Calpains participate in nerve terminal degeneration induced by spider and snake presynaptic neurotoxins

Elisa Duregotti a,1, Erik Tedesco b,1, Cesare Montecucco a,*, Michela Rigoni a,*

Abstract

Neurodegeneration a-latrotoxin and snake presynaptic phospholipases A2 neurotoxins target the presynaptic membrane of axon terminals of the neuromuscular junction causing paralysis. These neurotoxins display different biochemical activities, but similarly alter the presynaptic membrane permeability causing Ca2þ overload within the nerve terminals, which in turn induces nerve degeneration. Using different methods, here we show that the calciumactivated proteases calpains are involved in the cytoskeletal rearrangements that we have previously documented in neurons exposed to a-latrotoxin or to snake presynaptic phospholipases A2 neurotoxins. These results indicate that calpains, activated by the massive calcium influx from the extracellular medium, target fundamental components of neuronal cytoskeleton such as spectrin and neurofilaments, whose cleavage is functional to the ensuing nerve terminal fragmentation.

Keywords:
Presynaptic neurotoxins
Calpain
Ca2þ toxicity

1. Introduction

Many animal toxins target the neuromuscular junction (NMJ), interfering with its key function, the neurotransmission: among them the presynaptic toxins block acetylcholine (Ach) release by different, but often related, mechanisms of action, eventually leading to paralysis. The black widow spiders (genus Latrodectus) are largely diffused in many parts of the world. The venom of Latrodectus spp. contains at least 86 unique proteins, some of which play a role in its toxicity towards insects and crustaceans, with only one component, alpha-latrotoxin (aLtx), that specifically targets vertebrates. A large body of evidence shows that Ca2þ influx through membrane channels induced by a-Ltx in the presynaptic membrane accounts for a major part of its effects (Ushkaryov et al., 2004, 2008; Vassilevski et al., 2009).
Presynaptic snake neurotoxins endowed with PLA2 activity (SPANs) are major components of the venom of four families of venomous snakes (Crotalidae, Elapidae, Hydrophiidae and Viperidae). These neurotoxins play a major role in the envenomation of the prey by causing a persistent blockade of neurotransmitter release from nerve terminal (Harris, 1985; Kini, 1997; Pungercar and Krizaj, 2007). Once bound to the presynaptic membrane they hydrolyse phospholipids, leading to a progressive accumulation of lysophospholipids (LysoPLs) and fatty acids (FA) and the consequent degeneration of the NMJ (Montecucco and Rossetto, 2000; Rigoni et al., 2005; Rossetto and Montecucco, 2008).
A recent side by side comparative study of SPANs and a-Ltx action in primary cultures of central neurons and in mice showed that, despite the different molecular mechanisms of intoxication, both kind of toxins exert their degenerative activity via inducing a large calcium influx inside nerve terminals (Tedesco et al., 2009). Both SPANs and a-Ltx cause phenotypical alterations in neurites of cultured primary neurons called bulges, hallmarks of their neurotoxicity. Bulges are sites of unbalanced endo–exocytosis and of intracellular calcium overload, where the neuronal cytoskeleton appears dramatically affected, with accumulation of neurofilaments (NF) and actin staining (Rigoni et al., 2004, 2007; Tedesco et al., 2009).
In light of these observations we have considered the possible involvement of calcium-activated proteases in the cytoskeletal rearrangements observed in cultured neurons exposed to both classes of neurotoxins and have focused our attention on calpains, a class of cysteine-proteases involved in cell damage following Ca2þ accumulation in the cytosol (Vosler et al., 2008). The two ubiquitously expressed isoforms of the calpain superfamily, m-calpain and m-calpain, are the best-characterized ones and are conventionally termed calpains. These proteases are localized in the cytosol in their inactive form; in response to [Ca2þ] increase they translocate to membranes where they are activated by autolytic processing. The calpain isoforms differ in [Ca2þ] sensitivity: m-calpain requires micromolar while m-calpain requires millimolar [Ca2þ] for activation. Calpain physiological substrates include enzymes such as protein kinase C (PKC), as well as structural cytoskeletal proteins including spectrin, tubulin, microtubuleassociated protein 2 (MAP2) and neurofilament proteins. At resting physiological conditions, when [Ca2þ] oscillates around 100 nM, slight proteolytic modification of some calpain substrates is associated with normal cell functioning and protein turnover. Calpain inhibitors elicit accumulation of NF at the nerve terminal, suggesting that these proteases are indeed important in the normal regulation of synaptic structure (O’Hanlon et al., 2003). At pathological higher [Ca2þ] calpains were shown to act particularly in models of hypoxic and traumatic brain injury as well as in many neurodegenerative diseases (Schumacher et al., 1999; Vosler et al., 2008; Kilinc et al., 2009).
Neurofilaments are composed of three proteins with apparent molecular weights of 200, 160, and 68 kDa referred to as heavy (NF-H), medium (NF-M) and light neurofilaments (NF-L), respectively. The neurofilament proteins are phosphorylated in vivo, particularly at multiple repeats of the Lys-Ser-Pro (KSP) motif. Neurofilaments are good substrates for calpains and their susceptibility is enhanced by dephosphorylation (Geddes et al., 1995; Pant, 1988). Calpain targets also include spectrin, a major membrane-skeletal protein, whose cleavage leads to the generation of two unique and highly stable breakdown products, which is an early event in neural cell pathology (Czogalla and Sikorski, 2005).
We have tested calpain contribution to neuronal cytoskeletal fragmentation in primary neuronal cultures from cerebellum and cortex exposed to a-Ltx and notexin (Ntx), a prototype snake PLA2 presynaptic neurotoxin, in the absence or presence of calpain inhibitors, using different experimental approaches: i) analysis of spectrin and NF Western blotting profiles; ii) time-course of bulge formation; iii) live-imaging of calpain activity with a fluorigenic calpain substrate. Our results indicate that these proteases participate in the degeneration process initiated by these two classes of animal toxins.

