Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

brain research 1552 (2014) 1–16

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Research Report

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons Nathalia Delazeri de Carvalhoa, Raphael CaioTamborelli Garciad, Adilson Kleber Ferreirab, Daniel Rodrigo Batistae, Antonio Carlos Cassolae, Durvanei Mariab, Ivo Lebrunb, Sylvia Mendes Carneiroc, Solange Castro Afechea, Tania Marcourakisd, Maria Regina Lopes Sandovala,n a

Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brasil 1500, São Paulo, SP 05503 900, Brazil Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503 900, Brazil c Laboratory of Cellular Biology, Butantan Institute, São Paulo, SP 05503 900, Brazil d Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP 05508 000, Brazil e Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil b

art i cle i nfo

ab st rac t

Article history:

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in

Accepted 9 January 2014

rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-

Available online 27 January 2014

9 toxins, an increase in free cytosolic Ca2þ and a reduction in mitochondrial transmem-

Keywords:

brane potential (ΔΨm) became evident and occurred prior to the morphological changes

Micrurus lemniscatus venom

and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in

Neurotoxic phospholipases A2

the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images

Calcium

and ultrastructural analysis by scanning and transmission electron microscopy showed

Mitochondrial transmembrane

that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage,

potential

membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the

hippocampal cell culture

formation of apoptotic bodies were observed. The most striking alteration observed in the

Neuronal cell death

electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic

n

Corresponding author. E-mail addresses: [email protected], [email protected] (M.R.L. Sandoval).

0006-8993/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2014.01.008

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Ca2þ and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

In the Americas, the Elapidae family is represented by coral snakes that comprise 120 species and subspecies belonging to the genera Micruroides, Leptomicrurus and Micrurus (McDowell, 1987; Roze, 1996). Micrurus is the most representative genus as far as abundance and diversity are concerned, with a great number of species found in South America and Southern United States. However, the biochemistry and pharmacology of components from coral snake venoms have not been thoroughly studied so far. Recently, the Brazilian Ministry of Health has considered ofidism as a neglectful disease. Accidents involving Micrurus ssp. are considered a medical emergency due to the high risk of death which is usually due to respiratory failure. However, there are rare reports in literature aimed at understanding the mechanisms involved in the degeneration processes induced by pre and postsynaptic neurotoxins present in coral snake venoms. On the other side, venoms belonging to Elapidae family from the Old World have been extensively studied as sources of presynaptic phospholipases A2 (PLA2s) and postsynaptic neurotoxins in peripheral and central nervous system (Clapp et al., 1995; Da Silva Junior et al., 1991; Dorandeu et al., 2002; Khow et al., 2003; Kost et al., 2005). Snake presynaptic neurotoxins endowed with PLA2 activity, which are prominent components of elapid venoms, play an important role in envenomation of the prey by causing a persistent blockade of the neuromuscular transmission (Kini, 1997; Schiavo et al., 2000). Death usually follows due to respiratory paralysis with little or no damage to other organs (Connolly et al., 1995; Prasarnpun et al., 2005). The toxicity of PLA2s from Micrurus venoms in the peripheral nervous system showed that a presynaptic PLA2 isolated from Micrurus dumerilli venom also evoked neuromuscular blockade in vertebrate nerve–muscle preparations and triphasic changes in spontaneous neurotransmitter release from motor neuron terminals (Belo et al., 2005). Neurotoxic PLA2s have been isolated mainly from the venom of the two families of venomous snakes Elapidae and Viperidae (Lambeau and Lazdunski, 1999). Secreted PLA2s (sPLA2s) are small proteins of 13–19 kDa that comprise 17 groups (Ho et al., 2001; Rouault et al., 2003), however, neurotoxic sPLA2 have been found only in groups IA, IIA and IIIA (Kini, 1997; Pungercar and Krizaj, 2007). Those from elapids venoms belong to the group IA and no mammalian equivalent has been described so far (Lambeau and Lazdunski, 1999). Secreted PLA2s are enzymes that catalyze the hydrolysis of the sn-2 ester bond in 1, 2-diacyl-sn-3phosphoglycerides of glycerophospholipids to produce free fatty acids and lysophospholipids. Concerning the mechanisms of action of the sPLA2sneurotoxins, recent studies have made major contributions to understand their molecular mechanisms. Thus, data

from literature has claimed that snake presynaptic PLA2 neurotoxins block nerve terminals by binding to neuronal membranes receptors (Lambeau and Lazdunski, 1999) and by catalyzing phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate, with release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. At a later stage, other changes in nerve terminals take place such as increased plasma membrane permeability to ions and internalization of the toxins, markedly impairing the functional and structural integrity of nerve terminals (Pungercar and Krizaj, 2007; Rossetto et al., 2006; Rosso et al., 1996). Due to the complexity of the anatomically fine structure of the neuromuscular junction (NMJ) and to the inherent limited possibility of experimental approach at the molecular level of this tissue preparation, further progress has been achieved through the utilization of primary neuronal cultures from the central nervous system (Herkert et al., 2001; Paoli et al., 2009; Rigoni et al., 2004) and NSC34 cell line, a very similar model to actual motoneuron (Pungercar and Krizaj, 2007). Despite the target of neurotoxins sPLA2 is NMJ, it is well established that they are highly toxic when injected into the central nervous system (CNS) (Gandolfo et al., 1996; Kolko et al., 1999) or added to neuronal cell cultures (Bazan et al., 1995; Bazan, 1998; Kolko et al., 1999). Therefore, data obtained with cultured CNS neurons were relevant and it is accepted that replication would not be complete but it has contributed to the knowledge of the basic molecular events (Paoli et al., 2009; Pungercar and Krizaj, 2007). Considering neuronal injury, β-bungarotoxin (β-BuTX), the most investigated presynaptic PLA2 neurotoxin isolated from the elapid snake Bungarus ssp. venom, induces widespread neuronal cell death throughout the mammalian CNS (Francis et al., 1997). Moreover, intracerebroventricular (i.c.v.) injection of Naja mocambique-PLA2 provoked extensive cortical and subcortical injury to forebrain neurons and fiber pathway lesions (Brazil, 1972). This neuronal injury was also observed after i.c.v. injection of other PLA2 neurotoxins as paradoxin (Masroori et al., 2010), crotoxin (Masroori et al., 2010), β-bungarotoxin (Shakhman et al., 2003) and N. mocambique PLA2 (Clapp et al., 1995). In addition, we previously investigated the neurotoxicity of four sPLA2s toxins (Mlx-8, 9, 11 and 12) isolated from the venom of the elapid snake Micrurus lemniscatus after microinjection into the brain (Oliveira et al., 2008). This study showed the presence of isolated and clustered spikes on EEG records, behavioral alterations characterized mainly by forelimb clonus, compulsive scratching and severe neuronal damage. Thus, the present work was designed to investigate in detail the neurotoxic effects of two sPLA2 toxins (Mlx-8 and Mlx-9) isolated from M. lemniscatus venom on cultured primary hippocampal neurons. The integrity of mitochondria through the transmembrane potential


