25 publications

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“…This indicates that the major site of phospholipid hydrolysis by all the four SPANs tested here is the pre-synaptic membrane surface. This result should be considered in conjunction with several reports that observed SPANs inside cells (Herkert et al 2001;Neco et al 2003;Petrovic et al 2004;Pražnikar et al 2008;Rigoni et al 2008). The present findings indicate that the cytosolic SPAN hydrolysis contributes little, if any, to the overall phospholipid hydrolysis.…”

Section: Discussionsupporting

confidence: 89%

“…NSC34 cells differentiated with retinoic acid and exhibited neuronal projections and intercellular contacts. This cell line has been previously used in studies of the biological activity of snake neurotoxins (Petrovic et al 2004;Pražnikar et al 2008;Caccin et al 2009). cells when compared with CGNs; their activities levelled off after 20 min with a maximum hydrolysis of only 2.5% of the cellular PC content; no increased hydrolysis was found using a high toxin concentration (30 nM).…”

Section: Limited Span Hydrolysis Of a Neuroblastoma Cell Linementioning

confidence: 99%

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

Paoli

Rigoni

Koster

et al. 2009

Journal of Neurochemistry

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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...

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Section: Discussionsupporting

confidence: 89%

Section: Limited Span Hydrolysis Of a Neuroblastoma Cell Linementioning

confidence: 99%

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

Paoli

Rigoni

Koster

et al. 2009

Journal of Neurochemistry

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“…␤-Btx and ammodytoxin A were detected within hippocampal neurons (12,14), ammodytoxin A was recently found also in NSC34 cells (15), and Tpx staining by antibody labeling was reported within chromaffin cells (13). It was also previously established that endocytosis inside acidic intracellular compartments, as is the case of botulinum neurotoxins, is not involved in the intoxication by SPANs (31).…”

Section: Discussionmentioning

confidence: 86%

“…By antibody labeling, Tpx was found to localize inside chromaffin cells in culture (13). Fluorophore-conjugated ammodytoxin A (a 14-kDa PLA 2 neurotoxin isolated from the venom of Vipera ammodytes) was detected in the nucleus of hippocampal neurons (14) and in the cytosol of undifferentiated NSC34 cells (15), a mouse neuroblastoma ϫ spinal cord hybrid cell line (16). In addition, Tpx was reported to bind an endoplasmic reticulum-located protein in vitro (17), and ammodytoxin A was found to bind a variety of cytosolic proteins (18,19) and R25, an integral protein of mitochondria (20).…”

mentioning

confidence: 99%

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

Rigoni

Paoli

Milanesi³

et al. 2008

Journal of Biological Chemistry

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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...

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“…They may enable the enzymatic activity of Atxs in a reducing environment containing insufficient (nanomolar) concentrations of calcium. In support of this idea are (1) the high degree of stability of AtxA under conditions resembling those in the cytosol of eukaryotic cells (Kovačič et al, 2009;Petrovič et al, 2004), (2) the additional and very significant structural stabilization and augmentation of sPLA 2 enzymatic activity by CaM (Kovačič et al, 2009(Kovačič et al, , 2010, (3) the transient cytosolic microdomains of high local calcium concentrations (~100 μM) (Meldolesi et al, 2002) or calcium entry through the damaged plasmalemma due to sPLA 2 action prior to internalization , and (4) the presence of anionic phospholipids (PS) on the cytosolic face of the plasma membrane and internal cellular organelles (Okeley & Gelb, 2004). In conclusion, Atxs are effective enzymes that bind strongly to and hydrolyze rapidly both anionic and zwitterionic phospholipid aggregates, including mammalian plasma membranes, presenting a broad combination of properties characteristic of different mammalian sPLA 2 s. The potential for the high enzymatic activity of Atxs appears to be at odds with their specific neurotoxic action.…”

Section: Basic Structural Interfacial Kinetic and Binding Propertiesmentioning

confidence: 64%

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

Petan¹,

Žnidaršič²,

Pungerčar³

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

Cited by 37 publications

References 25 publications

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

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

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

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

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Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

Cited by 37 publications

References 25 publications

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

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

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

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

Contact Info

Product

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

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