2. Methods

2.1. Chemicals

BME (Basal Eagle Medium), Neurobasal A, B27, Glutamax and the fluorigenic calpain substrate t-BOC-L-leucyl-Lmethionine amide have been purchased from Invitrogen; AraC (cytosine b-D-arabinofuranoside), trypsin, trypsin inhibitor, DNAse I, poly-L-lysine, gentamycin, bovine serum albumin and oleic acid (OA) are Sigma–Aldrich; 1myristoil-2-lysophosphatidylcholine (mLysoPC) is Avanti Polar Lipids; FBS is Euroclone. For Western Blot detection we employed the ECL LUMINATA from Millipore.

2.1.1. Toxins

Notexin was obtained from Latoxan (France), a-latrotoxin from Alomone (Israel). The purity of these toxins was checked by SDS-PAGE and their neurotoxicity by ex-vivo mouse nerve-hemidiaphragm preparation as previously described (Rigoni et al., 2005). The lipid mixture (mLysoPC þ OA) was prepared following the procedure reported in Rigoni et al. (2005).

2.1.2. Calpain inhibitors
The following calpain inhibitors have been used at 20 mM final concentration: MDL28170 (Sigma–Aldrich), ALLN and PD150606 (Calbiochem). In experiments involving calpain inhibitors samples were preincubated 30 min to 1 h with the compounds and the inhibitors were kept in the medium throughout the experiment.

2.1.3. Antibodies

The following antibodies were used: mouse monoclonal anti-spectrin (Chemicon, working dilution in WB 1:1000); mouse monoclonal anti-neurofilaments (SMI31 and SMI32, Sternberg Monoclonals, 1:1000); mouse monoclonal antiHsc70 (Synaptic Systems, 1:5000); goat anti-mouse HRPconjugated (Calbiochem, 1:2000).

2.2. Primary neuronal cultures

All experiments were performed in accordance with Italian animal care guidelines, law no. 116/1992. Rat cerebellar granule neurons (CGNs) and cortical neurons were prepared from 6 or 2-days-old Wistar rats respectively as previously described (Levi et al., 1984; Rigoni et al., 2004) and used 6–8 days after plating.