determination and the intracellular calcium measurement were evaluated. The cell viability was analyzed by MTT and LDH assays. Morphological approach by transmission and scanning electron microscopy and fluorescence dyes was also used. Our data indicate that the early effects induced by the sPLA2s toxins were an increase in free cytosolic Ca2þ and a loss of mitochondrial transmembrane potential, and these actions of the toxins appear to be critical events involved in the neurotoxicity of Elapid Coral snake venom sPLA2s.

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increased from 0.3270.03 to 0.4970.13 (n ¼14). For Mlx-9 at concentration of 7.4 nM the results are shown in the right panel. For this toxin the delay in significant effects on intracellular Ca2þ concentration is higher. For seventeen different cells the ratios are 0.6170.11 in the control period and 0.8770.30 under the continuous effect of the toxin, after 60 min of observation and after 40 minutes of toxin application, respectively.

2.2. Measurement of mitochondrial transmembrane potential (ΔΨm)

2.

Results

2.1. Measurements of free intracellular Ca2þ concentrations by fluorescent indicator Fig.1 are average ratios of fluorescence at 510 nm emitted by cells loaded with fura-2, excited at wavelengths of 340 and 387 nm, against time. Left panels show the effect of Mlx-8 at concentration of 7.4 nM. After a control period of 20 min toxin was applied. With a very short delay the ratio starts to increase, reflecting a rise in free cytosolic Ca2þ concentration. Sixty minutes after starting the observations, the ratio has

Mitochondrial activity maintains a highly negative potential across their inner membrane which is essential for mitochondrial function and cell viability. Because of the large electrochemical Hþ gradient across the mitochondrial inner membrane, Rhodamine 123 accumulates in the mitochondrial matrix according to the ΔΨm. The present study demonstrated that the cells exposed for 1 or 3 h to 7.4 nM Mlx-8 (Fig. 2) or Mlx-9 (Fig. 3) show a reduction on the ΔΨm whereas the control cells presented a high ΔΨm. The preincubation of the cells with Rhodamine 123 and the analysis of the data by flow cytometry showed a percentage of no

Fig. 1 – Time series of ratios of fluorescence emitted by Fura-2 excited at two wavelengths (340 nm and 387 nm). The fluorescence ratios are due to modifications in free Ca2þ in the cytosol of cells defined in the images. When indicated, toxins (Mlx-8 or Mlx-9) in the concentration of 7.4 nM were applied to the cells. The histograms show average fluorescence ratios in control periods and under the specified toxin for a period of 40 min. The complete period of observation lasts 60 min.

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Fig. 2 – Mlx-8 neurotoxin (7.4 nM) reduces the mitochondrial transmembrane potential (Δψm) in cultured hippocampal neurons. Representative histograms obtained from FACS analysis of hippocampal neurons loaded with Rho123. The figure shows an overlay of histograms from samples analyzed by flow cytometry. Note that Mlx-8 (M2) reduces the percentage of cells with high Δψm (M1) in 1 h (A) and 3 h (B). Indeed, the depolarization of mitochondria is demonstrated by a reduction of fluorescence intensity in hippocampal neurons. The data are the means7SD ***po0.001 from at least three independent experiments.

Fig. 3 – Mlx-9 neurotoxin (7.4 nM) reduces the mitochondrial transmembrane potential (Δψm) in cultured hippocampal neurons. Representative histograms obtained from FACS analysis of hippocampal neurons loaded with Rho123. The figure shows an overlay of histograms from samples analyzed by flow cytometry. Note that Mlx-9 (M2) reduces the percentage of cells with high Δψm (M1) in 1 h (A) and 3 h (B). Indeed, the depolarization of mitochondria is demonstrated by a reduction of fluorescence intensity in hippocampal neurons. The data are the means7SD **po0.001 from at least three independent experiments.

viable cells after exposure to Mlx-8 for 1 or 3 h of 3.65% and 26.30%, respectively (Fig. 2) and after Mlx-9 exposure for 1 or 3 h of 2.74% and 38.99%, respectively (Fig. 3). The ability to reduce the ΔΨm was more accentuated in the cells exposed to Mlx-9.

2.3. Effects of Mlx-8 and Mlx-9 exposures on the survival of cultured hippocampal cells The MTT assay indicates changes in metabolic activity associated to cell survival (cell viability). The neuronal


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Fig. 4 – Neurotoxic effects caused by exposure of cultured hippocampal neurons to the two concentrations of Mlx-8 or Mlx-9 for 3 h (A), 6 h (B), 12 h (C) or 24 h (D). The results are represented as a percentage of MTT reduction, compared to control conditions (no toxins), and represent the means7S.E.M. of at least three independent experiments performed in triplicate. *Po0.05 – Significantly different from control (one-way ANOVA followed by Newman–Keuls test).

