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.

FIGURE 1.

Intracellular localization of SPANs in different primary neuronal cultures. A, left panel: the intracellular distribution of Alexa568-Ntx in spinal cord motor neurons after a 5-min incubation at 37 °C (50 nm) is shown. Right panel: the corresponding bright field is shown. The insets show selected areas at higher magnitude. Scale bar = 10 μm. B, a similar intracellular distribution was found also in cerebellar granular neurons and with another SPAN, i.e. Alexa568-Tpx. Scale bar = 2 μm.

FIGURE 2.

Accumulation of SPAN staining within toxin-induced membrane enlargements with time. Spinal cord motor neurons were incubated with 50 nm Alexa568-Tpx for 30 min at 37 °C and washed, and images were acquired. Alexa568-Tpx fluorescent signal accumulates within the toxin-induced membrane bulges, which can be better appreciated in B. The same results were obtained in cerebellar granular neurons and after Alexa568-Ntx and fluoresceinated β-Btx exposure (not shown). Scale bar = 10 μmin A and 5 μmin B.

FIGURE 3.

Colocalization between SPANs and mitochondria. Spinal cord motor neurons were incubated with 50 nm Alexa568-Tpx and 5 nm nonyl acridine orange (NAO) for 30 min at 37 °C and washed, and images were acquired. A and B show the fluorescence images at the single excitation wavelengths. C shows the superimposition between the two emitted wavelengths; D represents the corresponding differential interference contrast (DIC). Scale bar = 5 μm.

FIGURE 4.

SPANs specifically localize within neurons. Alexa568-Tpx (25 nm) intracellular localization in primary cultures of spinal cord motor neurons after a 30 min incubation at 37 °C is confined to neuronal cells, as demonstrated by the lack of fluorescent signal in fibroblasts, whose mitochondria are stained well with nonyl acridine orange (NAO). Scale bar = 5 μm. DIC, differential interference contrast.

FIGURE 5.

Influence of Ntx and CsA on mitochondrial CRC. Purified mitochondria were resuspended in the presence of Calcium Green-5N as described under “Experimental Procedures,” and CRC was tested. At 60-s intervals, 10 μm Ca²⁺ pulses were added until occurrence of the permeability transition, which is marked by a fast release of the previously accumulated Ca²⁺. A and B, reduced mitochondrial CRC in Ntx-treated mitochondria (B, 1 nm) compared with control (A). C and D, the ability of CsA to delay mitochondrial Ca²⁺ release of control (C) and Ntx-treated mitochondria (D, 1 nm). AU, arbitrary units.

FIGURE 6.

Effect of SPANs and PLA₂ activity products at different concentrations on Ca²⁺ uptake of purified rat brain mitochondria. A, mitochondria were resuspended as described under “Experimental Procedures,” and CRC was tested in the presence of the four snake neurotoxins at high (20 nm, white bars) and low (1 nm, gray bars) concentrations. CRC decrease of toxin-treated mitochondria is concentration-dependent. B, the hydrolytic products of PLA₂ activity (mLysoPC and OA) were tested both alone or in an equimolar mixture (1 μm). Data represent mean CRC values of intoxicated mitochondria normalized to control samples (black bars). For each condition, trials were performed in triplicate.

FIGURE 7.

Delayed SPAN-induced PTP opening by CsA. Calcium retention capacity of control and SPAN-treated (20 nm) mitochondria was measured in the absence or presence of CsA (0.8 μm). CRC values of CsA-treated mitochondria (in the absence or presence of toxins, respectively) are normalized to those of samples not treated with CsA (dotted line). An increased threshold for PTP opening is observed for both control and SPAN-treated mitochondria. For each condition, trials were performed in triplicate.