Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca²⁺ which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of β-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca²⁺ influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca²⁺ overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.     

Central effects of tetanus and botulinum neurotoxins

Tetanus neurotoxin (TeNT) and botulinum neurotoxins (BoNTs; from A to G) are metalloproteases that act on nerve terminals to prevent exocytosis. They are extensively exploited for the study of cellular physiology. Moreover, BoNTs are also employed in clinical neurology for the treatment of several disorders characterized by hyperexcitability of peripheral nerve terminals. This review summarizes recent studies that have provided a deeper understanding of the mode of action of TeNT and BoNTs. TeNT and BoNTs bind with extreme specificity and are internalized at the neuromuscular junction. We first examine the retrograde transport mechanisms by which TeNT gains access to the central nervous system. We also discuss recent findings indicating that, besides their well known local actions at the neuromuscular junction, BoNTs can also affect central circuits.     

Challenges in searching for therapeutics against Botulinum Neurotoxins

Introduction: Botulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes.

Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed.

Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged.     

The facilitatory actions of snake venom phospholipase A2 neurotoxins at the neuromuscular junction are not mediated through voltage-gated K channels

Electrophysiological investigations have previously suggested that phospholipase A₂ (PLA₂) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K⁺ channels in motor nerve terminals.We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely β-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K⁺ channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K⁺ channels. In addition, internal perfusion of the neurotoxins (100 μg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K⁺ currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K⁺ channels.     

Actions of hemicholinium (HC-3) on neuromuscular transmission

Hemicholinium No. 3 (HC-3; α,α'-dimethylaminoethanol-4, 4'-biacetophenone) produced neuromuscular block of the tibialis anterior muscle-sciatic nerve preparation of the anaesthetized cat which was of slow onset, of long duration and dependent on the nerve stimulus frequency. The failure of the compound to modify the response of the tibialis muscle to close-arterial injections of acetylcholine suggested that the sensitivity of the motor endplate was not changed by it. The neuromuscular block produced by hemicholinium was antagonized by choline but only partly relieved by anticholinesterase drugs. The response to tetanic stimulation of the nerve during the neuromuscular block was well sustained and was followed by a slight posttetanic potentiation. The neuromuscular blocking action of hemicholinium could be relieved by temporary suspension of stimulation. These results suggest that the mode of action of hemicholinium at the neuromuscular junction is different from that of tubocurarine, and indicate that the action of hemicholinium is presynaptic, probably arising from a reduction in acetylcholine released by nerve stimulation.     

Toxicity of botulinum neurotoxins in central nervous system of mice.

Botulinum neurotoxins (BoNTs) act specifically on cholinergic nerve terminals, where they cause a sustained block of acetylcholine release, and therefore they are powerful tools to study the role of cholinergic neurons in neuronal processes. Peripheral effects of BoNTs are widely documented while central effects have not been studied. Here, we report for the first time on the central toxicity of BoNT serotypes A and B following their direct intracerebroventricular (icv) injection in CD1 mice. The LD50 values were found to be in the range 0.5-1.0 x 10(-6)mg/kg. We recorded the following signs preceding animal death: piloerection and weight decrease appear first, followed by temperature decrease, eyelid closure, loss of sensorimotor reflexes, dehydration, dyspnea. Mice died of heart or respiratory failure. The surviving mice recovered completely within 4-6 days and regained the initial healthy conditions. At sub-lethal doses, the same clinical signs appear in a lighter form and with a longer time course.     

Comparison of pre-junctional α-adrenoceptors at the neuromuscular junction with vascular post-junctional α-receptors in cat skeletal muscle

1 Activation of pre-junctional α-adrenoceptors at the skeletal neuromuscular junction enhances acetylcholine release whereas activation of such receptors at autonomic nerve endings inhibits transmitter output. In the present study the characteristics of pre-junctional α-adrenoceptors at motor nerve terminals have been compared with post-junctional (vascular) α-adrenoceptors in the cat hind limb.