Fig. 5 – The MTT curves after 3, 6, 12 and 24 h of exposure to neurotoxins Mlx-8 (A) or Mlx-9 (B) in two concentrations (0.74 or 7.4 nM). Here, 250 mM KCl was used as the positive control. The results are represented as percentage of MTT reduction, compared to control conditions (no toxins), and represent the means7 S.E.M. of at least three independent assays performed in triplicate. *po0.005 compared to control (ANOVA and Newman–Keuls multiple comparison).

viability determination by MTT test after cell culture incubation with Mlx-8 showed a decrease in the mitochondrial metabolism. The neuronal injury was only evident at 24 h of exposure to Mlx-8 in the two concentrations used (cell survival: 0.74 nM: 73.3774.43%; 7.4 nM: 61.0975.55%) (Fig. 4D). The neuronal viability determination after cell incubation with Mlx-9 (0.74 or 7.4 nM) showed that there was a decrease in the mitochondrial metabolism that was evaluated through the percentage of cell survival after 6 h (0.74 nM: 77.0877.59% and 7.4nM: 69.0577.73% (Fig. 4B)), 12 h (0.74 nM: 71.8777.16%

and 7.4 nM: 44.3775.94% (Fig. 4C)) and 24 h (0.74 nM: 73.1273.27% and 7.4 nM: 70.0073.24% (Fig. 4D)) of exposure to the toxin. A clear dose-dependent reduction in cell viability was not observed. However, a time-dependent effect on cell viability of the hippocampal cultures incubated with Mlx-8 or Mlx-9 was clearly shown (Fig. 5). The toxic effect caused by Mlx-9 was even higher than for Mlx-8. The results obtained with the MTT assay, were confirmed by the LDH determination. LDH is released from cells as a result of loss of plasma membrane integrity. LDH was

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disintegrated or inexistent nucleus and the presence of cell debris (Fig. 7E and F). Necrotic cells and cell debris were present mainly in hippocampal neurons incubated with 74 nM of Mlx-9 toxin.

2.4.2.

Fig. 6 – Citotoxicity assay evaluated by LDH release to culture medium 24 h of exposure to Mlx-8 or Mlx-9 in two concentrations (0.74 or 7.4 nM) (n ¼three wells per group in each of the three independent assays). Here, 250mM KCl was used as the positive control. ***po0.001 compared to control (ANOVA and Newman–Keuls multiple comparison).

assayed only after 24 h of exposure to both toxins because at this time an evident neuronal death was observed. Increased LDH release to the medium was only observed when the hippocampal neurons were exposed to the Mlx-9 toxin for 24 h (Fig. 6) only with the highest concentration (7.4 nM) (31.3373.5 pmol of NADH consumption/min/cell, po0.001).

2.4. Morphological features of hippocampal neurons exposed to Mlx-8 or Mlx-9 2.4.1.

Ethidium bromide staining

We examined the neuronal morphology by confocal fluorescence microscopy after 3, 6, 12 or 24 h of exposure of hippocampal neurons to three different concentrations of Mlx-8 or Mlx-9 (0.74, 7.4 or 74 nM). The qualitative analysis of the data showed that Mlx-8, as well as Mlx-9, induce characteristic alterations of cell death as revealed by staining of the cells with the fluorescent dye ethidium bromide. Features of apoptotic, necrotic or viable cells were examined by employing the fluorescent dyes acridine orange (membrane permeable) and ethidium bromide (membrane impermeable), a method used to analyze chromatin condensation and/or fragmentation (Fig. 7). Viable cells were uniformly stained in green, without nuclear morphological alteration (Fig. 7A and B). The neurotoxicity was associated with the appearance of the hallmarks of apoptosis, such as early apoptotic cells that stained green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells also incorporated ethidium bromide and therefore stained orange but, in contrast to necrotic cells, the late apoptotic cells showed condensed and often fragmented nuclei (Fig. 7C and D). Necrotic cells (red arrow) were also identified and intensely and uniformly stained in orange or red, showing an increased cytoplasmic volume and a completely

Ultrastructural analysis

We examined the neuronal morphology after 24 h exposure of hippocampal neurons to Mlx-8 or Mlx-9 toxin (7.4 or 74 nM) by transmission electron microscopy. Control cultures presented well preserved neurons and a dense network of amielinic fibers (Fig. 8 A–C); the cytoplasm contained many free polyribosomes, profiles of rough and smooth endoplasmic reticulum, mitochondria, microtubules and Golgi complex (Fig. 8D). Occasionally neurons with more electrondense nuclei and cytoplasm were seen, but they were not degenerated (Fig. 8B). Synaptic buttons with microtubules and numerous microvesicles were frequently observed on cell bodies, and on the surrounding processes (Fig. 8E). Cultures incubated with Mlx-8 or Mlx-9 revealed diverse ultrastructural features of neuron and neuropile injury and these varied from mild alterations to severe disintegration (Figs. 9 and 10). Cultures exposed to Mlx-9 presented the more severe degenerative alterations. The most striking alteration was the fragmentation and rarefaction of the neuron processes network (Fig. 9 B, D, and E). Culture areas practically devoid of neuron processes were also observed (Fig. 9 F). Untypical and degenerated terminal synapses, cell debris and apoptotic bodies can be observed among the fragmented fibers (Fig. 9C and I). Among the diverse types of neurons modifications the following features can be observed: cells with cytoplasm filled with vacuoles containing filamentous or membranous cell remnants (Fig. 9A and D); cytoplasm with numerous swollen mitochondrias and dilated Golgi cisterns (Fig. 9H); neurons with electron lucid nuclei and cytoplasmic organelles largely confined in some regions leaving extensive fine granular homogeneous cytoplasmic areas (Fig. 9G); irregular nuclei with marginally condensed chromatin displaying prominent pore annuli (Fig. 10A); neurons with shrunken piknotic nuclei (Fig. 9E); neurons with aggregation of chromatin in the nuclear membrane, and severe cytoplasmic disintegration (Fig. 4E); apoptotic cells (Fig. 10F). Dendritic processes presenting vacuolated mitochondria and clusters of altered pyknotic or membrane whorled mitochondria were also occasionally observed (Fig. 10B). The cytoplasm of some degenerative neurons exhibited numerous microvilli-like projections in the process of extrusion from the cytoplasm (Fig. 10C). Phagocytic cells with internalized cell debris were also seen (Fig. 10D). With the aim to observe the toxicity of a higher concentration (74 nM) of both toxins, we employed it in the morphological experiments (Sections 2.4.1. and 2.4.2). However, since this concentration showed a high toxicity it was not used in the other experiments. In this regard, necrosis and intensity cell debris were observed.

