Calcium Influx and Mitochondrial Alterations at Synapses Exposed to Snake Neurotoxins or Their Phospholipid Hydrolysis Products

Michela Rigoni,Paola Pizzo,Giampietro Schiavo,Anne E Weston,Giancarlo Zatti,Paola Caccin,Ornella Rossetto,Tullio Pozzan,Cesare Montecucco

doi:10.1074/jbc.m610176200

Abstract

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.

Highlights

Toxins in general, and neurotoxins in particular, are invaluable tools in the molecular analysis of specific cellular processes, from the activation of G protein-coupled receptors to the characterization of the events controlling regulated exocytosis [1]
Whereas depletion of the already docked synaptic vesicles (SV) was expected [16], more surprising is the depletion of the “reserve” SV pool [21], since the membrane changes induced by LysoPC ϩ fatty acids (FA) are predicted to act predominantly on SV bound to the presynaptic membrane or which can rapidly enter in contact with its cytosolic leaflet
Cytosolic [Ca2ϩ]i Increases in Nerve Terminals Exposed to Snake Presynaptic phospholipase A2 (PLA2) Neurotoxins—A rise in intrasynaptic Ca2ϩ, such as that caused by prolonged nerve stimulation, is known to trigger SV mobilization from the reserve pool [21,22,23]

Summary

Introduction

Neurotoxins in particular, are invaluable tools in the molecular analysis of specific cellular processes, from the activation of G protein-coupled receptors to the characterization of the events controlling regulated exocytosis [1]. Using primary cultures of different types of neurons, we found that the synaptic bulging induced by SPANs and LysoPC ϩ FA is followed by a sustained increase in [Ca2ϩ]i.

Results

Conclusion