2 Reversal of partial (+)-tubocurarine blockade of contractions of the tibialis anterior muscle was used to monitor pre-junctional activity and increases in hindlimb vascular resistance to assess post-junctional actions at α-adrenoceptors.

3 Responses to intra-arterial injections of noradrenaline, adrenaline, phenylephrine, oxymetazoline, methoxamine and clonidine were monitored. Dose-response lines for all the compounds except clonidine were parallel. The latter agent produced only weak and inconsistent effects.

4 Ratios of the doses of the agents required to produce pre- and post-junctional effects indicated that oxymetazoline and adrenaline possessed some preferential activity at post-junctional sites, whereas the remaining agents were non-selective in their actions. If dose-ratios with respect to noradrenaline were compared at the two sites none of the compounds possessed a marked degree of selectivity.

5 In the presence of phentolamine or tolazoline, dose-response curves to the pre- and post-junctional effects of phenylephrine were shifted to a similar extent. Thymoxamine showed preferential activity as a pre-junctional α-receptor antagonist.

6 In comparing the results of this study with those of other authors, it is apparent that there are marked differences in the characteristics of pre-junctional α-receptors at the skeletal neuromuscular junction and at autonomic nerve endings. The pre- and post-junctional α-receptors in skeletal muscle show less divergence.

     

CLOSTRIDIAL NEUROTOXINS

Tetanus (TeNT) and botulinum (BoNTs) neurotoxins are powerful toxins endowed with a specific zinc-endopeptidase activity. Targets of these neurotoxins have been identified as synaptic members of the SNARE proteins, which are involved in the exocytosis of neurotransmitters at the synapse. Despite this identical intracellular mechanism of action, TeNT and BoNTs target different neurons in vivo. After binding at the neuromuscular junction, BoNTs block neurotransmitter release at this site, whereas TeNT is retrogradely transported through motor neurons and inhibits exocytosis in inhibitory interneurons. Recently, several studies have reported the structure of these neurotoxins and clarified important aspects of the intoxication process. However, important questions on the mechanism responsible for the binding specificity and for the targeting of TeNT and BoNTs remain to be addressed. Once elucidated, this novel information would enables us to use CNTs more efficiently as therapeutic tools in neuronal disorders.     

Different mechanisms of inhibition of nerve terminals by botulinum and snake presynaptic neurotoxins

The different mode of action on peripheral nerve terminals of the botulinum neurotoxins and of the snake presynaptic phospholipase A2 neurotoxins is reviewed here. These two groups of toxins are highly toxic because they are neurospecific and at the same time are enzymes that can modify many substrate molecules before being inactivated. The similarity of symptoms they cause in humans derives from the fact that both botulinum neurotoxins (seven serotypes named A-G) and snake presynaptic PLA2 neurotoxins block the nerve terminals and that peripheral cholinergic terminals are major targets. Given this general similarity of targets and clinical symptoms, the specific molecular and cellular mechanisms at the basis of their action are very different. This difference appears evident from the beginning of intoxication, i.e. neurotoxins binding to peripheral nerve terminals and proceeds with the different site of actions and molecular targets.     

Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL⁻¹ limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.     

Synaptic terminal degeneration and remodeling at the rat neuromuscular junction resulting from a single exposure to acrylamide

Repetitive exposure to low doses of acrylamide results in extensive pathological changes at the neuromuscular junction (NMJ), but it remains undetermined if a single exposure to a larger dose will produce a similar neuropathological outcome. In the present study, morphometric and ultrastructural analyses of rat soleus NMJ were performed to determine early pathological effects of an intraperitoneal injection of 100 mg/kg acrylamide. Widespread nerve terminal degeneration, terminal sprouting, and endplate lengthening were evident as early as 4 days after injection. Degenerating terminal branches were swollen and exhibited enhanced argyrophilia. Ultrastructurally, the majority of terminals exhibited axolemmal abnormalities, neurofilament accumulations, and a paucity of synaptic vesicles; occasional swollen terminals lacked neurofilaments but contained increased numbers of tubulovesicular profiles. This early morphological pattern of nerve terminal changes suggests that acrylamide may disrupt both synaptic vesicle recycling and neurofilament degradation. These findings indicate that a single high dose of acrylamide triggers pathological lesions and remodeling in motor nerve terminals virtually identical to those resulting from multiple low doses.     

