Search Results (12)

α-Latrotoxin stimulates neurotransmitter release by binding to a presynaptic receptor and then forming ion-permeable membrane pores and/or stimulating the receptor, latrophilin-1, or Adhesion G-protein-coupled receptor type L1 (ADGRL1). To avoid pore formation, we use the mutant α-latrotoxin (LTX^N4C), which does not form pores and only acts through ADGRL1. ADGRL1 is cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), which behave as independent cell-surface proteins, reassociating upon binding LTX^N4C. We investigated the role of the NTF-CTF association in LTX^N4C action, using perfluorooctanoic acid (PFOA). We demonstrate that at low concentrations (≤100 μM) PFOA does not adversely affect ADGRL1-expressing neuroblastoma cells or inhibit LTX^N4C binding. However, it causes the dissociation of the NTF-CTF complexes, independent redistribution of the fragments on the cell surface, and their separate internalization. PFOA also promotes the dissociation of NTF-CTF complexes induced by LTX^N4C binding. When applied to mouse neuromuscular junctions, PFOA inhibits LTX^N4C-induced neurotransmitter release in a concentration-dependent manner. Our results indicate that ADGRL1 can mediate LTX^N4C signaling only while its fragments remain associated. These findings explain some aspects of receptor-dependent toxin action and contribute to a mechanistic understanding of ADGRL1 functions in neurons. Full article

α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca²⁺ (Ca²⁺_e). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca²⁺-chelator demonstrates that Ca²⁺_e-independent effects of αLTX require an increase in cytosolic Ca²⁺ (Ca²⁺_cyt). We also show that thapsigargin, which depletes Ca²⁺ stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX’s effects on Ca²⁺_cyt using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca²⁺_e, αLTX forms membrane pores and allows the influx of Na⁺; this reverses the Na⁺-Ca²⁺ exchanger, leading to the release of stored Ca²⁺ and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca²⁺ store and induces transient opening of Ca²⁺ channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca²⁺ entry. These results indicate that Ca²⁺ release from intracellular stores and that Ca²⁺ influx through latrophilin-activated store-operated Ca²⁺ channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals. Full article

Latrophilins (LPHNs), a group of the G-protein–coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer. Full article

α-Latrotoxin (α-LTX) was found to form two-dimensional (2D) monolayer arrays in solution at relatively low concentrations (0.1 mg/mL), with the toxin tetramer constituting a unit cell. The crystals were imaged using cryogenic electron microscopy (cryoEM), and image analysis yielded a ~12 Å projection map. At this resolution, no major conformational changes between the crystalline and solution states of α-LTX tetramers were observed. Electrophysiological studies showed that, under the conditions of crystallization, α-LTX simultaneously formed multiple channels in biological membranes that displayed coordinated gating. Two types of channels with conductance levels of 120 and 208 pS were identified. Furthermore, we observed two distinct tetramer conformations of tetramers both when observed as monodisperse single particles and within the 2D crystals, with pore diameters of 11 and 13.5 Å, suggestive of a flickering pore in the middle of the tetramer, which may correspond to the two states of toxin channels with different conductance levels. We discuss the structural changes that occur in α-LTX tetramers in solution and propose a mechanism of α-LTX insertion into the membrane. The propensity of α-LTX tetramers to form 2D crystals may explain many features of α-LTX toxicology and suggest that other pore-forming toxins may also form arrays of channels to exert maximal toxic effect. Full article

This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50 years. It discusses the discoveries and studies of various animal toxins, including protein and peptide neurotoxins acting on the nicotinic acetylcholine receptors (nAChRs) and on other ion channels. Among the achievements are the determination of the primary structures of the α-bungarotoxin-like three-finger toxins (TFTs), covalently bound dimeric TFTs, glycosylated cytotoxin, inhibitory cystine knot toxins (ICK), modular ICKs, and such giant molecules as latrotoxins and peptide neurotoxins from the snake, as well as from other animal venoms. For a number of toxins, spatial structures were determined, mostly by ¹H-NMR spectroscopy. Using this method in combination with molecular modeling, the molecular mechanisms of the interactions of several toxins with lipid membranes were established. In more detail are presented the results of recent years, among which are the discovery of α-bungarotoxin analogs distinguishing the two binding sites in the muscle-type nAChR, long-chain α-neurotoxins interacting with α9α10 nAChRs and with GABA-A receptors, and the strong antiviral effects of dimeric phospholipases A2. A summary of the toxins obtained from arthropod venoms includes only highly cited works describing the molecules’ success story, which is associated with IBCh. In marine animals, versatile toxins in terms of structure and molecular targets were discovered, and careful work on α-conotoxins differing in specificity for individual nAChR subtypes gave information about their binding sites. Full article

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration. Full article

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca²⁺-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca²⁺-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K⁺-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K⁺-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release. Full article

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native wildlife, and in temperate regions it lives in synanthropic environments where it frequently encounters humans, subsequently leading to envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). S. nobilis is a sister group to Latrodectus which possesses the highly potent neurotoxins called α-latrotoxins that can induce neuromuscular paralysis and is responsible for human fatalities. However, and despite this close relationship, the venom composition of S. nobilis has never been investigated. In this context, a combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins without any identified function to date. Among the toxins, the most represented in numbers are α-latrotoxins (61), δ-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), δ-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. Such toxins are highly potent towards the nervous system of vertebrates and likely responsible for the array of symptoms occurring after envenomation by black widows and false widows. Thus, caution should be taken in dismissing S. nobilis as harmless. This work paves the way towards a better understanding of the competitiveness of S. nobilis and its potential medical importance. Full article

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration. Full article

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (β-Bungarotoxin, β-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body. Full article

Widow spiders have received much attention due to the frequently reported human and animal injures caused by them. Elucidation of the molecular composition and action mechanism of the venoms and toxins has vast implications in the treatment of latrodectism and in the neurobiology and pharmaceutical research. In recent years, the studies of the widow spider venoms and the venom toxins, particularly the α-latrotoxin, have achieved many new advances; however, the mechanism of action of the venom toxins has not been completely clear. The widow spider is different from many other venomous animals in that it has toxic components not only in the venom glands but also in other parts of the adult spider body, newborn spiderlings, and even the eggs. More recently, the molecular basis for the toxicity outside the venom glands has been systematically investigated, with four proteinaceous toxic components being purified and preliminarily characterized, which has expanded our understanding of the widow spider toxins. This review presents a glance at the recent advances in the study on the venoms and toxins from the Latrodectus species. Full article

The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K⁺ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca²⁺ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery. Full article