2.3. Sample preparation for Western blotting

CGNs (250,000/w, 24w plates) or cortical neurons (160,000/w, 24w plates) were exposed to a-Ltx, Ntx or mLysoPC þ OA (0.1 nM for 30 min, 25 nM for 45 min and 25 mM for 30 min, respectively) in Krebs Ringer Buffer (KRH: 125 mM NaCl, 5 mM KCl,1.2 mM MgSO4, 2 mM CaCl2, 1.2 mM KH2PO4, 6 mM glucose and 25 mM HEPES, pH 7.4), then washed twice with PBS 5 mM EDTA and lysed in Lysis Buffer (Hepes 10 mM, NaCl 150 mM, SDS 1%, EDTA 4 mM, protease inhibitors cocktail (Roche)). Protein concentration was quantified using the BCA assay (Protein Assay Kit, Pierce). 7–10 mg were loaded onto 10-well gels (Protogel, stacking gel 4%, resolving gel 6.5%, Minigel II Biorad) and then transblotted onto a nitrocellulose membrane. Following saturation (PBST 5% milk in the case of spectrin detection and PBS 5% BSA for phosphoneurofilaments), membranes were incubated o/n at 4 C with the primary antibodies and, after washings, for 1 h at RT with the corresponding secondary antibodies HRP-conjugated. For densitometric quantification the bands of interest were normalized with the housekeeping protein Hsc70.

2.4. Time course of bulging

CGNs or cortical neurons at 6 to 8 DIV plated onto polyL-lysine-coated 24 mm coverslips were washed and incubated in KRH at 37 C in the absence or presence of the toxins (a-Ltx 0.1 nM for 40 min, Ntx 25 nM for 45 min) or the lipid mixture (25 mM for 30 min) and w/o calpain inhibitors (inhibitors were added to the medium at 20 mM final concentration 45 min before toxin addition and maintained throughout the experiment). Brightfield images with Nomarsky optics were acquired at 5–10 min intervals for a maximum of 45 min (Leica DMI6000 epifluorescence microscope).

2.5. Live-imaging of calpain activity and quantification

To detect calpain activity in living neurons, cells were loaded with the fluorogenic calpain substrate 7amino-4-chloromethylcoumarin, t-BOC-L-leucyl-L-methionine amide (t-BOC). Non-fluorescent t-BOC freely diffuses into the cell and becomes membrane-impermeant after being conjugated to a thiol. Cleavage of t-BOC-thiol by calpain results in the release of fluorescent 7-amino-4methylcoumarin-thiol (MAC-thiol). The formation of MAC-thiol is not reversible. Calpain activity at steady state results in an increase in fluorescence due to the accumulation of MAC-thiols. Changes in calpain activity can thus be detected by considering the rate of change in the MAC-thiol fluorescent signal (Rosser et al., 1993). CGNs or cortical neurons were incubated in Krebs buffer containing 50 mM t-BOC for 15 min at 37 C, than the loading medium was washed out and fluorescence levels monitored in control conditions or following exposure to a-Ltx (0.1 nM for 30 min), Ntx (25 nM for 45 min) or to an equimolar mixture of mLysoPC þ OA that has been previously found to mimic the neurotoxic effects of SPANs (25 mM for 30 min). A selected area on the coverslip was imaged at 10 min intervals. An excitation/emission filter set suitable for DAPI was used to record t-BOC fluorescence (Ex ¼ 351 nm; Em ¼ 430 nm; Leica DMI6000, LAS AF software). Since tBOC is susceptible of photo-activation and this may turn out in cell photo-toxicity, exposure to ultraviolet light was kept at a minimum. The same experimental conditions were employed in the presence of calpain inhibitors; in this case cells were pre-treated with the inhibitors 45 min before intoxication and kept in the medium throughout the experiment. The changes in fluorescence with time within specific regions of interest at the level of cell bodies, neurites or bulges were measured (ImageJ software) and expressed as fold increase with respect to the fluorescence value at t ¼ 0 for each sample.

