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

Abstract

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...

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

“…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.…”

“…As almost all PS is confined inside cells, and snake PLA 2 s readily hydrolyse PS (Verkelij et al 1973;Napias and Heilbronn 1980;Rosenberg 1997), this finding is in keeping with the above conclusion. The present results do not conflict with previous observations that SPANs enter inside neurons (Pražnikar et al 2008;Rigoni et al 2008), but they indicate that the internalized toxins are not very active in phospholipid hydrolysis. However, if their hydrolytic activity is concentrated on defined organelles, even minor local phospholipid hydrolysis may be sufficient to alter dramatically the organelle physiology.…”

“…The present findings indicate that the cytosolic SPAN hydrolysis contributes little, if any, to the overall phospholipid hydrolysis. However, they do not exclude the possibility that limited localized hydrolysis may be of great relevance for intoxication as there are organelles very sensitive to the effect of LysoPLs and FA, such as the mitochondria to which Ntx, b-Btx and Tpx bind (Scorrano et al 2001;Rigoni et al 2008).In summary, the results presented here demonstrate the limited and the site-specific phospholipase activity of this panel of pre-synaptic neurotoxins. The discrepancy between the in vitro PLA 2 acitivities of the various SPANs, measured in a vesicular assay, with their activity against intact neuronal cells strongly suggests that the plasma membrane microenvironment of receptor-bound SPAN molecules imposes considerable constraints on their ability to hydrolyse PC.…”

“…The present findings indicate that the cytosolic SPAN hydrolysis contributes little, if any, to the overall phospholipid hydrolysis. However, they do not exclude the possibility that limited localized hydrolysis may be of great relevance for intoxication as there are organelles very sensitive to the effect of LysoPLs and FA, such as the mitochondria to which Ntx, b-Btx and Tpx bind (Scorrano et al 2001;Rigoni et al 2008).…”

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

“…The exact mechanism of their action is not yet fully understood, but it must include specific binding to receptor(s) on the presynaptic membrane and enzymatic activity . The binding to highly specific, and yet unknown, primary molecular targets of ß-neurotoxins (ß-ntxs) on the presynaptic membrane is most probably followed by entry of the toxin into the nerve cell (Logonder et al, 2009;Pražnikar et al, 2008;Rigoni et al, 2008). It has been proposed that different sPLA 2 -toxins exploit different internalization routes .…”