The facilitatory actions of snake venom phospholipase A(2) neurotoxins at the neuromuscular junction are not mediated through voltage-gated K(+) channels.

Electrophysiological investigations have previously suggested that phospholipase A₂ (PLA₂) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K⁺ channels in motor nerve terminals.

We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely β-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K⁺ channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K⁺ channels. In addition, internal perfusion of the neurotoxins (100 μg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K⁺ currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K⁺ channels.     

Botulinum neurotoxin for pain management: insights from animal models

The action of botulinum neurotoxins (BoNTs) at the neuromuscular junction has been extensively investigated and knowledge gained in this field laid the foundation for the use of BoNTs in human pathologies characterized by excessive muscle contractions. Although much more is known about the action of BoNTs on the peripheral system, growing evidence has demonstrated several effects also at the central level. Pain conditions, with special regard to neuropathic and intractable pain, are some of the pathological states that have been recently treated with BoNTs with beneficial effects. The knowledge of the action and potentiality of BoNTs utilization against pain, with emphasis for its possible use in modulation and alleviation of chronic pain, still represents an outstanding challenge for experimental research. This review highlights recent findings on the effects of BoNTs in animal pain models.     

Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.     

Snake Venom Neurotoxins: Pharmacological Classification

Neurotoxic proteins isolated from various snake venoms, because of their high affinity for a particular target site are used extensively as pharmacological tools to gain insights into the function of the nervous system. The potency of these molecules lies in their affinities towards the biomolecules involved in the functioning of neuromuscular transmission. Neuromuscular and pathophysiological effects of neurotoxic proteins result from their interaction with various microcompartments based on their similarities in mass and conformation to the types of amino acids and disulfide bridges in the normal ligands. Snake venom toxins can be broadly classified depending on whether their site of action is at the skeletal neuromuscular junction, or at sites other than the skeletal neuromuscular junction. Skeletal neuromuscular junction‐specific neurotoxins include the following: postsynaptic toxins, presynaptic toxins, presynaptic toxins with musculotropic or myonecrotic actions, presynaptic and postsynaptic, presynaptic and postsynaptic toxins with musculotropic or myonecrotic actions, myotoxic and antiAChE neurotoxins, etc. Snake venom neurotoxins with affinities selective to the sites other than the skeletal NMJ were categorised as non‐skeletal neuromuscular junction snake venom neurotoxins and they include toxins with affinity for muscarinic and neuronal receptors; toxins with affinity for K⁺ and Ca^{2 +} ion channels, toxins with affinity for enzymes and muscle elements, centrally‐acting neurotoxins, peptide neurotoxin and miscellaneous neurotoxins. There is an additional miscellaneous class of snake venom neurotoxins that includes weak neurotoxin, muscarinic toxin‐like proteins and vipoxin. The toxic mechanisms of well‐studied snake venom neurotoxins and their sites of action underlying neurotoxicity are discussed in this review, and they form the basis for classification of snake venom neurotoxins.     

C-Terminus of Botulinum A Protease Has Profound and Unanticipated Kinetic Consequences upon the Catalytic Cleft

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Facilitatory Neurotoxins and Transmitter Release