3. Results

3.1. Analysis of calpain proteolytic activity by Western blotting

As a first assay of the contribution of calpains to the phenotypical alterations observed in primary cultures of neurons treated with a-Ltx or SPANs, we performed a Western blotting analysis of cell lysates from intoxicated cerebellar and cortical neurons. Two major targets of these proteases, spectrin and neurofilaments, were detected with specific antibodies. In fact, a-spectrin cleavage is a reliable method to detect calpain activation and has extensively been used as a quantitative measure of calpain activity (Vanderklish and Bahr, 2000). The primary antibody used here recognizes the intact spectrin 280 kDa band as well as the 150–145 kDa doublet that corresponds to calpainspecific cleavage products. Treatment of neurons with staurosporine, a compound known to activate both calpains and caspases (Nath et al., 1996), gave rise to the expected pattern, whilst pretreatment with calpain inhibitors (MDL28170, ALLN and PD150606) protected against staurosporine-mediated spectrin proteolysis (Fig. 1, panel A). Both a-Ltx and Ntx led to a reduction of the 280 kDa band and an increase of the bands corresponding to the cleavage products, and this effect was reduced in the presence of calpain inhibitors (Fig. 1, panels B and C). Closely similar results were obtained with cortical neurons (not shown). Also a lipid mixture composed of myristoyllysophosphatidylcholine (LysoPC) and oleic acid (OA) in equimolar concentrations, the products of the PLA2 activity of SPANs that were previously shown to mimic their overall neurotoxic action in both neuronal cultures and isolated NMJs (Rigoni et al., 2005, 2007), caused a-spectrin cleavage (Supplementary Fig. S1, panel A). A small reduction in calpain basal activity was observed in control samples incubated with the inhibitors alone (not shown).
We next extended such analysis to another important calpain substrate, the neurofilament proteins. No signal was detected in toxins-treated lysates w/o calpain inhibitors when a primary antibody directed against the phosphorylated H subunit was employed (SMI31), suggesting the possibility that the activation of cellular phosphatases by toxins-induced calcium entry with consequent dephosphorylation of such epitope might have occurred during treatments (Fig. 2, panel A). Indeed, incubation of neurons with the toxins in the presence of phosphatase inhibitors restored the phospho-NF signal (not shown). Following a-Ltx or Ntx exposure a decrease of the 200 kDa band was observed when an anti-NF antibody raised against a non-phosphorylated epitope in neurofilament H was employed (SMI32). In both treatments a partial protection by pretreatment with calpain inhibitors was observed (Fig. 2, panels B and C). A decrease in NF staining was induced also by lipid mixture treatment (Fig.S1, panel B). Similar results were obtained in cortical neurons (not shown).

3.2. Calpain is not involved in the generation of neuronal bulges

The exposure of cerebellar and cortical neurons to a-Ltx or Ntx leads to the formation of characteristic swellings along neurites that we referred to as bulges (Rigoni et al., 2004, 2005). In the case of a-Ltx, bulges result mainly from the stimulation of exocytosis elicited by the toxin triggered by calcium influx, with consequent addition of vesicular membrane to the cell surface and neurotransmitter emptying of the terminal (Ceccarelli et al., 1972; Duchen et al., 1981; Ushkaryov et al., 2004). In the case of SPANs, the PLA2 activity exerted on the plasma membrane initiates the formation of bulges, with the ensuing unbalance between exocytosis and endocytosis, as shown by FRET and FM 1–43 experiments and by the surface exposure of the intraluminal domain of the vesicular protein synaptotagmin I (Rigoni et al., 2004, 2005; Bonanomi et al., 2005). Indeed, LysoPLs and FAs alone are capable of inducing neuronal bulging and this is reversed upon their removal (Caccin et al., 2006). However, with both classes of neurotoxins, vesicle exocytosis is not the only process involved in nerve terminal swelling, which nevertheless remains a good indicator of these presynaptic neurotoxins activity in vitro. We tested here the possibility that calpains are involved in nerve terminal bulging and found that preincubation of neurons with calpain inhibitors followed by toxins or lipid mixture addition did not decrease the rate of bulge formation nor affected their overall morphology (Fig. 3 and Supplementary Fig. S2). No toxicity was elicited by the inhibitors alone (not shown).

3.3. Live imaging of calpain activity in cultured neurons

To assay calpain involvement in later stages of toxinsinduced neurodegeneration, neurons were loaded with a fluorogenic calpain substrate (7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl-L-methionine amide, tBOC) which becomes fluorescent once specifically cleaved by these proteases. Using this approach, we expected not only to confirm the results obtained in Western blot, but also to obtain a picture of the spatial distribution of calpain activity. Once added to culture medium t-BOC freely diffuses across the membranes into the cell cytosol, where it is retained because it is modified and becomes membrane-impermeable. Control neurons loaded with t-BOC showed an increase in fluorescence with time due to the basal activity of these enzymes in the cells and the photo-activation of the fluorogenic substrate (Fig. 4, panel A). Using the same acquisition parameters, the samples treated with a-Ltx, Ntx or the mLysoPC þ OA lipid mixture (that was previously shown to induce Ca2þ influx, Rigoni et al., 2007) showed a more pronounced increase in fluorescence with respect to controls; in addition, the fluorescent signal was localized within swellings, indicating that bulges are indeed major sites not only of Ca2þ entry (Rigoni et al., 2007) but also of calpain activation (Fig. 4, panels B and C and Supplementary Fig. S3). Quantification of selected regions of interest (ROI) shows that the increase in fluorescence at the level of cell bodies was comparable between controls and treated samples, and that the mean fluorescence measured at the level of the neurites of toxintreated neurons was lower than that measured in the bulges. The mean fluorescence within bulges increased with time and, judging from the fluorescence signal, the potency of the two toxins was comparable among them and with the lipid mixture (Fig. S4). Pretreatment with calpain inhibitors reduced both the resting calpain activity in control neurons and that measured in treated samples, supporting the specificity of the phenomenon and confirming the results obtained by Western blotting (Fig. 5). Similar results were obtained using cortical neurons (not shown).