2.4.3.

Scanning electron microscopy

Degenerative alteration in hippocampal neurons following 24 h of Mlx-8 and Mlx-9 (7.4 nM) was observed. Severe shrinkage and condensation of cell bodies, extensive blebbing and axonal thinning and disruption were observed in all


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Fig. 7 – Representative micrographs from confocal fluorescence microscopy of cultured hippocampal neurons treated with sPLA2s isolated from Micrurus lemniscatus venom for 24 h co-labeled by acridine orange, AO, and ethidium bromide, EB. AO (green) penetrates even if the cell membrane has no damage; on the other hand, EB (red) only interacts with DNA if the cell membrane has an injury. Orange color may happen due to the potency of EB color, which enshrouds AO coloration. (A and B) Control cultures; (C and D) 7.4 nM and 74 nM Mlx-8, respectively; (E and F) 7.4 and 74 nM Mlx-9, respectively. Control group shows normal hippocampal cells (A and B). Morphological changes can be observed after Mlx-8 and Mlx-9 treatments (C–F) White arrows show apoptotic processes, while red arrows show necrotic processes.

treatments. These treatments resulted in cell swelling and membrane rupture (Fig. 11).

3.

Discussion

Despite numerous studies about the activity of sPLA2 neurotoxins from the venom of Old World elapid snakes, the effects of sPLA2s from the venom of the Brazilian coral snakes of the genus Micrurus ssp. are poorly investigated. To contribute to the understanding of the actions of these phospholipases, we have investigated the effects of Mlx-8 and Mlx-9 toxins isolated from M. lemniscatus venom. This is the first study

performed on the hippocampal cell culture model showing effects and mechanisms of actions of these sPLA2s. The early events that took place after the exposure of hippocampal neurons to Mlx-8 and Mlx-9 were an increase in free cytosolic Ca2þ and a decrease in the ΔΨm, suggesting that these actions of the toxins played a role in inducing, at least in part, the cell death. The cell viability compromising was observed by the reduction in the mitochondrial dehydrogenase activity and in the ΔΨm showing that mitochondrial impairment could be a cause of the observed cell death. Loss of ΔΨm is a crucial dysfunction of mitochondria, because loss of ΔΨm induces release of apoptogenic factors into cytoplasm and decrease in

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Fig. 8 – Ultrastructure of rat hippocampal neuron cultures from Control group. (A) Neuron presents a round nucleus (N) with dispersed chromatin. Well preserved cytoplasmic regions of other neuron (C) and initial dendritic processes (D). (B) Neuron with electron dense nucleus N, and processes surrounding it. (C) Neuropil area showing dense network of neuron processes. (D) Cytoplasmic area of a neuron with mitochondria (M), Golgi area (G), polyribossomes, microtubules and cisterns of rough endoplasmic reticulum. (E) Pre-synaptic terminal (S) and cytoplasmic area of a neuron (C).

ATP generation, leading to cell death (El Hakim et al., 2011; Wong et al., 2007). In fact, electron microscope imaging frequently displays mitochondria swelling and mitochondria degeneration. At early times of exposure to the toxins, there is an increase in free cytosolic Ca2þ concentration ([Ca2þ]i) that coincides with the decrease in mitochondrial ΔΨm. With the data available we cannot decide what is the cause or what is the effect: the primary effect could be a change in plasma membrane permeability to the ion, leading to the effect on mitochondria, as well as an effect on mitochondria, impairing ATP synthesis and consequently increasing the cytosolic free Ca2þ due to slowing down of the membrane pumps. As a speculation the first cause/effect relationship above, seems more likely, considering that the peptides hardly can enter the cell and that these molecules are phospholipases. Due to their enzymatic activity they could well change the bilayer structure, its Ca2þ permeability, leading primarily to a cytosolic free-Ca2þ accumulation. However, data in the literature evidence an opposite cause–effect relationship (see below).

Independently of the cause/effect relationship, however, both, early increase in free cytosolic Ca2þ concentration and mitochondrial function impairment would lead to gross structural changes and, at the end, to the cell death. Regarding the precocious increase in the free cytosolic Ca2þ there is a difference in the effect of the two toxins: the delay in the onset of changes in the ion concentration is longer for Mlx-9. We have currently no explanation for the fact. Our data are consistent with the studies performed with βbungarotoxin that show a decrease in the ΔΨm and ATP depletion caused by an excessive increase in the [Ca2þ]I (Tseng and Lin-Shiau, 2003). These results are in accordance with the findings of Rigoni et al. (2008) which indicate that sPLA2-neurotoxins play a direct role in the ensuing mitochondrial degeneration. They and other authors (Praznikar et al., 2008) demonstrated that β-bungarotoxin, taipoxin, notexin and textilotoxin are able to enter neurons and bind specifically to mitochondria leading to mitochondria depolarization and undergoing a profound shape change within regions of nerve terminals that swells and form round bulges


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Fig. 9 – Ultrastructural effects of Mlx-8 (A–C) and Mlx-9 (D–I) in rat hippocampal neuron cultures. In these figures we can observe diverse kind of neuron degeneration: vacuolization of the cytoplasm and swelling of cytoplasmic organelles (A, D, and H), segregation of cytoplasmic organelles (G), and apoptotic condensation of nuclear chromatin (E). The neuropil areas show fragmented network of neurofibers (neuron processes) (B, D, and E), apoptotic and dark cell debris (arrows in A and C), and vacuolated (C) or disrupted (I) synaptical boutons (S). There are culture areas practically devoid of neuron fibers (F). N¼ nucleus.