Abstract

Several protein neurotoxins from a variety of animals are known to increase the release of acetylcholine at the neuromuscular junction. Some toxins stimulate release in an uncontrolled fashion by inducing depolarization of the nerve terminal. Such toxins include those that alter the activation and inactivation characteristics of sodium channels, and those that create transmembrane pores. Toxins that facilitate evoked neurotransmitter release are more interesting because they can be used to explore the normal control mechanisms that modulate physiologically relevant release. Several snake toxins with phospholipase activity can block release, although they initially cause a stimulation. These include β-bungarotoxin, crotoxin, notexin, and taipoxin. The facilitation of transmitter release in mammalian nerve-muscle preparations is associated with a blockade of some of the potassium channels of the nerve terminal. This will slow the repolarization after an action potential, and thus allow calcium ions longer to enter the terminal and trigger release. Another type of facilitatory toxin is dendrotoxin from mamba snakes. It also blocks potassium channels, although a different subtype to those affected by the phospholipase toxins. Dendrotoxin causes repetitive firing of nerve terminals in addition to increasing acetylcholine release. More recently, the scorpion toxin, noxiustoxin, has been demonstrated to act prejunctionally to increase transmitter release. Another toxin with the potential to affect transmitter release is charybdotoxin, which blocks some potassium channels that are activated by internal calcium ions. Although charybdotoxin blocks calcium-activated potassium currents at motor nerve terminals, this does not normally affect acetylcholine release unless there is simultaneous block of voltage-dependent potassium currents.     

Botulinal neurotoxins: revival of an old killer

Botulinal neurotoxins (BoNTs) produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD(50) values in the range of 1-5 ng/kg. They are responsible for the pathophysiology of botulism. BoNTs are metalloproteinases that enter peripheral cholinergic nerve terminals, where they cleave one or two of the three core proteins of the neuroexocytosis apparatus and elicit persistent but reversible inhibition of neurotransmitter release. Their specificity of action has made them useful therapeutic agents for many human syndromes caused by hyperactivity of cholinergic nerve terminals. Their range of clinical applications is continuously growing, and BoNT/A is being used extensively as a pharmaco-cosmetic.     

Levocarnitine acetyl stimulates peripheral nerve regeneration and neuromuscular junction remodelling following sciatic nerve injury.

The effects of levocarnitine acetyl were investigated on both peripheral nerve regeneration and neuromuscular remodelling in male Sprague-Dawley rats, three months of age, following crush of their left sciatic nerve. Levocarnitine acetyl, 150 mg/kg/day in drinking water, was given from one week before to 5, 15, 20, and 60 days after nerve crush. The sciatic nerve was examined morphologically at all given times and morphometrically at 15, 20, and 60 days after the lesion. Morphology, at 5, 15, and 60 days, and morphometry, at 60 days after the nerve crush, were also performed on the neuromuscular junction in the soleus and extensor digitorum longus muscles. Five days after nerve crush, complete axonal degeneration was observed in both control and treated rats. At 15 and 20 days, recovery from injury in treated animals was better than in controls, as shown by a significantly higher increase in the number of regenerating axons. At the same times, denervated endplates were present in both groups. At 60 days, axonal regeneration restored the number of axons to normal values in all injured animals, while their size maturation was greater in treated rats than in controls. A markedly lower number of degenerating elements was found in treated animals. In the neuromuscular junctions of the soleus and extensor digitorum longus muscles, nerve terminal branch points were reduced in the lesioned rats in comparison with uninjured ones. However, morphometric analysis revealed a greater endplate complexity in treated animals in which, at 60 days after nerve crush, nerve terminal branching and sprouting index values were significantly higher than in controls. It is concluded that levocarnitine acetyl exerts a beneficial effect on nerve regeneration processes and synaptic remodelling in crush-induced neuropathies.     

Neuronal glia interactions at the vertebrate neuromuscular junction

Emerging studies demonstrate that perisynaptic Schwann cells (PSCs), which are the glia cells juxtaposed to the motor nerve terminal, actively participate in multiple aspects of the neuromuscular junction. During development, PSCs guide and promote synaptic growth. In adult muscles, PSCs can sense nerve stimulation by increasing intracellular calcium and are also capable of modulating transmitter release. Although adult PSCs are not required for acute synaptic maintenance and function, they are indispensable for long-term synaptic maintenance. Furthermore, PSC sprouts lead nerve terminal extension during synaptic remodeling. After nerve injury, PSCs sprout profusely and PSC processes guide regenerating nerve terminals. Future challenges will be to identify the molecular mechanisms by which PSCs interact with the nerve terminal and the muscle fiber.