4. Discussion

Neuronal Ca2þ homeostasis and Ca2þ signalling regulate multiple neuronal functions, including synaptic transmission, plasticity and cell survival. Disturbances in calcium homeostasis due to energy depletion have been described in acute neurodegenerative disorders, as well as in tissues from aged humans both with and without associated age-related pathologies such as Alzheimer’s disease. Alterations in Ca2þ homeostasis can affect the physiology of neurons in different ways and to various extents. A common observation from the pathologies mentioned above is the activation of the cysteine protease calpain, a calcium-dependent, non-lysosomal enzyme known to be widely expressed in animal tissues. The calcium sensitivity of calpains suggests that they are important effectors of changes in neurons brought about by calcium influx, a rather common pathological alteration in various forms of damage and/or degeneration of neurons (Nicotera et al., 1992; Bertipaglia and Carafoli, 2007). Alterations in calcium homeostasis during ischaemia, TBI and epilepsy result in the overwhelming activation of calpain in vitro, in vivo and in post-mortem brain. Chronic neurodegenerative diseases also show calcium disregulation with ensuing calpain activation (Vosler et al., 2008). Thus calciumdependent processes represent points of convergence among a heterogeneous set of degenerative mechanisms. Pathologic activation of calpain results in the cleavage of a number of neuronal substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms. Experimental findings suggest that caspase and/or calpain inhibitors can attenuate neuronal degeneration in models of these neurodegenerative disorders (Vanderklish and Bahr, 2000; Chan and Mattson, 1999).
SPANs and a-Ltx cause similar degeneration of nerve terminals (Chen and Lee, 1970; Ceccarelli et al., 1972; CullCandy et al., 1976; Ceccarelli and Hurlbut, 1980; Duchen et al., 1981; Lee et al., 1984; Gopalakrishnakone and Hawgood, 1984) with disappearance of neurofilaments and synaptic proteins (Dixon and Harris, 1999; Harris et al., 2000). We found previously that calcium overloading is the common eventual result of the different biochemical activities of these two classes of animal presynaptic neurotoxins (the PLA2 activity of SPANs and the ion channel activity of a-Ltx) (Rigoni et al., 2007, 2008; Tedesco et al., 2009).
Here, using different techniques, we have shown that calpains are indeed activated following intoxication with both classes of neurotoxins: their cleavage of spectrin and neurofilaments, two typical calpain substrates, detected by Western blot, is largely inhibited by preincubation with calpain inhibitors. Following toxins treatment, the NF heavy subunit is dephosphorylated and degraded; these results fit with previous observations that dephosphorylation enhances neurofilaments susceptibility to calpains (Pant, 1988).
Calpain involvement was further confirmed by liveimaging experiments of calpain activity in cultured neurons, showing a localized action within bulges, that are phenotypical hallmarks of intoxication by both a-Ltx and SPANs. Bulging derives mainly from membrane accumulation that follows unbalanced exo–endocytosis. In the case of a-Ltx, calcium entry is the major event responsible for nerve swelling from the beginning, as this toxin is a Ca2þ channel itself, whereas in the case of snake PLA2 the initial process in bulge formation is the production of LysoPLs, which favour synaptic vesicle exocytosis not followed by endocytosis. This is then followed by the entry of calcium mediated by transient LysoPLspores(Rigoni et al., 2004, 2005; Bonanomi et al., 2005). Indeed, bulge appearance preceeds intracellular calcium rise (Rigoni M., unpublished observations). Accordingly, calpain inhibitors did not prevent bulging, in linewith theobservationsthat bulges arenotthemereresult of cytoskeletal rearrangements and with electrophysiological experiments demonstrating that calpain inhibition does not prevent the induction of neuroexocytosis by a-Ltx (O’Hanlon et al., 2003).
Calpain activation appears to be a later event in the intoxication process by these two classes of presynaptic neurotoxins, which manifests itself as cytosolic protein cleavage, and particularly as the cleavage of cytoskeletal proteins. The fragmentation of the cytoskeleton components by calpains found here appears to be an important prerequisite for the following step which is the degeneration of the nerve terminal into fragments, necessary for the subsequent removal of the degraded terminals by phagocytosis. In turn, this is essential for the final regeneration of the neuromuscular junction which takes place in vivo (Grubb et al., 1991; Prasarnpun et al., 2005).
Axon degeneration with fragmentation of axonal cytoskeleton is a characteristic event in many chronic neurodegenerative conditions as well after acute nerve injuries. As early as 5–10 min after nerve transection, for example, the axonal segments immediately proximal and distal to the injury site rapidly degenerate by several hundred micrometres in either directions, and this process is thought to be driven by influx of extracellular calcium, with subsequent activation of calpains. Increased calpain cleavage of spectrin occurs as early as 30 min after injury in vivo, indicating that calpain activity is the primary effector of the acute axonal degeneration (AAD) (Wang et al., 2012).
The present study provides further insights into the molecular mechanisms of action of these two types of animal toxins, a-Ltx and Ntx, that have very different biological activities but block the NMJ functionality in a similar way, via the induction of the entry of calcium ions inside axon terminals. Our results demonstrate the involvement of the calcium-activated proteases calpains in the degeneration of the nerve terminals induced by both type of neurotoxins, and suggest that their study can provide useful information to the understanding of other neurodegenerative conditions.