of the plasma membrane. It is important to state that both elapid (beta-bungarotoxin, taipoxin, notexin, textilotoxin) and viperid (ammodytoxin) sPLA2-neurotoxins are actually internalized into neurons during the neurotoxic action (Praznikar et al., 2008, Rigoni et al., 2008) Regarding our toxins a direct effect on mitochondria after crossing the plasma membrane through some transporting process awaits for empirical evidences. Both neurotoxins have a time-dependent neurotoxic effect on cultured hippocampal neurons. Mlx-9 reduced the cell viability after 6–12 h and the maximal toxicity was achieved at 24 h. Mlx-8 required a longer time to produce neurotoxic effect and this was observed only 24 h after the treatment. These data suggest that Mlx-9 is more toxic than Mlx-8 in rat primary hippocampal cell culture. Despite the widespread use of the apoptosis-versusnecrosis paradigm, there is an increasing awareness of the complexity of processes occurring in dying cells that lead to the outcome of death (Fink and Cookson, 2005). Multiple types of death can be observed simultaneously in tissues or

cell cultures exposed to the same stimulus (Ankarcrona et al., 1995). The local intensity of a particular initial insult may influence the mechanism of demise taken by individual cells in a population (Bonfoco et al., 1995). Taking into consideration the morphological analysis, our data showed neurons in process of apoptosis, necrosis and autophagy. Inspection using fluorescent images of staining with ethidium bromide and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed was the fragmentation and rarefaction of the neuron processes network. Untypical and degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. In the cytoplasm, numerous large vacuoles were noted, as well as swollen mitochondria and dilated Golgi.

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Fig. 10 – Ultrastructural effects of Mlx-8 (B and E) and Mlx-9 (A, C, D, and F) in rat hippocampal neuron cultures. (A) Shrunk neuron nucleus with clumped heterochromatin displaying annuli of the nuclear envelope (arrows). (B) dendritic process with numerous mitochondria in degenerative process; arrows point to mitochondria double membrane. (C) Neuron cell releasing numerous cytoplasmic microvillus-like projections. (D) phagocytic cell (P) showing an engulfment of a cell in degeneration and a phagocytic cell with numerous phagocytic vacuoles in the cytoplasm. (E) Neuron cell presenting nucleus (N) with chromatin aggregation at the nuclear membrane, and loss of cytoplasmic organelles and membranes. (F) Neuron cell showing pyknotic nucleus and degeneration of cytoplasmic organelles.

Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to the higher doses of the PLA2-neurotoxins and this finding corroborates with the increase in LDH release observed in the present work. These results are in accordance to the morphological features observed during the 24 h-exposure for the highest concentration of Mlx-9, in which could be viewed a higher amount of cellular debris compared to the lower dose. Our results corroborate with those presented by Kolko et al. (2003) which showed that sPLA2-OS2 toxin induces an increase in LDH activity in cortical neurons culture, suggesting that one of the mechanisms underlying neuronal death is necrosis.

Apoptosis, a highly regulated process of cell death, has been observed when neuronal cultures are exposed to elapid snake venoms sPLA2 as β-bungarotoxin (Ankarcrona et al., 1995; Shakhman et al., 2003; Tseng and Lin-Shiau, 2002, 2003). Autophagy is an evolutionary conserved and highly regulated homeostatic process in which cytoplasmic macromolecules and organelles are degraded by the lysosomal system. Neurons exposed to Mlx-8 or Mlx-9 toxins showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presenting mitophagy-like structures that are typical of autophagy. The excess of activation of the autophagy


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Fig. 11 – Scanning electron micrograph of cultured hippocampal neurons. Neurons grown on glass coverslips were examined by scanning electron microscopy (SEM). Scanning electron microscope images of cultured hippocampal cells exposure to Mlx8 or Mlx-9 for 24 h revealed a number of morphologic alterations. (A) Control culture. Neurons exhibiting a round, smooth cell body with neuritic processes; (B and C) degenerative alteration in hippocampal neurons following 24 h of Mlx-8 and Mlx-9 (7.4 nM), respectively. Severe shrinkage of the cell body, and condensation of cell bodies, extensive blebbing of the membrane surface, axonal thinning, loss of neuritic processes and disruption were observed in all treatments. These treatments resulted in cell swelling and membrane rupture (see arrow). Bar¼1/um.

pathway may induce cell death directly by causing the collapse of cellular functions as a result of cellular toxicity.

Moreover, autophagy may accelerate the apoptotic cell destruction by initiating the self-digestion of the cells

