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

Rigoni, Michela; Caccin, Paola; Gschmeissner, Steve; Koster, Grielof; Postle, Anthony D.; Rossetto, Ornella; Schiavo, Giampietro; Montecucco, Cesare

doi:10.1126/science.1120640

Cited by 183 publications

(176 citation statements)

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“…These data support the idea that PA might promote fusion via a biophysical mechanism. In this regard, it should be noted that a recent study by Rigoni et al (36) found that the biological effects of snake phospholipase A2 neurotoxins, which induce massive exocytosis of neurotransmitters and depletion of synaptic vesicles, can be mimicked by the incubation of nerve terminals with an equimolar mixture of lysophospholipids and fatty acids. They concluded that local lipid changes such as the accumulation of positive curvaturepromoting lipids (such as LPC) in the outer leaflet of the plasma membrane or negative curvature-promoting lipids (such as PA) in the inner leaflet of the plasma membrane might be of functional significance in synaptic vesicle release.…”

Section: Discussionmentioning

confidence: 99%

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Zeniou-Meyer

Zabari

Ashery

et al. 2007

Journal of Biological Chemistry

191

228

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Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNAmediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion. Phosphatidic acid (PA)2 is a pleiotropic bioactive lipid that has been proposed to activate selected enzymes (1), recruit proteins to membrane surfaces (2), and serve as a substrate for the formation of other signaling lipids (3). Most intriguingly, PA has also been shown to promote negative curvature in bi-layer membranes due to its small polar head-group in combination with two fatty-acyl side chains (4). The bulk of cellular PA is synthesized via two different acylation pathways, the glycerol 3-phosphate pathway and the dihydroxy acetone phosphate pathway, which are named according to their respective precursors. However, PA is also produced via hydrolysis of phosphatidylcholine by phospholipase D (PLD) (5) on a much faster time scale, and this latter source is thought to underlie the dynamic regulation of PA that allows it to function as a signaling lipid in agonist-stimulated cell biological responses such as secretion and changes in cellular morphology.In mammals, the classic PLD family is composed of a pair of membrane-associated proteins, PLD1 and PLD2. Both PLD isoforms require phosphatidylinositol 4,5-bisphosphate for their enzymatic activity. However, whereas PLD2 exhibits relatively high basal activity in isolation, full activation of PLD1 requires its stimulation by small GTPases of the ADP-ribosylation factor (ARF), Rho and Ral families, and protein kinase C (3, 6). PLD enzymes have been proposed to be involved in a number of cellular processes, including cell growth and survival, cell differentiation, and vesicular trafficking (3)....

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Section: Discussionmentioning

confidence: 99%

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Zeniou-Meyer

Zabari

Ashery

et al. 2007

Journal of Biological Chemistry

191

228

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“…Principally, different pathways can generate PA: (1) hydrolysis of phospholipids via PLD (Bader and Vitale, 2009), (2) phosphorylation of diacylglycerol (DAG) by DAG kinase (Yang et al, 2011), and (3) acylation of lysophospholipids by lysophospholipid acyltransferases ). Lysophospholipids, substrates of the latter reaction, are generated under various physiological and pathophysiological situations at various cell organelles, e.g., the endoplasmic reticulum, Golgi apparatus, endosomes, and presynaptic terminals (de Figueiredo et al, 2001;Chambers et al, 2003;Ong et al, 2005;Rigoni et al, 2005;Rossetto and Montecucco, 2008;Schmidt and Brown, 2009;Oliveira et al, 2010;Vitale et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

Schwarz¹,

Natarajan²,

Kassas³

et al. 2011

J. Neurosci.

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Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.

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“…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 addition of an equimolar mixture of the PLA 2 hydrolysis products, lysophosphatidylcholine (LysoPC) + oleic acid, indicating that these molecules are the biochemical mediators of SPAN action (Rigoni et al 2005;Caccin et al 2006). Of the two lipid molecules, LysoPC was shown to be most effective (Caccin et al 2006(Caccin et al , 2009.…”

mentioning

confidence: 93%

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

Cited by 183 publications

References 19 publications

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

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

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

Cited by 183 publications

References 19 publications

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

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

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