References

Bertipaglia, I., Carafoli, E., 2007. Calpains and human diseases. Subcell. Biochem. 45, 29–53.
Bonanomi, D., Pennuto, M., Rigoni, M., Rossetto, O., Montecucco, C., Valtorta, F., 2005. Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2. Mol. Pharmacol. 67, 1901–1908.
Caccin, P., Rigoni, M., Bisceglie, A., Rossetto, O., Montecucco, C., 2006. Reversible skeletal neuromuscular paralysis induced by different lysophospholipids. FEBS Lett. 580, 6317–6321.
Ceccarelli, B., Hurlbut, W.P., 1980. Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60, 396–441.
Ceccarelli, B., Hurlbut, W.P., Mauro, A., 1972. Depletion of vesicle from frog neuromuscular junction by prolonged tetanic stimulation. J. Cell. Biol. 50, 30–38.
Chan, S.L., Mattson, M.P., 1999. Caspase and calpain substrates: roles in synaptic plasticity and cell death. J. Neurosci. Res. 58, 167–190.
Chen, I.L., Lee, C.Y., 1970. Ultrastructural changes in the motor nerve terminals caused by beta-bungarotoxin. Virchows Arch. B Cell. Pathol.6, 318–325.
Cull-Candy, S.G., Fohlman, J., Gustavsson, D., Lullmann-Rauch, R., Thesleff, S., 1976. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1, 175–180.
Czogalla, A., Sikorski, A.F., 2005. Spectrin and calpain: a ‘target’ and a ‘sniper’ in the pathology of neuronal cells. Mol. Life Sci. 62, 1913– 1924.
Dixon, R.W., Harris, J.B., 1999. Nerve terminal damage by betabungarotoxin: its clinical significance. Am. J. Pathol. 154, 447–455.
Duchen, L.W., Gomez, S., Queiroz, L.S., 1981. The neuromuscular junction of the mouse after black widow spider venom. J. Physiol. 316, 279–291.
Geddes, J.W., Bondada, V., Tekirian, T.L., Pang, Z., Siman, R.G., 1995. Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain. Neurobiol. Aging 16, 651–660.
Gopalakrishnakone, P., Hawgood, B.J., 1984. Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon 22, 791–804.
Grubb, B.D., Harris, J.B., Schofield, I.S., 1991. Neuromuscular transmission at newly formed neuromuscular junctions in the regenerating soleus muscle of the rat. J. Phisiol. 441, 405–421.
Harris, J.B., Grubb, B.D., Maltin, C.A., Dixon, R., 2000. The neurotoxicity of the venom phospholipases A2, notexin and taipoxin. Exp. Neurol. 161, 517–526.
Harris, J.B., 1985. Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol. Ther. 31, 79–102.
Kilinc, D., Gallo, G., Barbee, K.A., 2009. Mechanical membrane injury induces axonal beading through localized activation of calpain. Exp.Neurol. 219, 553–561.
Kini, R.M., 1997. Venom Phospholipase A2 Enzymes. John Wiley & Sons.
Lee, C.Y., Tsai, M.C., Chen, J.M., Ritonja, A., Gubensek, F., 1984. Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom. Arch. Int. Pharmacodyn. Ther. 268, 313– 324.
Levi, G., Aloisi, F., Ciotti, M.T., Gallo, V., 1984. Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures. Brain Res. 290, 77–86.
Montecucco, C., Rossetto, O., 2000. How do presynaptic PLA2 neurotoxins block nerve terminals? Trends Biochem. Sci. 25, 266–270.
Nath, R., Raser, K.J., Stafford, D., Iradj Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R.V., Yuen, P., Gilbertse, R.B., Wang, K.K.W., 1996. Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319, 683–690.