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destined to die. Paradoxically, autophagy may also represent a form of non apoptotic cell death, which has been implicated in the demise of a variety of cell types during development, tissue homeostasis, and in response to toxic stimulus (Lockshin and Zakeri, 2004). Transmission electron microscopy revealed the formation of autophagosomes in cultured hippocampal neurons after Mlx-8 or Mlx-9 exposure. The biogenesis of autophagosomes begins with the formation of a pre-autophagosome, a membranous structure containing all the proteins necessary for the creation of an autophagosome, assembled from membranes of preexisting organelles (endoplasmic reticulum, Golgi, mitochondria, and plasma membrane). However, the relationship of autophagy with neuronal survival or death remains unclear. The autophagy pathways may play important roles in neuronal survival and neuronal function under physiological and pathological conditions (Lee, 2012; Son et al., 2012). Recent studies have shown that in several diseases autophagy is induced and activated to protect cells against cell death by removing toxic components in damaged cells. Moreover, Lin et al. (2012) showed that the induction of autophagy may constitute a cytoprotective response after methamphetamine-induced neurotoxicity. The morphological data observed in the present work can be compared with those from β-bungarotoxin once it induced a hybrid cell death characterized by apoptosis and necrosis in cell culture with the presence of apoptotic bodies and loss of the plasmatic membrane integrity (Montecucco et al., 2008; Rigoni, 2004; Rigoni et al., 2008). Moreover, morphologic alterations observed in a section of neuromuscular junction exposed to β-bungarotoxin are also characterized by destroyed terminal buttons and intact terminal buttons containing fewer synaptic vesicles, swollen and flocular mitochondria with loss of the internal structure of cristae (Prasarnpun et al., 2005). The particular prevalence of morphological phenotypes depends heavily on the severity of the induced injury and the specific sensitivity of the neuronal subtypes to the different excitotoxic stimulus. The relationship between the differential expression of receptors, trophic factors and antioxidant molecules and the cell death phenotypes and sensitivity is still not well understood. Neurotoxicity was also observed in vivo after intrahippocampal administration of the Mlx-8 or Mlx-9 sPLA2s. These toxins induced behavioral and electroencephalographic seizures and neuronal degeneration (Oliveira et al., 2008). The same neuronal degeneration was observed after intracerebroventricular injection of paradoxin (Gandolfo et al., 1996), βbungarotoxin (Hanley and Emson, 1979) and N. mocambiquePLA2 (Masroori et al., 2010) or after intrastriatal injection of PLA2-OS2 (Kolko et al., 1999). There is high homology among presynaptic sPLA2neurotoxins belonging to elapid snakes venoms from the Old World and components identified in the Micrurus ssp. venoms, a genera representative of the family Elapidae in Americas (Ciscotto et al., 2011; Da Silva Junior et al., 1991; Fernandez et al., 2011). It is also known that PLA2neurotoxins isolated from different elapid venoms induce neurotransmitter release from motor nerve terminal (Belo et al., 2005; Prijatelj et al., 2006) and neuronal cultures (Rigoni et al., 2005, 2006). When taking these facts into consideration,

it could be suggested that Mlx-8 and Mlx-9 toxins could have its toxic action like other sPLA2-neurotoxins such as, notexin, taipoxin and textilotoxin by the induction of glutamate release (Rigoni, 2004). Thus, hippocampal neurons, being mainly glutamatergic, could release glutamate in the cell culture medium by the action of Mlx-8 or Mlx-9 toxin. Glutamate could induce an increase in the intracellular Ca2þ and consequently lead to a cascade of events that could culminate in cell death. In addition, it has also been demonstrated the involvement of ionotropic glutamate receptors in the neuronal death induced by sPLA2-OS2 in primary neuronal cultures or injected into the rat striatum (Kolko et al., 2002). We could also consider the participation of inflammatory mediators on the outcomes induced by Mlx-8 and Mlx-9 since this cell death pathway was also observed (Kolko et al., 2003). The authors showed that the neuronal death induced by sPLA2-OS2 toxin is partially mediated by neuronal signaling cascade that initiates with the binding of sPLA2 to a membrane receptor, activating cytosolic PLA2, the release of arachidonic acid, the production of PAF, and the induction of COX-2. In spite of the fact that sPLA2s isolated from elapid venoms belonging to IA group do not have a correspondent in mammalian tissues, these phospholipases A2 are active and/or neurotoxic in mammalian preparations as rat primary neuronal cultures. In this way, Mlx-8 and Mlx-9 are sPLA2neurotoxins from snake venom that could be a useful tool for the knowledge of the sPLA2s role in physiologic and/or neuropathologic processes. The findings of the present work evidenced that the utilization of hippocampal neurons for investigating the PLA2 neurotoxicity could be a good model. It is important to emphasize that isolated neurons in culture has been considered a model used to investigate the pharmacological mechanisms at the basis of the toxins actions in vivo but we must be careful with this extrapolation. In conclusion, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the M. lemniscatus snake venom induce early increase in free cytosolic Ca2þ concentration and mitochondrial function impairment that would lead to structural changes and could explain the toxicity to hippocampal neurons. Furthermore, the morphological approaches showed features of hybrid cell death with apoptotic, autophagic and necrotic signs. Further investigations should show which cell death pathways are involved in the outcomes showed in the present work.

4.

Experimental procedures

4.1.

Animals

Pregnant Wistar rats, weighing 230–250 g, were obtained from Butantan Institute, São Paulo, Brazil. They were housed in plastic cages and maintained in a room with constant temperature (2271 1C) on a 12:12 h light/dark cycle (lights on at 7:00 a.m.). Food and water were provided ad libitum. This study was performed according to NIH guidelines and approved by the Animal Use Ethic Committee of Butantan Institute. All efforts were made to minimize the number of animals used and their suffering.


4.2.

Materials

4.2.1.

Venom

Freeze-dried and fresh M. lemniscatus venom was obtained from the Laboratory of Venomous Animals, Federal University of Bahia, Brazil.

4.2.2.

Micrurus lemniscatus purification venom

PLA2 neurotoxins, Mlx-8 and Mlx-9, were obtained by M. lemniscatus venom purification (RP-HPLC) as described previously. Mass spectrometry and PLA2 activity were also determined (Oliveira et al., 2008). Molecular masses: Mlx8¼ 13,531.3 Da; Mlx-9¼ 13,568.0 Da.

4.3.

Preparation of rat primary hippocampal cell cultures

Hippocampal neurons were dissociated from hippocampi of E18-E19 Wistar rat fetuses as described previously (Banker and Cowan, 1977; Huettner and Baughman, 1986; Jahr and Stevens, 1987). Briefly, pregnant rats were anesthetized (sodium pentobarbitone 55 mg/Kg) and the fetuses were rapidly decapitated to remove their hippocampi. The tissue was placed into a Petri dish containing 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco) in a cooled Neurobasal medium (Gibco). Hippocampi were washed with Hank's Balanced Salt Solution (HBSS) and were carefully triturated with a pair of appropriate scissors and transferred to a 0.25% trypsin in Earl's Balanced Salt Solution (EBSS) pH 7.2–7.4 and incubated for 10 min at 37 1C. Following trypsinization, cells were transferred to EBSS containing DNAse 277.5 U/mL and 10% fetal bovine serum (FBS) and centrifuged at 300g (Eppendorf 5804R) for 2 min at 20 1C. Neurons were isolated by mechanical dissociation using Pasteur pipettes with three different diameter sizes, in an EBSS solution containing DNAse 277.5 U/mL and 10% FBS and centrifuged for 5 min (300g). The cells were re-suspended in Neurobasal medium (GIBCO, USA) containing 0.5 mM L-glutamine, 25 mM L-glutamic acid, 100 U/mL penicillin, 100 mg/mL streptomycin and 2% B27 supplement (GIBCO) and then seeded onto 0.01% poli-L-lysine-coated multi-well culture plate or 22-mmdiameter coverslips. Cultures were maintained at 37 1C in a humidified atmosphere of 5% CO2. On the second day, 50% of the medium was exchanged. On the seventh day, cells were incubated with Mlx-8 or Mlx-9 in different concentrations for 3, 6, 12 or 24 h, according to the experiments. This primary neuronal culture is relatively homogeneous and, after a few days in culture (6–7 days in vitro), it forms a network of functional synaptic contacts, acquiring the characteristics of mature neurons (Rigoni, 2004). Previously, the culture cells were immune-histochemically characterized with MAP2 (neuronal marker) and GFAP (astrocytic marker) and a predominance of neurons (92%) and 8% of astrocytes was observed (Garcia et al., 2012). The cell viability analyzed immediately after dissociation (0 day) as determined by the FACScan flow cytometry system (Scalibur-Becton Dickinson, San Jose, CA) showed that 98% and 89% of the cells preserved the membrane and DNA integrity, respectively. For the assessment of neuronal injury with the 3-(4-5dimethylthiazol-2-yl)-2-,5-diphenyltetrazolim bromide (MTT)

13

assay, or to measure the lactate dehydrogenase (LDH) released, hippocampal neurons were plated on poli-Llysine-coated 96-well culture plate at a density of 2 104 cells/well. For morphological studies with ethidium bromide staining and ultra structural analysis, cells were plated at a density of 2 104 cells/mL on poly-L-lysine-coated 22-mmdiameter coverslips. For scanning electron microscopy cells were plated at a density of 1 106 cells/mL.

4.4. Measurement of mitochondrial transmembrane potential (ΔΨm) Rhodamine 123 (Rho123) is a lipophilic fluorescent dye that has been used to estimate the electrical potential across the inner mitochondrial membrane (ΔΨm). It accumulates in the mitochondrial matrix because of their charge and solubility in both the inner mitochondrial membrane and matrix space (Scaduto and Grotyohann, 1999) and is able to produce images of high fluorescence in live mitochondria. In the present study, the ΔmΨ was measured by the Rho123 assay, monitored by flow cytometry. Hippocampal cells at density of 105 cells were plated in 6-well plates, and then treated for 1 h and 3 h with 7.4 nM MIx-8 or Mlx-9. Rho 123 was added at 100 mg/L 30 min before the end of the treatment. After washing with PBS, the cells were analyzed using a FACScan flow cytometry system (Scalibur-Becton Dickinson, San Jose, CA). A total of 10,000 cells/samples were analyzed and the mean fluorescence intensity and percentage of cells in each population (M1 and M2) was recorded.

4.5. Measurements of free intracellular Ca2þ concentrations by fluorescent indicator Hippocampal cells at a density of 2 104 grown on poly-Llysine-coated, 4 4-mm cover-slips were loaded with fura-2 for 15 min at 35 1C, AM, a ratiometric indicator dye. Imaging was done exciting by two wavelengths 340 and 387 nm – and the fluorescence emission was detected at 510 nm. Ratio imaging was performed, and is shown in Fig. 1. A Leica System – Leica AF6000 Series – was used. The chamber containing the cells was continuously perfused with a physiological solution, for control imaging, and with the same solution containing the toxin, for exploring its effects on intracellular [Ca2þ]. The solution was warmed to 35 1C at the entrance of the chamber. Time series images were recorded, at a frequency of 0.2 Hz, and ratios were calculated for regions selected in the images, corresponding to the cells. Control periods lasted minutes, before toxin was applied. The figures represent time averages of the ratios against time.

4.6.

Assessment of neuronal viability

4.6.1.

MTT reduction assay

Cell viability was evaluated by the3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) reduction assay (Liu et al., 1997; Mosmann, 1983). Briefly, the yellow compound was reduced to formazan, a purple product, by mitochondrial reductase enzymes of viable cells, and absorbance was measured at 570 nm.

14


After each period of incubation with Mlx-8, Mlx-9 (0.74 or 7.4 nM) or 250 mM KCl all the medium was removed and 100 mL of MTT solution [5 mg/mL MTT in phosphate buffer solution (PBS)] and Neurobasal medium without phenol red (1:9 v/v) was added. After 3 h of incubation with MTT at 37 1C in a humidified atmosphere of 5% CO2, 100 mL of dimethyl sulfoxide (DMSO) was also added to each one. After 30 min of shaking, the absorbance was measured at 570 nm in a multiwell plate reader (Biotek Power Wave XS2s), which has direct correlation with cell viability (Abe and Saito, 1999; Ioudina et al., 2004). The results were expressed as percentage of control value.

4.6.2.

Lactate dehydrogenase (LDH) release assay

The LDH assay is a measure of membrane integrity. The intracellular enzyme lactate dehydrogenase (LDH) is rapidly released to the medium with the injury of the cellular membrane. This enzyme converts pyruvate to lactate with NADH consumption, which is kinetically measured in the supernatant and it is directly correlated to LDH released (Clem et al., 1996; Jin et al., 2007; Silva et al., 2006). Therefore, cell viability is inversely proportional to LDH released. LDH release was measured in hippocampal cells treated for 24 h with 7.4 nM MIx-8 or Mlx-9. Briefly, 20 mL of the supernatant medium and 170 mL of buffer solution (120 mM Tris–HCl, 3.4 mM NADH, 50 mg/mL antimycin-A in ethanol, 1% triton-X 100 and Milli-Q water; 50:5:1:5:32) were added to a 96 well plate. To make sure that all the pyruvate present in the sample had been consumed, the absorbance was measured at 340 nm in a multiwell plate reader (Spectra Max Plus – Molecular Devicess) for 15–20 min, with intervals of 20 s, or until complete sample reading stability. Then, 10 mL of 20 mM pyruvate solution was added to each well and the absorbance was immediately measured at 340 nm for at least 5 min, with intervals of 20 s. The kinetic curve was traced at a linear location of the graph with at least 3 min (Jayalakshmi et al., 2005). The results were expressed as pmol of NADH consumption/minute/cell.