Nicotera, P., Bellomo, G., Orrenius, S., 1992. Calcium-mediated mechanisms in chemically induced cell death. Annu. Rev. Pharmacol. Toxicol. 32, 449–470.
O’Hanlon, G.M., Humphreys, P.D., Goldman, R.S., Halstead, S.K., Bullens, R.W.M., Plomp, J.J., Ushkaryov, Y., Willison, H.J., 2003. Calpain inhibitors protect against axonal degeneration in a model of antiganglioside antibody-mediated motor nerve terminal injury. Brain 126, 2497–2509.
Pant, H.C., 1988. Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. Biochem. J. 256, 665– 668.
Prasarnpun, S., Walsh, J., Awad, S.S., Harris, J.B., 2005. Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis. Brain 128, 2987–2996.
Pungercar, J., Krizaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892.
Rigoni, M., Schiavo, G., Weston, A.E., Caccin, P., Allegrini, F., Pennuto, M., Valtorta, F., Montecucco, C., Rossetto, O., 2004. Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles. J. Cell. Sci. 117, 3561–3570.
Rigoni, M., Caccin, P., Gschmeissner, S., Koster, G., Postle, D.A., Rossetto, O., Schiavo, G., Montecucco, C., 2005. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures. Science 210, 1678–1680.
Rigoni, M., Pizzo, P., Schiavo, G., Weston, A.E., Zatti, G., Caccin, P., Rossetto, O., Pozzan, T., Montecucco, C., 2007. Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products. J. Biol. Chem. 282, 11238–11245.
Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., Montecucco, C., 2008. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores. J. Biol. Chem. 283, 34013–34020.
Rosser, B.G., Powers, S.P., Gores, G.J., 1993. Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach. J. Biol. Chem. 268, 23593–23600.
Rossetto, O., Montecucco, C., 2008. Presynaptic neurotoxins with enzymatic MDL-28170 activities. Handb. Exp. Pharmacol. 184, 129–170.
Schumacher, P.A., Eubanks, J.H., Fehlings, M.G., 1999. Increased calpain Imediated proteolysis, and preferential loss of dephosphorylated NF200, following traumatic spinal cord injury. Neuroscience 91, 733–744.
Tedesco, E., Rigoni, M., Caccin, P., Grishin, E., Rossetto, O., Montecucco, C., 2009. Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins. Toxicon 54, 138–144.
Ushkaryov, Y.A., Volynski, K.E., Ashton, A.C., 2004. The multiple actions of black widow spider toxins and their selective use in neurosecretion studies. Toxicon 43, 527–542.
Ushkaryov, Y.A., Rohou, A., Sugita, S., 2008. alpha-Latrotoxin and its receptors. Handb. Exp. Pharmacol. 184, 171–206.
Vanderklish, P.W., Bahr, B.A., 2000. The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states. Int. J. Exp. Pathol. 81, 323–339.
Vassilevski, A.A., Kozlov, S.A., Grishin, E.V., 2009. Molecular diversity of spider venom. Biochemistry (Mosc.) 74, 1505–1534.
Vosler, P.S., Brennan, C.S., Chen, J., 2008. Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol. Neurobiol. 38, 78–100.
Wang, J.T., Medress, Z.A., Barres, B.A., 2012. Axon degeneration: molecular mechanisms of a self-destruction pathway. J. Cell. Biol. 196, 7–18.