4.7.

Morphological analysis

4.7.1.

Ethidium bromide staining

Cell death induced by Mlx-8 and Mlx-9 (0.74, 7.4 and 74 nM) was also evaluated by morphological examination of the acridine orange (AO) – ethidium bromide (EB) stained cells using a fluorescence microscopy equipped with a blue filter (LSM510, Zeiss). Both AO and EB are able to interact with cellular DNA. However, AO permeates the cell membrane even if it has no damage; on the other hand, EB only interacts with DNA if the cell membrane has an injury. Through ultraviolet radiation, the cells stained with AO present a green color under blue filter whereas those cells stained with EB exhibit a red color. Orange color may happen due to the potency of EB color, which enshrouds AO coloration (Mercille and Massie, 1994). Cell samples cultivated in coverslips were mixed with equal volumes of a solution containing 100 mg/mL of both AO and EB. The samples were analyzed in duplicates (2 coverslips per group) and 10 pictures of different locations were taken per group. The hippocampal cells morphology

were analyzed by using a confocal fluorescence microscope (LSM 510 META, ZEISS).

4.7.2.

Ultrastructural analysis

Hippocampal cells seeded onto glass coverslips in 24-well plates were exposed to the neurotoxins Mlx-8, Mlx-9 at a concentration of 7.4 or 74 nM for 24 h at 37 1C in culture medium. After washing in 0.1 M PBS (phosphate buffer solution) at a pH 7.4 the cultured cells were fixed for 1 h with paraformaldehyde 1.0%, aldeydeglutaric 1.5% in 0.1 M, pH 7.3 cacodilate buffer, and post-fixed in 1% osmium tetroxide in the same buffer. After brief washing, the cells were prestained with 0.5% uranyl acetate in sucrose 13% during 30 min. Following dehydration in a graded ethanol series, and two quick passages in propylene oxide, the cells were covered with a mixture 1:1 of propylene oxide and Epon resin for 5 min. Gelatin capsules filled with Epon embedding medium were then inverted on top of the coverslips containing the preparations and placed into a 601 C oven for 24–48 h, as described in Bozzola and Russell (1998). Following resin polymerization, the capsules were snapped off of the glass substrate after been cooled with liquid nitrogen. Ultrathin sections from selected areas on the resin capsules were contrasted with uranyl acetate and lead citrate. The grids were examined in a LEO 906E transmission electron microscope (Zeiss, Germany) at 80 kV acceleration voltage. Images were acquired by a CCD camera MegaView III through the iTEM – Universal TEM Imaging Platform program (Olympus Soft Imaging Solutions GMBh, Germany), and saved in TIF extension.

4.7.3.

Scanning electron microscopy

The hippocampal cells control treated by Mlx-8 or Mlx-9 (7.4 nM), were fixed with 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer containing 1% sucrose in PBS overnight at room temperature. After washing with PBS three times, the samples were fixed with 1% osmium tetroxide (pH 7.4) at 4 1C for 1 h. The samples were dehydrated in an acetone gradient (30–100%), cells were critical point-dried in a CO2 system (Critical Point Dryer CPD 030, BAL-TEC GmbH, Germany). Samples were then sputtercoated with gold (Sputter Coating Device SCD 050, BAL-TEC GmbH) and examined at 10 kV accelerating voltage in an environmental scanning electron microscope LEO 435 VP SEM (Carl Zeiss –Germany).

4.8.

Statistical analysis

Data were analyzed by variance analysis (ANOVA) followed by Newman–Keuls post-test. The values were considered significant when Pr 0.05.

Acknowledgments This work was supported by a Grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – 04/ 01817-1) and Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTtox). We are in debit with Cássia Maria Braga Nunes, Felipe Alves Morais, Eduardo Osório Frare, Lívia Rocha


and Suelen Fukuda for assistant help. The skillful technical assistance of Alexsander Seixas de Souza to operate the "Zeiss LSM 510 META Laser Scanning Microscope (FAPESP – 00/11624-5) is gratefully acknowledged.

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Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2.

Neuroprotective effects of ginsenoside Rb1 on high glucose-induced neurotoxicity in primary cultured rat hippocampal neurons.

Desensitization of GABA-induced currents in cultured rat hippocampal neurons.

Computed potential responses of small cultured rat hippocampal neurons.

Spleen phospholipases A2.

Neutralization of myotoxic phospholipases A2 from the venom of the snake Bothrops asper by monoclonal antibodies.

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies.

Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: identification of directly hemolytic phospholipases A2.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom.

Evidence for coupling of different receptors for gonadotropin-releasing hormone to phospholipases C and A2 in cultured rat luteal cells.

Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus.

Neuroprotective effects of diarylpropionitrile against β-amyloid peptide-induced neurotoxicity in rat cultured cortical neurons.

Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons.

Phospholipases A1 and A2 in bovine thyroid.

Neurotoxicity evaluation of three root canal sealers on cultured rat trigeminal ganglion neurons.

Preparation of antibodies to phospholipases A2.

Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

Purification of mammalian nonpancreatic extracellular phospholipases A2.

B1 by a BDNF-dependent mechanism in cultured hippocampal neurons.

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity.

Blockade of ionotropic quisqualate receptor desensitization by wheat germ agglutinin in cultured postnatal rat hippocampal neurons.

Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons.

Physiological modulation of the GABA receptor by convulsant and anticonvulsant barbiturates in cultured rat hippocampal neurons.